U.S. patent application number 11/671951 was filed with the patent office on 2007-09-13 for methods for treating lentivirus infections.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Carlos M.C. deNoronha, Warner C. Greene, Kimberely S. Stopak.
Application Number | 20070212756 11/671951 |
Document ID | / |
Family ID | 34068095 |
Filed Date | 2007-09-13 |
United States Patent
Application |
20070212756 |
Kind Code |
A1 |
Greene; Warner C. ; et
al. |
September 13, 2007 |
METHODS FOR TREATING LENTIVIRUS INFECTIONS
Abstract
The present invention provides methods of identifying an agent
that inhibits an activity of a lentiviral Vif protein. The present
invention provides methods of identifying an agent that increases
the level of active APOBEC3G in a cell. The present invention
provides agents identified by a subject screening method; and
further provides methods for treating lentivirus infections.
Inventors: |
Greene; Warner C.;
(Hillsborough, CA) ; Stopak; Kimberely S.; (San
Francisco, CA) ; deNoronha; Carlos M.C.;
(Slingerlands, NY) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
34068095 |
Appl. No.: |
11/671951 |
Filed: |
February 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10865663 |
Jun 9, 2004 |
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11671951 |
Feb 6, 2007 |
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60477549 |
Jun 10, 2003 |
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60505738 |
Sep 24, 2003 |
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Current U.S.
Class: |
435/69.1 ;
435/345; 435/5 |
Current CPC
Class: |
A61P 31/18 20180101;
G01N 33/5008 20130101; G01N 2500/00 20130101; A61P 31/12 20180101;
C12Q 1/702 20130101; G01N 2333/155 20130101; A61P 43/00 20180101;
G01N 33/56983 20130101 |
Class at
Publication: |
435/069.1 ;
435/345; 435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 21/06 20060101 C12P021/06; C12N 5/06 20060101
C12N005/06; C12N 5/16 20060101 C12N005/16 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The U.S. government may have certain rights in this
invention, pursuant to grant nos. P01 HD40543, R01 CA86814, and
2P30 A127763-1 awarded by the National Institutes of Health.
Claims
1. A method for identifying an agent that inhibits Vif-mediated
degradation of APOBEC3G in a cell, the method comprising the steps
of: (a) contacting a cell that produces a Vif protein and an
APOBEC3G protein with a test agent; and (b) determining the effect,
if any, of the test agent on the level of APOBEC3G protein in the
cell; wherein a higher level of the APOBEC3G protein in the cell
contacted with the test agent compared to the level of APOBEC3G
protein in a cell not contacted with the test agent indicates that
the test agent inhibits Vif-mediated degradation of APOBEC3G in the
cell.
2. The method of claim 1, further comprising the step of: (c)
lysing the cell.
3. The method of claim 1, wherein the step of determining the level
of APOBEC3G protein in the cell comprises determining an
interaction between the Vif protein and the APOBEC3G protein.
4. The method of claim 1, wherein the step of determining the level
of APOBEC3G protein in the cell comprises determining binding of
the Vif protein to the APOBEC3G protein.
5. The method of claim 1, wherein the step of determining the level
of APOBEC3G protein in the cell comprises detecting ubiquitylated
APOBEC3G protein.
6. The method of claim 1, wherein the step of determining the level
of APOBEC3G protein in the cell comprises determining the ability
of a lentivirus to replicate in the cell, wherein an increase in
the level of APOBEC3G protein identifies the test agent as one that
inhibits lentiviral replication.
7. The method of claim 1, wherein the step of determining the level
of APOBEC3G protein in the cell comprises determining the number of
infectious lentiviral particles produced by the cell.
8. The method of claim 1, wherein the step of determining the level
of APOBEC3G protein in the cell comprises determining APOBEC3G
protein packaged in lentiviral particles.
9. The method of claim 1, wherein the step of determining the level
of APOBEC3G protein in the cell comprises detecting an APOBEC3G
mRNA.
10. The method of claim 1, wherein the step of determining the
level of APOBEC3G protein in the cell comprises determining an
enzymatic activity of the APOBEC3G protein.
11. The method of claim 10, wherein the step of determining the
level of APOBEC3G protein in the cell comprises determining the
number of C.fwdarw.U mutations in the minus strand of a lentiviral
nucleic acid in the cell.
12. The method of claim 1, wherein the step of determining the
level of APOBEC3G protein in the cell comprises detecting an
APOBEC3G protein comprising a label that provides a detectable
signal.
13. The method of claim 12, wherein the label is selected from
group of a radioisotope, a fluorescer, a chemiluminescer, an
epitope tag, an enzyme, a fusion partner, and a binding
partner.
14. The method of claim 13, wherein the label is a
radioisotope.
15. The method of claim 13, wherein the label is selected from the
group consisting of .beta.-galactosidase, luciferase, horse radish
peroxidase, glutathione-S-transferase, hemagglutinin (HA), FLAG,
c-myc, (His).sub.n, a fluorescent protein from an Anthozoa species,
a green fluorescent protein (GFP) from Renilla reniformis, a GFP
from Renilla mulleri, a GFP from Ptilosarcus guernyi, a GFP from
Aequoria Victoria, and a humanized GFP.
16. The method of claim 1, wherein the step of determining the
level of APOBEC3G protein in the cell comprises an immunological
assay.
17. The method of claim 16, wherein the immunological assay
comprises detecting APOBEC3G protein using an anti-APOBEC3G
antibody.
18. The method of claim 16, wherein the immunological assay
comprises detecting a detectable fusion partner in the cell that
produces an APOBEC3G fusion protein comprising the APOBEC3G protein
and the fusion partner.
19. The method of claim 18, wherein the fusion partner is selected
from the group consisting of HA, FLAG, c-myc, and (His).sub.n.
20. The method of claim 1, wherein the step of determining the
level of APOBEC3G protein in the cell comprises a fluorimetric
assay.
21. The method of claim 20, wherein the fluorimetric assay
comprises detecting a detectable fusion partner in the cell that
produces an APOBEC3G fusion protein comprising the APOBEC3G protein
and the fusion partner.
22. The method of claim 21, wherein the fusion partner is a green
fluorescent protein (GFP).
23. The method of claim 22, wherein the green fluorescent protein
is selected from the group of GFPs from Renilla reniformis, Renilla
mulleri, Ptilosarcus guernyi, Aequoria Victoria, a humanized green
fluorescent protein and fluorescent mutants thereof.
24. The method of claim 1, wherein the APOBEC3G protein is an
endogenous APOBEC3G protein produced in the cell.
25. The method of claim 1, wherein the cell is a cell culture.
26. The method of claim 1, wherein the cell is a eukaryotic
cell.
27. The method of claim 26, wherein the cell is a yeast cell.
28. The method of claim 26, wherein the cell is a mammalian
cell.
29. The method of claim 26, wherein the cell is an immortalized
cell.
30. The method of claim 28, wherein the cell is a non-permissive
cell to infection with a human immunodeficiency virus.
31. The method of claim 30, wherein the cell is a H9 cell.
32. The method of claim 28, wherein the cell is a permissive cell
to infection with a human immunodeficiency virus.
33. The method of claim 32, wherein the cell is a 293T cell.
34. The method of claim 1, wherein the cell is genetically modified
with a nucleic acid comprising a nucleotide sequence encoding a Vif
protein, wherein the Vif protein is produced in the cell.
35. The method of claim 34, wherein Vif is expressed from a
vector.
36. The method of claim 35, wherein the Vif protein is expressed
from a lentiviral vector.
37. The method of claim 34, wherein the cell is genetically
modified with a nucleic acid comprising a nucleotide sequence
encoding a Vif protein and a fusion partner, wherein a Vif fusion
protein is produced in the cell.
38. The method of claim 34, wherein the cell is transiently
transfected with the nucleic acid.
39. The method of claim 34, wherein the cell is stably transfected
with the nucleic acid.
40. The method of claim 1, wherein the cell is genetically modified
with a nucleic acid comprising a nucleotide sequence encoding an
APOBEC3G protein, wherein the APOBEC3G protein is produced in the
cell.
41. The method of claim 40, wherein APOBEC3G protein is expressed
from a vector.
42. The method of claim 41, wherein the APOBEC3G protein is
expressed from a lentiviral vector.
43. The method of claim 40, wherein the cell is genetically
modified with a nucleic acid comprising a nucleotide sequence
encoding an APOBEC3G protein and a fusion partner, wherein an
APOBEC3G fusion protein is produced in the cell.
44. The method of claim 40, wherein the cell is transiently
transfected with the nucleic acid.
45. The method of claim 40, wherein the cell is stably transfected
with the nucleic acid.
46. The method of claim 1, wherein the eukaryotic cell is
genetically modified with one or more nucleic acids comprising a
nucleotide sequence encoding an APOBEC3G protein and a nucleotide
sequence encoding a Vif protein, wherein the APOBEC3G protein and
the Vif protein are produced in the cell.
47. The method of claim 46, wherein the APOBEC3G protein and the
Vif protein are expressed from a vector.
48. The method of claim 47, wherein the APOBEC3G protein and the
Vif protein are expressed from a lentiviral vector.
49. The method of claim 46, wherein the cell is genetically
modified with (i) a nucleic acid comprising a nucleotide sequence
encoding an APOBEC3G protein and a first fusion partner; wherein an
APOBEC3G fusion protein is produced in the cell, and (ii) a nucleic
acid comprising a nucleotide sequence encoding a Vif protein and a
second fusion partner; wherein a Vif fusion protein is produced in
the cell.
50. The method of claim 46, wherein the cell is transiently
transfected with the nucleic acid.
51. The method of claim 46, wherein the cell is stably transfected
with the nucleic acid.
52. The method of claim 1, wherein the cell is infected with a
lentivirus.
53. The method of claim 52, wherein the lentivirus is selected from
the group consisting of human immunodeficiency virus-1 (HIV-1),
human immunodeficiency virus-2 (HIV-2), simian immunodeficiency
virus and feline immunodeficiency virus.
54. The method of claim 52, wherein the lentivirus is HIV-1 or
HIV-2.
55. The method of claim 1, wherein the step of determining the
level of APOBEC3G protein in the cell comprises using a high
throughput technique.
56. The method of claim 1, wherein the test agent comprises a
synthetic, semi-synthetic, or naturally-occurring inorganic or
organic molecule.
57. The method of claim 1, wherein the test agent is selected from
the group of biomolecules consisting of a peptides, a
peptidomimetic, a peptide aptamer, a saccharide, a fatty acid, a
steroid, a purines, a pyrimidine, a derivative, structural analog
and combination thereof.
58. The method of claim 57, wherein the test agent comprises a
peptide or peptidomimetic.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/477,549 filed Jun. 10, 2003, and U.S.
Provisional Patent Application No. 60/505,738, filed Sep. 24, 2003,
which applications are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0003] This invention is in the field of treating lentivirus
infections, particularly immunodeficiency virus infections.
BACKGROUND OF THE INVENTION
[0004] Human immunodeficiency virus (HIV) is the etiologic agent of
acquired immunodeficiency syndrome (AIDS). HIV infection leads to
depletion of CD4.sup.+ T lymphocytes. AIDS is characterized by
various pathological conditions, including immune incompetence,
opportunistic infections, neurological dysfunctions, and neoplastic
growth.
[0005] HIV-1 relies on Vif (virion infectivity factor), a protein
encoded by HIV-1 and many related primate lentiviruses, to evade
the potent innate antiviral function of APOBEC3G (apolipoprotein B
mRNA-editing enzyme, catalytic polypeptide-like 3G, also known as
CEM15). APOBEC3G is a DNA cytidine deaminase that is incorporated
into virions and produces extensive hypermutation in newly
synthesized viral DNA formed during reverse transcription.
[0006] Several drugs have been approved for treatment of AIDS,
including azidovudine (AZT), didanosine (dideoxyinosine, ddI), d4T,
zalcitabine (dideoxycytosine, ddC), nevirapine, lamivudine (epivir,
3TC), saquinavir (Invirase), ritonavir (Norvir), indinavir
(Crixivan), and delavirdine (Rescriptor). However, none of the
available drugs used to combat HIV is completely effective, and
treatment frequently gives rise to drug-resistant virus.
[0007] Despite the availability of a number of drugs to combat HIV
infections, there is a need in the art for additional drugs that
inhibit HIV replication, and which are suitable for treating HIV
and other lentiviral infections. The present invention addresses
this need by providing methods for identifying agents that target
the HIV Vif protein, as well as agents that interfere with Vif
action in a manner that preserves the potent innate anti-lentiviral
properties of APOBEC3G.
Literature
[0008] Fisher et al. (1987) Science 237, 888-893; von Schwedler et
al. (1993) J Virol 67, 4945-4955; Desrosiers et al. (1998) J Virol
72, 1431-1437; Gabuzda et al. (1992) J Virol 66, 6489-6495; Sova
and Volsky (1993) J Virol 67, 6322-6326; Harris et al. (2003)
(2003) Cell 113:803-809; Harris et al. (2004) Cell 116:629;
Lecossier et al. (2003) Science 300:1112; Mangeat et al. (2003)
Nature 424:99-103; Zhang et al. (2003) Nature 424:94-98.
SUMMARY OF THE INVENTION
[0009] The present invention provides methods of identifying an
agent that inhibits an activity of a lentiviral Vif protein. The
present invention provides methods of identifying an agent that
increases the level of active APOBEC3G in a cell. The present
invention provides agents identified by a subject screening method;
and further provides methods for treating lentivirus
infections.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1A and 1B depict expression and localization of
APOBEC3G.
[0011] FIG. 2 depicts the inhibitory effect of Vif on incorporation
of full-length endogenous APOBEC3G into virions.
[0012] FIGS. 3A and 3B depict intracellular depletion of APOBEC3G
induced by HIV wild type but not HIV.DELTA.Vif viruses following
infection of human H9 cells.
[0013] FIG. 4 depicts APOBEC3G mRNA levels in the presence of
Vif.
[0014] FIGS. 5A-5C depict intracellular depletion of APOBEC3G
induced by Vif alone.
[0015] FIGS. 6A and 6B depict Vif association with APOBEC3G/RNA
high molecular weight complexes.
[0016] FIGS. 7A-C depict HIV-1 Vif induction of APOBEC3G
degradation.
[0017] FIGS. 8A-C depict the effect of Vif on the half-life of
APOBEC3G; and the effect of proteasome inhibitors on depletion of
intracellular APOBEC3G.
[0018] FIGS. 9A-B depict the effect of Vif on APOBEC3G-HA mRNA.
[0019] FIG. 10 is a schematic representation depicting how Vif
targets ubiquitylated APOBEC3G for degradation in a virus-producing
cell, thus blocking virion incorporation of APOBEC3G; and the
potent mutagenic action of APOBEC3G's cytidine deaminase activity
during viral DNA synthesis.
[0020] FIG. 11 depicts the effect of phytohemagglutinin (PHA),
anti-CD3 antibody, anti-CD28 antibody, and IL-15 on the level of
APOBEC3G in peripheral blood lymphocytes (PBL).
[0021] FIG. 12 depicts the effect of IL-2 on APOBEC3G levels in
PBL.
[0022] FIG. 13 depicts the effect of
phorbol-12-myristate-13-acetate (PMA), PHA, anti-CD3 antibody,
anti-CD28 antibody, and prostratin on the level of APOBEC3G in
PBL.
[0023] FIG. 14 depicts size-exclusion FPLC analysis of endogenous
APOBEC3G. Endogenous APOBEC3G is assembled into a high molecular
weight, RNase-sensitive complex in nonpermissive H9 cells.
[0024] FIG. 15 depicts results showing that transfection of
HA-APOBEC3G DNA into permissive cells leads to HMW APOBEC3G complex
formation.
[0025] FIG. 16 depicts results showing that the low molecular
weight, but not the high molecular weight, APOBEC3G complex
exhibits cytidine deaminase activity.
[0026] FIG. 17 depicts results showing that APOBEC3G in resting PBL
is present in a LMW complex.
[0027] FIG. 18 depicts results showing that PHA/IL-2 stimulation of
CD4.sup.+ PBL promotes assembly of HMW APOBEC3G complexes.
[0028] FIG. 19 depicts results showing that APOBEC3G in monocytes
is present in a LMW complex.
[0029] FIG. 20 depicts results showing that differentiation of
monocytes promotes assembly of HMW APOBEC3G complexes.
DEFINITIONS
[0030] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse affect attributable to the disease. "Treatment," as
used herein, covers any treatment of a disease in a mammal,
particularly in a human, and includes: (a) preventing the disease
from occurring in a subject which may be predisposed to the disease
but has not yet been diagnosed as having it; (b) inhibiting the
disease, i.e., arresting its development; and (c) relieving the
disease, e.g., causing regression of the disease, e.g., to
completely or partially remove symptoms of the disease. In the
context of lentivirus infection, the term "treatment" encompasses
prevention of establishment of a systemic infection following
initial contact with the virus; and prophylactic treatment of an
individual not yet infected with the virus.
[0031] The terms "individual," "host," "subject," and "patient,"
used interchangeably herein, refer to a mammal, including, but not
limited to, murines, felines, simians, humans, mammalian farm
animals, mammalian sport animals, and mammalian pets. The term
includes mammals that are susceptible to infection by a lentivirus
that encodes Vif. In many embodiments, treatment of humans is of
interest.
[0032] By "genetic transformation" is meant a permanent or
transient genetic change induced in a cell following incorporation
of new DNA (i.e., DNA exogenous to the cell). Genetic change can be
accomplished either by incorporation of the new DNA into the genome
of the host cell, or by transient or stable maintenance of the new
DNA as an episomal element. Where the cell is a mammalian cell, a
permanent genetic change is generally achieved by introduction of
the DNA into the genome of the cell.
[0033] A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents; washed; or enrichment for certain cell
populations, such as CD4.sup.+ T lymphocytes, glial cells,
macrophages, tumor cells, peripheral blood mononuclear cells
(PBMC), and the like. The term "biological sample" encompasses a
clinical sample, and also includes cells in culture, cell
supernatants, tissue samples, organs, bone marrow, and the
like.
[0034] The term "lentivirus" as used herein, refers to human
immunodeficiency virus-1 (HIV-1); human immunodeficiency virus-2
(HIV-2); simian immunodeficiency virus (SIV); and feline
immunodeficiency virus (FIV).
[0035] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0036] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0037] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0038] It must be noted that as used herein and in the appended
claims, the singular forms "a," "and," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a Vif protein" includes a plurality of such
proteins and reference to "the agent" includes reference to one or
more agents and equivalents thereof known to those skilled in the
art, and so forth.
[0039] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention provides methods of identifying an
agent that inhibits an activity of a lentiviral Vif protein. The
invention further provides methods of identifying an agent that
increases the level of active APOBEC3G in a cell; as well as
methods of identifying an agent that converts an inactive form of
APOBEC3G to an active form of APOBEC3G. The present invention
provides agents identified by a subject screening methods; and
further provides methods for treating lentivirus infections.
[0041] Human immunodeficiency virus type 1 (HIV-1) relies on
Vif(virion infectivity factor), a protein encoded by HIV-1 and many
related primate lentiviruses, to evade the potent innate antiviral
function of APOBEC3G (apolipoprotein B mRNA-editing enzyme,
catalytic polypeptide-like 3G, also known as CEM15). APOBEC3G is a
DNA cytidine deaminase that is incorporated into virions and
produces extensive hypermutation in newly synthesized viral DNA
formed during reverse transcription.
[0042] The present invention is based in part on the following
observations: 1) Vif impairs the incorporation of native APOBEC3G
protein into virions by targeting it for degradation within
infected cells (including, e.g., T cells, macrophages, glial cells,
etc.) infected with a Vif-encoding lentivirus; 2) Vif alone is
sufficient to induce the loss of APOBEC3G, indicating that Vif does
not require the activity of other viral proteins; 3) Vif either
directly or indirectly assembles with APOBEC3G; 4) APOBEC3G and Vif
co-fractionate in a >700 kD complex in infected H9 cells, and
this large APOBEC3G complex disassembles following treatment with
RNAse; 5) APOBEC3G is polyubiquitylated; 6) Vif targets modified
forms of APOBEC3G protein, such as polyubiquitylated APOBEC3G, for
degradation; 7) Vif inhibits translation of APOBEC3G mRNA; 8)
APOBEC3G is found in a high molecular weight (HMW) complex in cells
permissive for HIV infection; 9) certain factors can increase the
level of APOBEC3G protein in a cell; 10) Low molecular weight (LMW)
APOBEC3G complexes exhibit cytidine deaminase activity, while HMW
APOBEC3G complexes do not.
Screening Methods
[0043] The present invention provides methods of identifying an
agent that inhibits a lentivirus Vif activity. The methods
generally involve contacting a sample containing a Vif protein and
an APOBEC3G protein with a test agent; and determining the effect,
if any, of the test agent on a Vif activity. A test agent that
affects Vif activity is a candidate agent for treating lentiviral
infections.
[0044] The present invention further provides methods of
identifying an agent that increases the level of active APOBEC3G
protein in a cell. The methods generally involve contacting a cell
with a test agent; and determining the effect, if any, of the test
agent on the level of active APOBEC3G protein in the cell. A test
agent of interest increases the level of active APOBEC3G in a cell,
regardless of the mechanism by which the increase is achieved. For
example, an agent that increases a level of active APOBEC3G in a
cell includes i) an agent that increases production of active
APOBEC3G by the cell, regardless of the mechanism; ii) an agent
that reduces formation of an inactive form of APOBEC3G in a cell;
iii) an agent that induces a shift from an inactive form of
APOBEC3G to an active form of APOBEC3G in a cell; and the like.
[0045] The present invention provides methods of identifying an
agent that converts an inactive form of APOBEC3G protein to an
active form of an APOBEC3G protein in a cell. The methods generally
involve contacting a cell with a test agent; and determining the
effect, if any, of the test agent on converting an inactive form of
APOBEC3G to an active form of APOBEC3G.
[0046] As used herein, the term "active APOBEC3G" refers to a form
of APOBEC3G that is enzymatically active and induces one or more
mutations in a lentivirus genome. An "active" form of APOBEC3G is a
form that can induce mutations in a lentivirus genome. In general,
active APOBEC3G is a low molecular weight form of APOBEC3G, e.g., a
form that exists in a low molecular weight complex. In some
embodiments, a low molecular weight form of APOBEC3G is a form of
APOBEC3G that exists in a complex that has a molecular weight in a
range of from about 50 kD to about 100 kD, as determined by
size-exclusion fast performance liquid chromatography (FPLC). An
agent of interest that increases the level of APOBEC3G is in many
embodiments an agent that increases a level of APOBEC3G that
induces mutations into a lentivirus genome, such that the active
APOBEC3G induces a sufficient number of mutations such that viral
replication is inhibited and/or production of infectious virus is
reduced.
[0047] As used herein, the term "inactive APOBEC3G" refers to a
form of APOBEC3G that is relatively inactive, e.g., is a form that
does not induce mutations in a lentivirus genome in the cytoplasm
of a cell that would result in disabling the lentivirus, e.g., a
form that does not induce a number of mutations in a lentivirus
genome that would be sufficient to adversely affect production of
infectious virus; or that is not incorporated into a lentivirus
virion. In some embodiments, an inactive form of APOBEC3G is a form
that exists in a high molecular weight complex. In some
embodiments, a high molecular weight form of APOBEC3G is a form of
APOBEC3G that exists in a complex that has a molecular weight in a
range of greater than about 700 kD, as determined by size-exclusion
FPLC.
[0048] As used herein, the term "Vif activity" includes, but is not
limited to, Vif binding to APOBEC3G; Vif-mediated reduction of
intravirion incorporation of endogenous APOBEC3G protein; Vif
association with a high-molecular weight complex comprising
APOBEC3G and cellular RNA; Vif inhibition of translation of
APOBEC3G mRNA; and Vif induction of APOBEC3G degradation.
[0049] A subject assay for identifying agents that inhibit Vif
activity can be designed in a number of ways. In some embodiments,
the assay provides for determining the effect of a test agent on
the level of APOBEC3G protein in a cell in the presence of Vif. In
other embodiments, the assay provides for determining the effect of
a test agent on Vif association with APOBEC3G or an APOBEC3G high
molecular weight complex. These embodiments are described in detail
below.
[0050] A subject assay for identifying agents that increase the
level of active APOBEC3G protein in a cell can be designed in a
number of ways. In some embodiments, a subject assay for
identifying an agent that increases the level of active APOBEC3G
involves determining the effect of a test agent on the level of
APOBEC3G protein that induces a C.fwdarw.U mutation in minus strand
DNA, resulting in a G.fwdarw.A mutation in viral plus strand DNA.
In other embodiments, a subject assay for identifying an agent that
increases the level of active APOBEC3G involves determining the
effect of the agent on packaging of APOBEC3G into a lentivirus
particle.
[0051] Methods of identifying an agent that converts an inactive
form of APOBEC3G protein to an active form of an APOBEC3G protein
in a cell can be designed in a number of ways. For example, in some
embodiments, a method of identifying an agent that converts an
inactive form of APOBEC3G to an active form of APOBEC3G involves
contacting a cell with a test agent; and determining the molecular
weight of a complex with which APOBEC3G is associated in the cell.
Generally, an agent of interest is a test agent that converts a
high molecular weight APOBEC3G-containing complex to a low
molecular weight APOBEC3G-containing complex. In other embodiments,
a method of identifying an agent that converts an inactive form of
APOBEC3G to an active form of APOBEC3G involves contacting a cell
with a test agent; and determining the effect, if any, of the test
agent on the number of APOBEC3G-induced mutations in a lentivirus
genome present in the cell.
[0052] The subject screening methods are carried out in vitro. In
some embodiments, the methods are cell-based methods. In other
embodiments, the methods are cell-free methods.
[0053] The terms "candidate agent," "agent," "substance," "test
agent," and "compound" are used interchangeably herein. Candidate
agents encompass numerous chemical classes, and are generally
synthetic, semi-synthetic, or naturally-occurring inorganic or
organic molecules.
[0054] In some embodiments, a candidate agent is a small organic
compound having a molecular weight of more than 50 daltons and less
than about 2,500 daltons.
[0055] Candidate agents may comprise functional groups necessary
for structural interaction with proteins, particularly hydrogen
bonding, and may include at least an amine, carbonyl, hydroxyl or
carboxyl group, and may contain at least two of the functional
chemical groups. The candidate agents may comprise cyclical carbon
or heterocyclic structures and/or aromatic or polyaromatic
structures substituted with one or more of the above functional
groups. Candidate agents are also found among biomolecules
including peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations
thereof.
[0056] In other embodiments, a candidate agent has a molecular
weight of greater than 2,500 daltons. In these embodiments, a
candidate agent may be a peptide, an oligopeptide, a polypeptide, a
carbohydrate, a polysaccharide, a lipid, a lipopolysaccharide, a
glycoprotein, a proteoglycan, a lipoprotein, or other
macromolecule.
[0057] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
[0058] A candidate agent is assessed for any cytotoxic activity it
may exhibit toward the cell used in the assay, using well-known
assays, such as trypan blue dye exclusion, an MTT
([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium
bromide]) assay, and the like. Agents that do not exhibit
significant cytotoxic activity are considered candidate agents.
[0059] Assays of the invention usually include one or more
controls. Thus, a test sample includes a test agent, and a control
sample has all the components of the test sample except for the
test agent. Generally a plurality of assay mixtures is run in
parallel with different agent concentrations to obtain a
differential response to the various concentrations. Typically, one
of these concentrations serves as a negative control, i.e. at zero
concentration or below the level of detection. Where the screening
assay is a binding assay, one or more of the molecules may be
joined to a label, where the label can directly or indirectly
provide a detectable signal. Various labels include radioisotopes,
fluorescers, chemiluminescers, enzymes, specific binding molecules,
particles, e.g. magnetic particles, and the like. Specific binding
molecules include pairs, such as biotin and streptavidin, digoxin
and antidigoxin, etc. For the specific binding members, the
complementary member would normally be labeled with a molecule that
provides for detection, in accordance with known procedures.
[0060] A variety of reagents may be included in the screening
assay. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc that are used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Reagents that improve the efficiency of the assay,
such as nuclease inhibitors, anti-microbial agents, etc. may be
used. The components may be added in any order. Incubations are
performed at any suitable temperature, typically between 4.degree.
C. and 40.degree. C. Incubation periods are selected for optimum
activity, but may also be optimized to facilitate rapid
high-throughput screening. Typically between 0.1 and 1 hour will be
sufficient.
[0061] The screening methods may be designed a number of different
ways, where a variety of assay configurations and protocols may be
employed, as are known in the art. For example, one of the
components may be bound to a solid support, and the remaining
components contacted with the support bound component. As another
example, a protein whose activity is being assayed is in some
embodiments an endogenous protein (e.g., a protein that is normally
produced by the cell); and in other embodiments, a protein whose
activity is being assayed is encoded on an expression construct and
introduced into the cell, such the encoded protein is produced in
the cell. The above components of the method may be combined at
substantially the same time or at different times.
[0062] Where the assay is a binding assay, following the contact
and incubation steps, the subject methods will generally, though
not necessarily, further include a washing step to remove unbound
components, where such a washing step is generally employed when
required to remove label that would give rise to a background
signal during detection, such as radioactive or fluorescently
labeled non-specifically bound components. Following the optional
washing step, the presence of bound complexes will then be
detected.
[0063] In many embodiments, for safety reasons, where a subject
assay method involves testing for viral infectivity, encapsidation
of APOBEC3G in a viral particle, etc., instead of using an
infectious lentivirus, a subject assay will employ a construct that
includes nucleotide sequences encoding viral structural proteins
(e.g., Gag) and other proteins necessary for encapsidation of a
viral particle; however, the viral particles so produced will not
be pathogenic for laboratory personnel. Thus, in the present
application, the term "lentivirus-infected cell" and similar terms
are understood to include cells infected with pathogenic virus, as
well as (e.g., for testing purposes) cells transfected with viral
constructs that do not lead to production of viral particles that
are pathogenic for humans.
[0064] Furthermore, it should be understood that a subject method
that involves determining the effect of a test agent on the number
of APOBEC3G-induced mutations in a lentivirus genome need not be
carried out using the entire lentivirus genome. Instead, a portion
of a lentivirus genome can be used to determine the effect of a
test agent on the number of APOBEC3G-induced mutations in a
lentivirus genome. Thus, the term "lentivirus nucleic acid," as
used herein, thus includes all or a portion of a lentivirus genome,
where the nucleic acid may be single stranded or double stranded.
The term "lentivirus nucleic acid" includes nucleic acids that
comprise, in addition to a lentivirus nucleotide sequence, a
heterologous nucleotide sequence (e.g., non-lentivirus nucleotide
sequences, such as nucleotide sequences encoding detectable
markers; non-lentivirus control elements; and the like). The term
"lentivirus nucleic acid" includes nucleic acids that comprise
naturally-occurring lentivirus nucleotide sequences; variants of
lentivirus nucleotide sequences; and synthetic lentivirus
nucleotide sequences. The term "lentivirus nucleic acid" includes a
nucleic acid that comprises lentivirus minus strand DNA. In many
embodiments, for assays involving determining the number of
mutations in a lentivirus genome, and in particular determining the
number of APOBEC3G-induced mutation in a lentivirus genome, a
lentivirus nucleic acid that includes lentivirus minus strand DNA
is used.
[0065] In some embodiments, a test agent of interest is one that
reduces a Vif activity by at least about 10%, at least about 20%,
at least about 25%, at least about 30%, at least about 35%, at
least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about 60%, at least about 65%, at least about
70%, at least about 80%, at least about 90%, or more, when compared
to the level of the Vif activity in a control in the absence of the
test agent.
[0066] In other embodiments, a test agent of interest is one that
increases the level of active APOBEC3G in a cell by at least about
10%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 80%, at least about 90%, at
least about 2-fold, at least about 5-fold, at least about 10-fold,
or at least about 25-fold, or more, when compared to the level of
active APOBEC3G in a cell in the absence of the test agent.
[0067] In other embodiments, a test agent of interest is one that
increases the number of mutations (e.g., APOBEC3G-induced
mutations) in a lentivirus nucleic acid by at least about 10%, at
least about 20%, at least about 25%, at least about 30%, at least
about 35%, at least about 40%, at least about 45%, at least about
50%, at least about 55%, at least about 60%, at least about 65%, at
least about 70%, at least about 80%, at least about 90%, at least
about 2-fold, at least about 5-fold, at least about 10-fold, or at
least about 25-fold, or more, when compared to the number of
mutations in the lentivirus nucleic acid in the absence of the test
agent.
[0068] In other embodiments, a test agent of interest is one that
converts at least about 10%, at least about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
80%, or at least about 90%, or more, of an enzymatically inactive
form of APOBEC3G to an enzymatically active form of APOBEC3G in a
eukaryotic cell, when compared to the level of inactive APOBEC3G in
the cell in the absence of the test agent.
Level of APOBEC3G
[0069] In some embodiments, a subject screening method involves
determining the effect of a test agent on the level of APOBEC3G
protein in a cell in the presence of Vif protein. Thus, in some
embodiments, the method involves contacting a cell that produces
Vif and APOBEC3G with a test agent; and determining the effect, if
any, of the test agent on the level of APOBEC3G protein in the
cell.
[0070] In these embodiments, a test agent of interest increases the
level of APOBEC3G in the presence of Vif protein by at least about
10%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 80%, at least about 90%, at
least about 2-fold, at least about 4-fold, at least about 5-fold,
at least about 10-fold, at least about 15-fold, at least about
20-fold, or more, compared the level of APOBEC3G in the presence of
Vif protein and in the absence of the test agent.
[0071] The APOBEC3G protein that is produced by the cell may have
the amino acid sequence that is provided under GenBank Accession
No. NP.sub.--068594, or fragments or variants thereof. For example,
the APOBEC3G protein can have truncations of one to 50 amino acids
from the amino terminus and/or the carboxyl terminus of the
sequence provided under NP.sub.--068594. The APOBEC3G protein that
is used can vary from the sequence provided under NP-068594 by from
one to about 20 amino acids, e.g., the sequence can include from
one to about 20 amino acid substitutions. Typically, the APOBEC3G
protein that is used retains cytidine deaminase activity of the
native, naturally-occurring protein. Suitable APOBEC3G proteins
also include fusion proteins that include APOBEC3G and a
heterologous protein (a "fusion partner") fused in-frame to the
amino terminus or carboxyl terminus of the APOBEC3G protein.
Suitable fusion partners include peptides and polypeptides that
provide ease of purification, e.g., (His).sub.n, e.g., 6His, and
the like; provide an epitope tag, e.g., glutathione-S-transferase
(GST), hemagglutinin (HA; e.g., CYPYDVPDYA; SEQ ID NO:1), FLAG
(e.g., DYKDDDDK; SEQ ID NO:2), c-myc (e.g., CEQKLISEEDL; SEQ ID
NO:3), and the like; peptides and polypeptides provide a detectable
signal, e.g., an enzyme that generates a detectable product (e.g.,
.beta.-galactosidase, luciferase, horse radish peroxidase, alkaline
phosphatase, etc.), or a protein that is itself detectable, e.g., a
fluorescent protein (e.g., a green fluorescent protein), etc.; and
the like. In some embodiments, an APOBEC3G protein comprises the
amino acid sequence set forth in SEQ ID NO:4.
[0072] The Vif protein that is produced by the cell can have the
amino acid sequence of any known lentivirus Vif protein. For
example, the amino acid sequences of HIV-1 Vif proteins are
provided under GenBank Accession Nos. AAP33677; AAN74526; AAN74517;
AAP29646; AAD10945; AAD10937; AAD10929; etc.
[0073] In many embodiments, the effect of a test agent on APOBEC3G
protein levels in the presence of Vif is determined by introducing
expression vectors, which include (typically on separate vectors)
Vif and APOBEC3G coding sequences, into suitable eukaryotic cells
in in vitro culture, generating genetically modified cells that
produce Vif and APOBEC3G proteins; contacting the genetically
modified cells with a test agent; and determining the effect, if
any, of the test agent on the level of APOBEC3G protein produced by
the genetically modified cell.
[0074] Expression vectors that are suitable for expression in
eukaryotic cells are constructed to include a coding region for a
Vif protein (for production of Vif protein in the cell) or a coding
region for APOBEC3G (for production of APOBEC3G in the cell). An
expression vector comprising a Vif or an APOBEC3G coding region
will generally include regulatory sequences ("control sequences" or
"control regions") which are necessary to effect the expression of
a Vif- or an APOBEC3G-coding polynucleotide to which they are
operably linked. Expression vectors typically comprise a
transcription initiation region, a promoter region, a Vif- or an
APOBEC3G-coding nucleotide sequence, and a transcriptional
termination region. Suitable promoters include constitutive
promoters and inducible promoters, a number of which are well known
in the art. Expression vectors generally have convenient
restriction sites located near the promoter sequence to provide for
the insertion of nucleic acid sequences encoding Vif or APOBEC3G
proteins. A selectable marker operative in the expression host may
be present.
[0075] Expression vectors are introduced into eukaryotic cells
using any convenient means, including calcium phosphate
precipitation; electroporation; infection (where the expression
vectors are packaged into viral particles); liposome-mediated
transfection; and the like. Suitable cells are eukaryotic cells,
typically mammalian cells, including primary cells, immortalized
cell lines, etc., including, but not limited to, COS cells, 293T
cells, Jurkat cells, H9 cells, and the like.
[0076] Whether a test agent inhibits Vif-induced reduction of
APOBEC3G protein levels can be determined by any known method for
determining the level of a particular protein in a cell. In some
embodiments, the assay is an immunological assay, using a
APOBEC3G-specific antibody. Such methods include, but are not
limited to, immunoprecipitating APOBEC3G from a cellular extract,
and analyzing the immunoprecipitated APOBEC3G by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE); detecting a
detectable fusion partner in a cell that produces a fusion protein
that includes APOBEC3G and a fusion partner that provides a
detectable signal; standard SDS-PAGE and immunoblotting (e.g.,
transfer of proteins from a gel generated during SDS-PAGE to a
membrane, and probing the membrane with detectably labeled
antibodies) of APOBEC3G from cells producing APOBEC3G.
[0077] In other embodiments, the assay is an assay that detects a
fusion partner of a APOBEC3G fusion protein. Thus, e.g., where the
APOBEC3G is part of a fusion protein that includes APOBEC3G, and,
as a fusion partner, a protein that provides a detectable signal,
the assay detects the fusion partner. Fusion partners include, but
are not limited to, a green fluorescent protein (GFP); a
fluorescent protein from an Anthozoa species (see, e.g., Matz et
al. (1999) Nat. Biotechnol. 17:969-973); luciferase;
.beta.-galactosidase; and the like. A construct that includes a
nucleotide sequence that encodes the APOBEC3G fusion protein is
introduced into a eukaryotic cell. The level of APOBEC3G protein
that is produced in the cell is determined by detecting the fusion
partner. Immunological assays (protein blots, ELISAs, etc.) are
used where the fusion partner is an immunological tag. Enzymatic
assays are used where the fusion partner is an enzyme (e.g.,
.beta.-galactosidase, luciferase, horse radish peroxidase, etc.)
that produces a detectable product. Fluorimetric assays are used to
detect fusion partners that are fluorogenic. A luminometer is used
to detect fusion partners that yield a luminescent product. The
fusion partner is detected by any known method appropriate to the
fusion partner. The fusion partner is detected in a cell extract,
using an assay appropriate to the fusion partner (e.g., an
enzymatic assay, an immunological assay, etc.); or the fusion
partner is detected in an intact cell, e.g., using flow cytometry.
For example, where the fusion partner provides a fluorescent
signal, the level of APOBEC3G is in some embodiments determined
using a flow cytometer.
[0078] The following is one non-limiting example of a suitable
assay for identifying agents that reduce Vif-induced degradation of
APOBEC3G. Mammalian cells (e.g., an immortalized cell line) are
stably transfected with an expression vector that includes a coding
sequence for a luciferase-APOBEC3G fusion protein, such that the
cells synthesize the luciferase-APOBEC3G (luc-APOBEC3G) fusion
protein. A substrate for luciferase is provided in the cell medium,
such that the substrate enters the cell, and is acted on by the
luciferase portion of the luciferase-APOBEC3G to yield a
luminescent product. The level of luciferase in the cell is
detected using a luminometer, and the level of luc-APOBEC3G is
expressed as relative light units. The cells are transfected with
an expression vector that includes a coding sequence for Vif to
yield a Vif/luc-APOBEC3G cell. Because Vif induces degradation of
luc-APOBEC3G, the presence of Vif protein in the Vif/luc-APOBEC3G
cell reduces the level of luc-APOBEC3G, compared to the level of
luc-APOBEC3G in the absence of Vif. When the Vif/luc-APOBEC3G cell
is contacted with a test agent that reduces Vif-induced APOBEC3G
degradation, the level of luc-APOBEC3G increases relative to the
level of luc-APOBEC3G in the Vif/luc-APOBEC3G cell in the absence
of the test agent.
[0079] Thus, in some embodiments, a method of identifying an agent
that reduces Vif-induced APOBEC3G degradation involves contacting a
cell, which cell produces Vif and an APOBEC3G protein fused to a
protein that provides a detectable signal, with a test agent (and,
if necessary, a substrate for the protein that provides a
detectable signal); and determining the effect, if any, of the test
agent on the level of APOBEC3G in the cell. In some embodiments, a
method of identifying an agent that reduces Vif-induced APOBEC3G
degradation involves contacting a cell that produces Vif and an
APOBEC3G-luciferase protein with a test agent and a substrate for
luciferase; and determining the effect, if any, of the test agent
on the level of APOBEC3G-luciferase in the cell, compared to the
level of APOBEC3G-luciferase in the cell in the absence of the test
agent. An agent that increases the level of APOBEC3G-luciferase
protein in the cell by at least about 5%, at least about 10%, at
least about 20%, at least about 30%, at least about 50%, at least
about 75%, at least about 100%, at least about 2.5-fold, at least
about 3-fold, at least about 4-fold, at least about 5-fold, at
least about 10-fold, at least about 20-fold, or at least about
50-fold, or more, is an agent that inhibits Vif-induced APOBEC3G
degradation.
Methods of Identifying Agents that Increase a Level of Active
APOBEC3G in a Cell
[0080] In some embodiments, the invention provides methods of
identifying an agent that increases a level of active APOBEC3G in a
cell. In these embodiments, a subject screening method involves
determining the effect of a test agent on the level of active
APOBEC3G protein in a cell. The method generally involves
contacting a cell that produces APOBEC3G with a test agent; and
determining the effect, if any, of the test agent on the level of
active APOBEC3G protein in the cell.
[0081] In these embodiments, a test agent of interest is one that
increases the level of active APOBEC3G in a cell by at least about
10%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 80%, at least about 90%, at
least about 2-fold, at least about 5-fold, at least about 10-fold,
or at least about 25-fold, or more, when compared to the level of
active APOBEC3G in a cell in the absence of the test agent.
[0082] In some embodiments, a test agent of interest is one that
increases production (e.g., synthesis) of active APOBEC3G in a cell
by at least about 10%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 80%, at
least about 90%, at least about 2-fold, at least about 5-fold, at
least about 10-fold, or at least about 25-fold, or more, when
compared to the level of production of active APOBEC3G in the cell
in the absence of the test agent.
[0083] In other embodiments, a test agent of interest is one that
increases the number of mutations (e.g., APOBEC3G-induced
mutations) in a lentivirus nucleic acid by at least about 10%, at
least about 20%, at least about 25%, at least about 30%, at least
about 35%, at least about 40%, at least about 45%, at least about
50%, at least about 55%, at least about 60%, at least about 65%, at
least about 70%, at least about 80%, at least about 90%, at least
about 2-fold, at least about 5-fold, at least about 10-fold, or at
least about 25-fold, or more, when compared to the number of
mutations in the lentivirus nucleic acid in the absence of the test
agent.
[0084] In some embodiments, a test agent of interest is one that
increases the number of C.fwdarw.U mutations in a minus strand of a
lentivirus by at least about 5%, at least about 10%, at least about
20%, at least about 25%, at least about 30%, at least about 35%, at
least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about 60%, at least about 65%, at least about
70%, at least about 80%, at least about 90%, at least about 2-fold,
at least about 5-fold, at least about 10-fold, or at least about
25-fold, or more, when compared to the number of C.fwdarw.U
mutations in a minus strand of a lentivirus in the absence of the
test agent.
[0085] In some embodiments, a test agent of interest is one that
increases the proportion of lentivirus particles produced by a cell
that have encapsidated APOBEC3G by at least about 5%, at least
about 10%, at least about 20%, at least about 25%, at least about
30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 80%, at least about
90%, at least about 2-fold, at least about 5-fold, at least about
10-fold, or at least about 25-fold, or more, when compared to the
proportion of lentivirus particles produced by a cell that have
encapsidated APOBEC3G in the absence of the test agent.
[0086] In some embodiments, a test agent of interest is one that
decreases the infectivity of lentiviruses produced by a cell by at
least about 5%, at least about 10%, at least about 20%, at least
about 25%, at least about 30%, at least about 35%, at least about
40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%, at least about 65%, at least about 70%, at least
about 80%, or at least about 90%, or more, when compared to the
infectivity of the viruses produced by the cell in the absence of
the test agent.
[0087] In other embodiments, a test agent of interest is one that
promotes disassembly of an inactive high molecular weight APOBEC3G
complex, to yield an active low molecular weight APOBEC3G complex.
In these embodiments, a test agent of interest is one that promotes
disassembly of at least about 10%, at least about 20%, at least
about 25%, at least about 30%, at least about 35%, at least about
40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%, at least about 65%, at least about 70%, at least
about 80%, or at least about 90%, or more, of an inactive high
molecular weight APOBEC3G complex to an enzymatically active low
molecular weight APOBEC3G complex in a eukaryotic cell, when
compared to the level of inactive high molecular weight APOBEC3G
complex in the cell in the absence of the test agent.
[0088] In some embodiments, a test agent of interest is one that
converts at least about 10%, at least about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
80%, or at least about 90%, or more, of an enzymatically inactive
form of APOBEC3G to an enzymatically active form of APOBEC3G in a
eukaryotic cell, when compared to the level of inactive APOBEC3G in
the cell in the absence of the test agent.
[0089] In some embodiments, the cell is one that produces
endogenous APOBEC3G. In other embodiments, the effect of a test
agent on the level of active APOBEC3G protein in a cell is
determined by introducing an expression vector, which includes an
APOBEC3G coding sequence, into suitable eukaryotic cells in in
vitro culture, generating genetically modified cells that produce
APOBEC3G protein; contacting the genetically modified cells with a
test agent; and determining the effect, if any, of the test agent
on the level of active APOBEC3G protein produced by the genetically
modified cell.
[0090] The level of active APOBEC3G in a cell can be determined in
various ways. In some embodiments, a subject assay for identifying
an agent that increases production of active APOBEC3G in a cell
involves determining the effect of the agent on the level of
APOBEC3G production. In some embodiments, a subject assay for
identifying an agent that increases the level of active APOBEC3G
involves determining the effect of a test agent on the level of
APOBEC3G protein that induces a C.fwdarw.U mutation in a minus
strand, resulting in a G.fwdarw.A mutation in viral reverse
transcripts and/or that induces a C.fwdarw.T mutation in a DNA
copy. In other embodiments, a subject assay for identifying an
agent that increases the level of active APOBEC3G involves
determining the effect of the agent on packaging of APOBEC3G into a
lentivirus particle. In other embodiments, a subject assay for
identifying an agent that increases the level of active APOBEC3G
involves determining the effect of the agent viral infectivity. In
other embodiments, a subject assay involves determining the effect
of a test agent on converting an inactive high molecular weight
APOBEC3G complex to an active low molecular weight APOBEC3G
complex.
[0091] Where a subject assay for identifying an agent that
increases the level of active APOBEC3G in a cell involves
determining the effect of a test agent on the level of synthesis of
APOBEC3G by the cell, the assay can be conducted in a number of
ways. For example, synthesis of APOBEC3G by the cell can be
detected using a cell that produces APOBEC3G; contacting the cell
with a test agent; and detecting the level of APOBEC3G protein
using an immunoblot assay with antibody specific for APOBEC3G, or
an ELISA assay with antibody specific for APOBEC3G. The cells are
typically lysed to release the APOBEC3G. As another example,
synthesis of APOBEC3G by the cell can be detected using a cell that
includes an expression construct that comprises a nucleotide
sequence that encodes APOBEC3G under control of an APOBEC3G
promoter; contacting the cell with the test agent; and detecting
the level of APOBEC3G protein using an immunoblot assay with
antibody specific for APOBEC3G, or an ELISA assay with antibody
specific for APOBEC3G. As another non-limiting example, synthesis
of APOBEC3G by the cell can be detected using a cell that includes
an expression construct that comprises a nucleotide sequence that
encodes a fusion protein comprising all or a portion of APOBEC3G
and a detectable heterologous protein that provides for a
detectable signal (e.g., luciferase, .beta.-galactosidase, a green
fluorescent protein, an epitope tag such as hemagglutinin, and the
like), under the control of an APOBEC3G promoter; contacting the
cell with the test agent; and detecting the level of APOBEC3G
protein by detecting the heterologous protein, using standard
assays well known in the art.
[0092] In some embodiments, the level of APOBEC3G in the cytoplasm
is detected. In some embodiments, it is advantageous to increase
the level of active APOBEC3G in the cytoplasm of a cell that is not
yet infected with a lentivirus. Should such a cell become infected
with a lentivirus, the increased level of active APOBEC3G in the
cytoplasm of the cell will result in an increased number of
APOBEC3G-induced mutations in the lentivirus genome, and thereby
reduce the level of infectious virus produced by the cell. In these
embodiments, a test agent of interest increases the level of active
APOBEC3G in the cytoplasm of an uninfected cell (e.g., a cell not
infected by a lentivirus) by at least about 5%, at least about 10%,
at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 80%, at least about 90%, at
least about 2-fold, at least about 5-fold, at least about 10-fold,
or at least about 25-fold, or more, when compared to the level of
active APOBEC3G in the cytoplasm of the uninfected cell in the
absence of the test agent. Whether a test agent increases the level
of active APOBEC3G in the cytoplasm can be determined using any
known assay (e.g., a cytidine deaminase assay). Whether a test
agent increases the level of active APOBEC3G in the cytoplasm is
readily determined by, e.g., immunoblotting nuclear and cytoplasmic
fraction with APOBEC3G-specific antibody, as described in the
Examples.
[0093] Where a subject assay for identifying an agent that
increases the level of active APOBEC3G involves determining the
effect of a test agent on the level of APOBEC3G protein that
induces a C.fwdarw.U mutation in a lentivirus minus strand,
resulting in a G.fwdarw.A mutation in viral reverse transcripts
and/or that induces a C.fwdarw.T mutation in a DNA copy, the assay
can be conducted in a number of ways. For example, the number of
C.fwdarw.U mutations induced in a lentivirus minus strand is
determined by detecting polymerase chain reaction (PCR) products
amplified using Taq polymerase or Pfu polymerase, using as a
template a cDNA copy of a lentivirus RNA produced by a cell in the
presence or absence of a test agent. Taq polymerase will copy
templates containing uracil; Pfu polymerase does not readily copy
templates containing uracil. PCR performed with Taq polymerase will
yield a product of the expected size (based on the location of the
primers used); while PCR performed with Pfu polymerase will be
produced at a much lower level than the Taq-produced product. As
another example, PCR products amplified using Taq polymerase and,
as template, a cDNA copy of a lentivirus RNA produced by a cell in
the presence or absence of a test agent, are sequenced, and the
presence of G.fwdarw.A substitutions is detected. Thus, e.g., in
some embodiments, the level of active APOBEC3G in a cell is
determined by detecting the number of G.fwdarw.A substitutions in a
cDNA copy of a lentivirus RNA produced by a cell in the presence or
absence of a test agent. Where a test agent increases the level of
active APOBEC3G in a cell, the number of G.fwdarw.A substitutions
in a cDNA copy of a lentivirus RNA produced by the cell in the
presence of the test agent would be expected to be higher than the
number of G.fwdarw.A substitutions in a cDNA copy of a lentivirus
RNA produced by the cell in the absence of the test agent.
[0094] In some embodiments, the number of APOBEC3G-induced
mutations in a lentivirus nucleic acid is determined using an in
vitro cytidine deaminase assay. For example, a radiolabeled
oligonucleotide substrate for deamination is incubated with test
samples lacking or containing APOBEC3G. A suitable substrate
contains a target deoxycytidine(s) for APOBEC3G action. APOBEC3G
deaminates a cytosine base, converting it to uracil. Uracil is
susceptible to the activity of uracil DNA glycosylase (UDG or UNG).
Addition of UDG/UNG to the enzymatic reaction results in the
removal of the uracil base from the oligonucleotide, creating an
abasic lesion at the site of cytidine deamination. The
phosphate-sugar backbone of the oligonucleotide at the abasic site
is, in turn, susceptible to alkaline hydrolysis. Addition of NaOH
to the reaction thus results in cleavage at the site of
deamination. Cleavage is monitored by electrophoresis through
acrylamide-urea gels, and APOBEC3G activity is thereby scored by
the appearance of a shorter cleavage product derived from the
longer substrate oligonucleotide, with cleavage occurring
specifically at the site of deamination.
[0095] Where a subject assay for identifying an agent that
increases the level of active APOBEC3G involves determining the
level of virus encapsidation of APOBEC3G protein, the assay can be
conducted in a number of ways. For example, a test cell (e.g.,
cells that are susceptible to infection by a lentivirus, e.g., HIV;
e.g., cells that express CD4.sup.+, CCR5 and CXCR4 on the cell
surface) that produces lentivirus particles is contacted with a
test agent; and the amount of APOBEC3G in viral particles is
determined by analyzing viral particles in the cell supernatants
for the presence of APOBEC3G. For example, viral particles present
in the cell supernatant are harvested 24-48 hours after
transfection with a viral construct; cellular debris is removed
(e.g., by centrifugation); and the viral particles are
concentrated. APOBEC3G is detected in viral particles using an
immunological assay. For example, APOBEC3G is detected in an
immunoblot assay in which viral particles are lysed, the proteins
separated on a gel, then transferred to a solid support such as a
nylon membrane or other type of membrane, and APOBEC3G is detected
on the membrane using an antibody specific for APOBEC3G.
[0096] Where a subject assay for identifying an agent that
increases the level of active APOBEC3G in a cell involves
determining the level of lentivirus infectivity, the assay can be
conducted in a number of ways. For example, viral infectivity can
be determined using a single-round titration assay on CD4.sup.+
cells, and measuring the viral titer in the presence and in the
absence of the test agent. A suitable assay may involve contacting
a permissive test cell (e.g., cells that are susceptible to
infection by a lentivirus, e.g., HIV; e.g., cells that express
CD4.sup.+, CCR5 and CXCR4 on the cell surface) with supernatant
from a lentivirus-producing cell (e.g., a cell transfected with a
lentivirus construct), wherein the test cells include a coding
sequence for a detectable marker (e.g., luciferase,
.beta.-galactosidase, a fluorescent protein (e.g., a green
fluorescent protein) and the like) operably linked and under
transcriptional control of an HIV long terminal repeat (LTR), such
that infection of the cell by an infectious HIV virus particle
results in activation of the HIV LTR and production of the
detectable marker. Cell supernatants containing viral particles are
used to infect the test cells; and infection of the test cells is
detected by assaying for the detectable marker. Cell supernatants
from lentivirus-producing cells contacted with a test agent are
compared with cell supernatants from the same cells not contacted
with the test agent. The number of infected test cells is an
indication of the infectivity of the viral particles in the cell
supernatant. Where the detectable marker is a green fluorescent
protein, infectivity can be scored using a fluorescence activated
cell sorter. Where the detectable marker is .beta.-galactosidase,
infectivity can be scored by
5-bromo-4-chloro-3-indoyl-.beta.-D-galactoside staining. See, e.g.,
Mangeat et al. ((2003) Nature 424: 99-103. The infectivity of HIV
viral particles in the supernatant of HIV infected cells having a
high level of active APOBEC3g would be expected to be lower than
the infectivity of HIV viral particles in the supernatant of HIV
infected cells having a low level of active APOBEC3G.
[0097] Where a subject method involves determining the effect of a
test agent on converting an inactive high molecular weight APOBEC3G
complex to an active low molecular weight APOBEC3G complex, the
levels of high molecular weight APOBEC3G complex and low molecular
weight APOBEC3G complex in a cell can be determined by, e.g.,
lysing a cell that has been contacted with a test agent; and
performing size exclusion FPLC on the lysate.
Methods of Identifying Agents that Inhibit Vif Binding to
APOBEC3G
[0098] In some embodiments, the invention provides methods of
identifying an agent that inhibits the interaction of a Vif
polypeptide with APOBEC3G or an APOBEC3G complex. The methods
generally involve contacting a Vif polypeptide and an APOBEC3G
polypeptide or an APOBEC3G complex with a test agent, and
determining the effect, if any, of the test agent on the
interaction of Vif with APOBEC3G polypeptide or APOBEC3G complex.
In some embodiments, methods of identifying an agent that inhibits
the interaction of Vif polypeptide with an APOBEC3G polypeptide or
an APOBEC3G complex are cell-based methods. In other embodiments,
methods of identifying an agent that inhibits the interaction of a
Vif polypeptide with an APOBEC3G polypeptide or an APOBEC3G complex
are cell-free methods.
[0099] Formation of a binding complex between Vif and an APOBEC3G
polypeptide or an APOBEC3G complex can be detected using any known
method. Suitable methods include: a yeast two-hybrid method; a FRET
assay; a BRET assay; a fluorescence quenching assay; a fluorescence
anisotropy assay; an immunological assay; and an assay involving
binding of a detectably labeled protein to an immobilized protein
(e.g., GST-Vif).
[0100] Immunological assays, and assays involving binding of a
detectably labeled protein to an immobilized protein can be
arranged in a variety of ways. Immunoprecipitation assays can be
designed, wherein the Vif/APOBEC3G polypeptide complex is detected
by precipitating the complex with antibody specific for Vif,
antibody specific for APOBEC3G, or antibody specific for a fusion
partner of a Vif fusion protein and/or an antibody specific for a
fusion partner of an APOBEC3G fusion polypeptide. In some
embodiments, a APOBEC3G protein or a Vif protein is immobilized
directly on an insoluble support. In other embodiments, a APOBEC3G
protein or a Vif protein is fused or bound to a second protein, and
the second protein is immobilized on an insoluble support.
Insoluble supports include, but are not limited to, plastic
surfaces (e.g., polystyrene, and the like) such as a multi-well
plate; beads, including magnetic beads, plastic beads, and the
like; membranes (e.g., polyvinylpyrrolidone, nitrocellulose, and
the like); etc.
[0101] In other embodiments, the assay is a binding assay which
detects binding of Vif to immobilized APOBEC3G, or which detects
binding of a APOBEC3G to immobilized Vif protein. In some
embodiments, the Vif polypeptide is labeled with a detectable
label, and binding to an immobilized APOBEC3G polypeptide is
detected. In other embodiments, the APOBEC3G polypeptide is labeled
with a detectable label, and binding to immobilized Vif polypeptide
is detected. In other embodiments, the Vif polypeptide is
immobilized, and binding of the APOBEC3G polypeptide to the Vif
polypeptide is detected using an antibody specific for the APOBEC3G
polypeptide, where the antibody is either directly labeled or a
secondary antibody that is labeled is used. In other embodiments,
the APOBEC3G polypeptide is immobilized, and binding of the Vif
polypeptide to the APOBEC3G polypeptide is detected using an
antibody specific for the Vif polypeptide, where the antibody is
either directly labeled or a secondary antibody that is labeled is
used.
[0102] Formation of a binding complex between a Vif polypeptide and
an APOBEC3G polypeptide can also be detected using fluorescence
resonance energy transfer (FRET), bioluminescence resonance energy
transfer (BRET), anisotropy measurements, and fluorescence
quenching measurements.
[0103] FRET involves the transfer of energy from a donor
fluorophore in an excited state to a nearby acceptor fluorophore.
For this transfer to take place, the donor and acceptor molecules
must in close proximity (e.g., less than 10 nanometers apart,
usually between 10 and 100 .ANG. apart), and the emission spectra
of the donor fluorophore must overlap the excitation spectra of the
acceptor fluorophore. In these embodiments, a fluorescently labeled
Vif protein serves as a donor and/or acceptor in combination with a
second fluorescent protein or dye, e.g., a fluorescent protein as
described in Matz et al., Nature Biotechnology (October 1999)
17:969-973; a green fluorescent protein (GFP), including a
"humanized" GFP; a GFP from Aequoria victoria or fluorescent mutant
thereof, e.g., as described in U.S. Pat. No. 6,066,476; 6,020,192;
5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445;
5,874,304, the disclosures of which are herein incorporated by
reference; a GFP from another species such as Renilla reniformis,
Renilla mulleri, or Ptilosarcus guernyi, as described in, e.g., WO
99/49019 and Peelle et al. (2001) J. Protein Chem. 20:507-519;
"humanized" recombinant GFP (hrGFP) (Stratagene); other fluorescent
dyes, e.g., coumarin and its derivatives, e.g.
7-amino-4-methylcoumarin, aminocoumarin, bodipy dyes, such as
Bodipy FL, cascade blue, fluorescein and its derivatives, e.g.
fluorescein isothiocyanate, Oregon green, rhodamine dyes, e.g.
texas red, tetramethylrhodamine, eosins and erythrosins, cyanine
dyes, e.g. Cy3 and Cy5, macrocyclic chelates of lanthanide ions,
e.g. quantum dye, etc., chemilumescent dyes, e.g., luciferases.
[0104] BRET is a protein-protein interaction assay based on energy
transfer from a bioluminescent donor to a fluorescent acceptor
protein. The BRET signal is measured by the amount of light emitted
by the acceptor to the amount of light emitted by the donor. The
ratio of these two values increases as the two proteins are brought
into proximity. The BRET assay has been amply described in the
literature. See, e.g., U.S. Pat. Nos. 6,020,192; 5,968,750; and
5,874,304; and Xu et al. (1999) Proc. Natl. Acad. Sci. USA
96:151-156. BRET assays may be performed by analyzing transfer
between a bioluminescent donor protein and a fluorescent acceptor
protein. Interaction between the donor and acceptor proteins can be
monitored by a change in the ratio of light emitted by the
bioluminescent and fluorescent proteins. In this application, the
Vif protein serves as donor and/or acceptor protein.
[0105] Fluorescent Vif can be produced by generating a construct
comprising a Vif and a fluorescent fusion partner, e.g., a
fluorescent protein as described in Matz et al. ((1999) Nature
Biotechnology 17:969-973), a green fluorescent protein from any
species or a derivative thereof, e.g., a GFP from another species
such as Renilla reniformis, Renilla mulleri, or Ptilosarcus
guernyi, as described in, e.g., WO 99/49019 and Peelle et al.
(2001) J. Protein Chem. 20:507-519; "humanized" recombinant GFP
(hrGFP) (Stratagene); a GFP from Aequoria Victoria or fluorescent
mutant thereof, e.g., as described in U.S. Pat. No. 6,066,476;
6,020,192; 5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713;
5,919,445; 5,874,304. Generation of such a construct, and
production of a Vif/fluorescent protein fusion protein is well
within the skill level of those of ordinary skill in the art.
[0106] Alternatively, binding may be assayed by fluorescence
anisotropy. Fluorescence anisotropy assays are amply described in
the literature. See, e.g., Jameson and Sawyer (1995) Methods
Enzymol. 246:283-300.
[0107] In some embodiments, a subject method involves detecting Vif
binding to a high molecular weight complex (>700 kD) comprising
APOBEC3G and cellular RNA. In these embodiments, the screening
methods are typically cell-based methods. Cells are genetically
modified with expression vectors that provide for production of Vif
and APOBEC3G in a suitable eukaryotic cell, as described above.
After allowing time for production of Vif and APOBEC3G, cells are
lysed, and lysates are examined for the presence of Vif in the
APOBEC3G complex. Any known method for detecting Vif association
with the APOBEC3G complex is suitable. A non-limiting example is
size-exclusion fast performance liquid chromatography (FPLC), as
described in Example 1.
Active Agents
[0108] The present invention further provides agents that reduce or
inhibit Vif activity; agents that increase the level of active
APOBEC3G in a cell; and agents that convert an inactive high
molecular weight APOBEC3G complex to an active low molecular weight
APOBEC3G complex. Of particular interest in many embodiments are
agents identified using a screening method of the invention. The
subject agents are useful for inhibiting lentiviral replication,
and are therefore useful for treating lentiviral infections.
[0109] In many embodiments, the agent is a small molecule, e.g., a
small organic or inorganic compound having a molecular weight of
more than 50 and less than about 2,500 daltons. Agents may comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and may include at least
an amine, carbonyl, hydroxyl or carboxyl group, and may contain at
least two of the functional chemical groups. The agents may
comprise cyclical carbon or heterocyclic structures and/or aromatic
or polyaromatic structures substituted with one or more of the
above functional groups. Agents are also found among biomolecules
including peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations
thereof.
[0110] In some embodiments, an active agent is a peptide (e.g.,
peptide inhibitors of Vif activity; peptides that increase the
level of active APOBEC3G; peptides that convert an inactive HMW
form of APOBEC3G to an active LMW form of APOBEC3G; etc.). Suitable
peptides include peptides of from about 3 amino acids to about 50
amino acids, from about 5 amino acids to about 30 amino acids, from
about 10 amino acids to about 25 amino acids, from about 25 amino
acids to about 50 amino acids, from about 50 amino acids to about
75 amino acids, or from about 75 amino acids to about 100 amino
acids in length. In some embodiments, the peptide is linear; in
other embodiments, the peptide is cyclized. In some embodiments,
the peptide is modified, e.g., comprises one or more non-peptide
moieties covalently or non-covalently linked to the peptide.
Suitable non-peptide moieties include, but are not limited to,
polyethylene glycol (PEG) moieties; carbohydrate moieties; lipid
moieties; fatty acid moieties; polysaccharide moieties; phosphate
groups; and the like. In some embodiments, the active peptide is
linked to a heterologous peptide, e.g., a heterologous peptide that
confers increased stability or residence time in vivo; a
heterologous peptide that facilitates crossing a cell membrane; a
heterologous peptide that binds to a cell surface receptor; a
heterologous peptide that provides for dimerization; a heterologous
peptide that provides an epitope tag; a heterologous peptide that
provides a detectable signal; and the like.
[0111] Peptides can include naturally-occurring and non-naturally
occurring amino acids. Peptides may comprise D-amino acids, a
combination of D- and L-amino acids, and various "designer" amino
acids (e.g., .beta.-methyl amino acids, C.alpha.-methyl amino
acids, and Nu-methyl amino acids, etc.) to convey special
properties to peptides. Additionally, peptide may be a cyclic
peptide. Peptides may include non-classical amino acids in order to
introduce particular conformational motifs. Any known non-classical
amino acid can be used. Non-classical amino acids include, but are
not limited to, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate;
(2S,3S)-methylphenylalanine, (2S,3R)-methyl-phenylalanine,
(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine;
2-aminotetrahydronaphthalene-2-carboxylic acid;
hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate;
.beta.-carboline (D and L); HIC (histidine isoquinoline carboxylic
acid); and HIC (histidine cyclic urea). Amino acid analogs and
peptidomimetics may be incorporated into a peptide to induce or
favor specific secondary structures, including, but not limited to,
LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a .beta.-turn
inducing dipeptide analog; .beta.-sheet inducing analogs;
.beta.-turn inducing analogs; .alpha.-helix inducing analogs;
.gamma.-turn inducing analogs; Gly-Ala turn analog; amide bond
isostere; tretrazol; and the like.
[0112] A peptide may be a depsipeptide, which may be a linear or a
cyclic depsipeptide. Kuisle et al. (1999) Tet. Letters
40:1203-1206. "Depsipeptides" are compounds containing a sequence
of at least two alpha-amino acids and at least one alpha-hydroxy
carboxylic acid, which are bound through at least one normal
peptide link and ester links, derived from the hydroxy carboxylic
acids, where "linear depsipeptides" may comprise rings formed
through S-S bridges, or through an hydroxy or a mercapto group of
an hydroxy-, or mercapto-amino acid and the carboxyl group of
another amino- or hydroxy-acid but do not comprise rings formed
only through peptide or ester links derived from hydroxy carboxylic
acids. "Cyclic depsipeptides" are peptides containing at least one
ring formed only through peptide or ester links, derived from
hydroxy carboxylic acids.
[0113] Peptides may be cyclic or bicyclic. For example, the
C-terminal carboxyl group or a C-terminal ester can be induced to
cyclize by internal displacement of the --OH or the ester (--OR) of
the carboxyl group or ester respectively with the N-terminal amino
group to form a cyclic peptide. For example, after synthesis and
cleavage to give the peptide acid, the free acid is converted to an
activated ester by an appropriate carboxyl group activator such as
dicyclohexylcarbodiimide (DCC) in solution, for example, in
methylene chloride (CH.sub.2Cl.sub.2), dimethyl formamide (DMF)
mixtures. The cyclic peptide is then formed by internal
displacement of the activated ester with the N-terminal amine.
Internal cyclization as opposed to polymerization can be enhanced
by use of very dilute solutions. Methods for making cyclic peptides
are well known in the art.
[0114] The term "bicyclic" refers to a peptide in which there
exists two ring closures. The ring closures are formed by covalent
linkages between amino acids in the peptide. A covalent linkage
between two nonadjacent amino acids constitutes a ring closure, as
does a second covalent linkage between a pair of adjacent amino
acids which are already linked by a covalent peptide linkage. The
covalent linkages forming the ring closures may be amide linkages,
i.e., the linkage formed between a free amino on one amino acid and
a free carboxyl of a second amino acid, or linkages formed between
the side chains or "R" groups of amino acids in the peptides. Thus,
bicyclic peptides may be "true" bicyclic peptides, i.e., peptides
cyclized by the formation of a peptide bond between the N-terminus
and the C-terminus of the peptide, or they may be "depsi-bicyclic"
peptides, i.e., peptides in which the terminal amino acids are
covalently linked through their side chain moieties.
[0115] A desamino or descarboxy residue can be incorporated at the
terminii of the peptide, so that there is no terminal amino or
carboxyl group, to decrease susceptibility to proteases or to
restrict the conformation of the peptide. C-terminal functional
groups include amide, amide lower alkyl, amide di(lower alkyl),
lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives
thereof, and the pharmaceutically acceptable salts thereof.
[0116] In addition to the foregoing N-terminal and C-terminal
modifications, a peptide or peptidomimetic can be modified with or
covalently coupled to one or more of a variety of hydrophilic
polymers to increase solubility and circulation half-life of the
peptide. Suitable nonproteinaceous hydrophilic polymers for
coupling to a peptide include, but are not limited to,
polyalkylethers as exemplified by polyethylene glycol and
polypropylene glycol, polylactic acid, polyglycolic acid,
polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose
and cellulose derivatives, dextran and dextran derivatives, etc.
Generally, such hydrophilic polymers have an average molecular
weight ranging from about 500 to about 100,000 daltons, from about
2,000 to about 40,000 daltons, or from about 5,000 to about 20,000
daltons. The peptide can be derivatized with or coupled to such
polymers using any of the methods set forth in Zallipsky, S.,
Bioconjugate Chem., 6:150-165 (1995); Monfardini, C, et al.,
Bioconjugate Chem., 6:62-69 (1995); U.S. Pat. Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417; 4,791,192; 4,179,337 or WO
95/34326.
[0117] An active peptide (e.g., peptide inhibitors of Vif activity;
peptides that increase the level of active APOBEC3G; peptides that
convert an inactive HMW form of APOBEC3G to an active LMW form of
APOBEC3G; etc.) will in some embodiments be conjugated to
decapeptides comprised of Arginine residues to allow uptake across
the plasma membrane by protein transduction. Such modifications
allow peptides to enter cells (e.g., cross the plasma membrane)
with high efficiency.
[0118] In some embodiments, an active peptide (e.g., peptide
inhibitors of Vif activity; peptides that increase the level of
active APOBEC3G; peptides that convert an inactive HMW form of
APOBEC3G to an active LMW form of APOBEC3G; etc.) is a peptide
aptamer. Peptide aptamers are peptides or small polypeptides that
act as dominant inhibitors of protein function. Peptide aptamers
specifically bind to target proteins, blocking their function
ability. Kolonin and Finley (1998) Proc. Natl. Acad. Sci. USA
95:14266-14271. Due to the highly selective nature of peptide
aptamers, they may be used not only to target a specific protein,
but also to target specific functions of a given protein (e.g. a
protein binding function). Further, peptide aptamers may be
expressed in a controlled fashion by use of promoters which
regulate expression in a temporal, spatial or inducible manner.
[0119] Peptide aptamers that bind with high affinity and
specificity to a target protein may be isolated by a variety of
techniques known in the art. Peptide aptamers can be isolated from
random peptide libraries by yeast two-hybrid screens (Xu et al.,
(1997) Proc. Natl. Acad. Sci. USA 94:12473-12478). They can also be
isolated from phage libraries (Hoogenboom et al., Immunotechnology
(1998) 4:1-20) or chemically generated peptides/libraries.
[0120] Agents that increase the level of active APOBEC3G in a
eukaryotic cell include agents that prevent or reduce inclusion of
APOBEC3G in high molecular weight complexes; agents that mediate
disassembly of a high molecular weight APOBEC3G complex; and agents
that increase production of active APOBEC3G protein by a cell.
[0121] Agents that increase production of active APOBEC3G include,
but are not limited to, antibodies, e.g., antibodies to CD3 (e.g.,
Orthoclone OKTe.RTM. (muromonab-CD3); etc.); cytokines, e.g., IL-2
(e.g., Proleukin.RTM. IL-2 (aldesleukin)); IL-15; phorbol esters,
e.g., prostratin (12-deoxyphorbol-13-acetate),
phorbol-12-myristate-13-acetate, etc.; lectins, e.g.,
phytohemagglutinin; mitogens; etc. Combinations of two or more of
the foregoing agents will in some embodiments be used to increase
the level of active APOBEC3G.
[0122] Agents that promote conversion of an inactive high molecular
weight APOBEC3G complex to an active low molecular weight APOBEC3G
complex include agents that disrupt protein-protein interactions;
RNAse; peptide fragments of APOBEC3G that inhibit APOBEC3G
homodimerization; peptide fragments of APOBEC3G that inhibit
association of APOBEC3G with RNA; peptide fragments of APOBEC3G
that inhibit association of APOBEC3G with other polypeptides
present in a high molecular weight APOBEC3G complex; short
interfering RNA (siRNA) or antisense RNA targeting the RNA in the
HMW APOBEC3G complex; siRNA or antisense RNA targeting mRNA that
encodes a protein component of the HMW APOBEC3G complex; and the
like.
[0123] Peptide agents that inhibit APOBEC3G homodimerization and/or
that inhibit formation of HMW APOBEC3G complexes and/or that
promote conversion of an inactive high molecular weight APOBEC3G
complex to an active low molecular weight APOBEC3G complex and/or
that inhibit a Vif activity include, but are not limited to,
peptides corresponding to the amino terminus of APOBEC3G; and
peptides corresponding to the carboxyl terminus of APOBEC3G.
Exemplary, non-limiting peptides that are suitable for use include,
e.g., peptides comprising from about 5 to about 10, from about 10
to about 15, from about 15 to about 20, from about 20 to about 30,
or from about 30 to about 40, contiguous amino acids of the
amino-terminal forty amino acids of APOBEC3G. For example, a
suitable peptide comprises from about 5 to about 10, from about 10
to about 15, from about 15 to about 20, from about 20 to about 30,
or from about 30 to about 40, contiguous amino acids of the
following peptide: NH.sub.2-MKPHFRNTVE RMYRDTFSYN FYNRPILSRR
NTVWLCYEVK--COOH (SEQ ID NO:5); and variants thereof. Other
exemplary peptides include peptides comprising from about from
about 5 to about 10, from about 10 to about 15, from about 15 to
about 20, from about 20 to about 25 contiguous amino acid of the
following peptide: NH.sub.2--CPFQP WDGLDEHSQD LSGRLRAILQ NQEN--COOH
(SEQ ID NO:6), up to the entire length of a peptide having the
sequence set forth in SEQ ID NO:6; and variants of any of the
foregoing peptides (e.g., peptides that differ in amino acid
sequence by from about 1 amino acid to about 10 amino acids from a
foregoing peptide). Also suitable for use are any of the
above-mentioned peptides in a cyclized form; and any of the
above-mentioned peptides that have one or more modifications, e.g.,
modification by addition of one or more polyethylene glycol
moieties covalently linked to the peptide (e.g., PEGylated
peptides), modification by addition of one or more carbohydrate
moieties, etc., as discussed above.
[0124] Peptide agents that that reduce Vif-induced degradation of
APOBEC3G include peptides comprising from about 5 to about 100
contiguous amino acids of the amino-terminal portion of APOBEC3G,
e.g., peptides that comprise from about 5 to about 10, from about
10 to about 20, from about 20 to about 30, from about 30 to about
40, from about 40 to about 50, from about 50 to about 60, from
about 60 to about 70, from about 70 to about 80, from about 80 to
about 90, or from about 90 to about 100 contiguous amino acids of
the amino-terminal portion of APOBEC3G. For example, a suitable
peptide agent that reduces Vif-induced degradation of APOBEC3G
includes a peptide comprising from about 5 to about 10, from about
10 to about 20, from about 20 to about 30, from about 30 to about
40, from about 40 to about 50, from about 50 to about 60, from
about 60 to about 70, from about 70 to about 80, from about 80 to
about 90, or from about 90 to about 100 contiguous amino acids of a
peptide having the sequence set forth in SEQ ID NO:7. Also suitable
for use are variants of any of the foregoing peptides (e.g.,
peptides that differ in amino acid sequence by from about 1 amino
acid to about 10 amino acids from a foregoing peptide). Also
suitable for use are any of the above-mentioned peptides in a
cyclized form; and any of the above-mentioned peptides that have
one or more modifications, e.g., modification by addition of one or
more polyethylene glycol moieties covalently linked to the peptide
(e.g., PEGylated peptides), modification by addition of one or more
carbohydrate moieties, etc., as discussed above.
Method of Treating a Lentivirus Infection
[0125] The present invention provides methods of treating a
lentivirus infection in an individual. In some embodiments, the
methods generally involve administering to an individual having a
lentivirus infection, or at risk of having a lentivirus infection,
a subject agent in an amount effective to inhibit Vif activity in a
lentivirus-infected cell in the individual, thereby treating the
lentivirus infection. In other embodiments, the methods generally
involve administering to an individual having a lentivirus
infection, or at risk of having a lentivirus infection, an
effective amount of an agent that increases the level of active
APOBEC3G in a cell that is susceptible to lentivirus infection. In
other embodiments, the methods generally involve administering to
an individual having a lentivirus infection, or at risk of having a
lentivirus infection, an effective amount of an agent that converts
a HMW APOBEC3G complex to a LMW APOBEC3G complex in a cell that is
susceptible to lentivirus infection.
[0126] In some embodiments, a therapeutically effective amount of
an agent that inhibits Vif activity is an amount that reduces
lentivirus load in the individual and/or reduces lentivirus
replication in a lentivirus-infected cell in the individual by at
least about 10%, at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, or at least about 95%
or more, compared to the lentivirus load or lentivirus replication
in a lentivirus-infected cell of the individual not treated with
the agent.
[0127] In some embodiments, a therapeutically effective amount of
an agent that increases the level of active APOBEC3G protein in a
cell that is susceptible to lentivirus infection is an amount that
reduces lentivirus load in the individual and/or reduces lentivirus
replication in a lentivirus-infected cell in the individual by at
least about 10%, at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, or at least about 95%
or more, compared to the lentivirus load or lentivirus replication
in a lentivirus-infected cell of the individual not treated with
the agent.
[0128] In some embodiments, a therapeutically effective amount of
an agent that converts a HMW APOBEC3G complex to a LMW APOBEC3G
complex in a cell that is susceptible to lentivirus infection is an
amount that reduces lentivirus load in the individual and/or
reduces lentivirus replication in a lentivirus-infected cell in the
individual by at least about 10%, at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, or
at least about 95% or more, compared to the lentivirus load or
lentivirus replication in a lentivirus-infected cell of the
individual not treated with the agent.
[0129] In some embodiments, a therapeutically effective amount of
an agent that inhibits Vif activity is an amount that increases the
CD4.sup.+ T cell count in an individual by at least about 10%, at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 100%, at least about
2.5-fold, at least about 3-fold, at least about 3.5-fold, at least
about 4-fold, at least about 5-fold, or at least about 10-fold, or
more, compared to the CD4.sup.+ T cell count of the individual not
treated with the agent. In some embodiments, a therapeutically
effective amount of an agent that inhibits Vif activity is an
amount that restores the CD4.sup.+ T cell count to within a normal
range. In human blood, the number of CD4.sup.+-T cells which is
considered to be in a normal range is from about 600 to about 1500
CD4.sup.+-T cells/mm.sup.3 blood.
[0130] In some embodiments, a therapeutically effective amount of
an agent that increases the level of active APOBEC3G in a cell is
an amount that increases the CD4.sup.+ T cell count in an
individual by at least about 10%, at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 100%, at least about 2.5-fold, at least about 3-fold,
at least about 3.5-fold, at least about 4-fold, at least about
5-fold, or at least about 10-fold, or more, compared to the
CD4.sup.+ T cell count of the individual not treated with the
agent. In some embodiments, a therapeutically effective amount of
an agent that increases the level of active APOBEC3G in a cell is
an amount that restores the CD4.sup.+ T cell count to within a
normal range.
[0131] In some embodiments, a therapeutically effective amount of
an agent that converts a HMW APOBEC3G complex to a LMW APOBEC3G
complex in a cell that is susceptible to lentivirus infection is an
amount that increases the CD4.sup.+ T cell count in an individual
by at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, at least about
100%, at least about 2.5-fold, at least about 3-fold, at least
about 3.5-fold, at least about 4-fold, at least about 5-fold, or at
least about 10-fold, or more, compared to the CD4.sup.+ T cell
count of the individual not treated with the agent. In some
embodiments, a therapeutically effective amount of an agent that
converts a HMW APOBEC3G complex to a LMW APOBEC3G complex in a cell
that is susceptible to lentivirus infection is an amount that
restores the CD4.sup.+ T cell count to within a normal range.
[0132] Treating a lentivirus infection, includes, but is not
limited to, preventing lentivirus infection, reducing the
probability of lentivirus infection, reducing the spread of
lentivirus from an infected cell to a susceptible cell, reducing
viral load in an lentivirus-infected individual, reducing an amount
of virally-encoded polypeptide(s) in an lentivirus-infected
individual, and increasing CD4 T cell count in an
lentivirus-infected individual.
[0133] The amount of subject agent which is administered will vary
with the nature of the drug. As one non-limiting example, a subject
agent can be administered in the range of about 0.2 mg/kg/day to
about 20 mg/kg/day. The determination of how large a dose is to be
used may be determined using an animal model (e.g., a non-human
primate model) and relating the dosage based on pharmacokinetics,
e.g. with equations predictive of interspecies scaling. Usually,
the lowest effective dose will be used.
[0134] Any of a variety of methods can be used to determine whether
a treatment method is effective. For example, methods of
determining whether the methods of the invention are effective in
reducing lentivirus load, and/or treating an lentivirus infection,
are any known test for indicia of lentivirus infection, including,
but not limited to, measuring viral load, e.g., by measuring the
amount of lentivirus in a biological sample, e.g., using a
polymerase chain reaction (PCR) with primers specific for a
lentivirus polynucleotide sequence; detecting and/or measuring a
polypeptide encoded by lentivirus, e.g., p24, gp120, reverse
transcriptase, using, e.g., an immunological assay such as an
enzyme-linked immunosorbent assay (ELISA) with an antibody specific
for the polypeptide; and measuring the CD4.sup.+ T cell count in
the individual.
[0135] Methods of assaying an lentivirus infection (or any indicia
associated with an lentivirus infection) are known in the art, and
have been described in numerous publications such as HIV Protocols
(Methods in Molecular Medicine, 17) N. L. Michael and J. H. Kim,
eds. (1999) Humana Press.
Formulation, Dosages, and Routes of Administration
[0136] In general, an active agent (e.g., an agent that inhibits a
Vif activity; an agent that increases the level of active APOBEC3G
in a cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) is prepared in a pharmaceutically
acceptable composition for delivery to a host. The terms "active
agent," "drug," "agent," "therapeutic agent," and the like are used
interchangeably herein. Pharmaceutically acceptable carriers
preferred for use with a subject agent may include sterile aqueous
of non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, and
microparticles, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. A composition comprising a subject agent
may also be lyophilized using means well known in the art, for
subsequent reconstitution and use according to the invention.
Formulations
[0137] An active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) is administered to an individual in need
thereof in a formulation with a pharmaceutically acceptable
excipient(s). A wide variety of pharmaceutically acceptable
excipients are known in the art and need not be discussed in detail
herein. Pharmaceutically acceptable excipients have been amply
described in a variety of publications, including, for example, A.
Gennaro (2000) "Remington: The Science and Practice of Pharmacy",
20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical
Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al.,
eds 7.sup.th ed., Lippincott, Williams, & Wilkins; and Handbook
of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds.,
3.sup.rd ed. Amer. Pharmaceutical Assoc.
[0138] In the subject methods, an active agent (e.g., an agent that
inhibits a Vif activity; an agent that increases the level of
active APOBEC3G in a cell; an agent that converts a HMW APOBEC3G
complex to a LMW APOBEC3G complex; etc.) may be administered to the
host using any convenient means capable of resulting in the desired
reduction in a Vif activity and/or increase in the level of active
APOBEC3G. Thus, an active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) can be incorporated into a variety of
formulations for therapeutic administration. More particularly, an
active agent (e.g., an agent that inhibits a Vif activity; an agent
that increases the level of active APOBEC3G in a cell; an agent
that converts a HMW APOBEC3G complex to a LMW APOBEC3G complex;
etc.) can be formulated into pharmaceutical compositions by
combination with appropriate, pharmaceutically acceptable carriers
or diluents, and may be formulated into preparations in solid,
semi-solid, liquid or gaseous forms, such as tablets, capsules,
powders, granules, ointments, solutions, suppositories, injections,
inhalants and aerosols.
[0139] In pharmaceutical dosage forms, an active agent (e.g., an
agent that inhibits a Vif activity; an agent that increases the
level of active APOBEC3G in a cell; an agent that converts a HMW
APOBEC3G complex to a LMW APOBEC3G complex; etc.) may be
administered in the form of its pharmaceutically acceptable salts,
or it may also be used alone or in appropriate association, as well
as in combination, with other pharmaceutically active compounds.
The following methods and excipients are merely exemplary and are
in no way limiting.
[0140] For oral preparations, an active agent (e.g., an agent that
inhibits a Vif activity; an agent that increases the level of
active APOBEC3G in a cell; an agent that converts a HMW APOBEC3G
complex to a LMW APOBEC3G complex; etc.) can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0141] An active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) can be formulated into preparations for
injection by dissolving, suspending or emulsifying them in an
aqueous or nonaqueous solvent, such as vegetable or other similar
oils, synthetic aliphatic acid glycerides, esters of higher
aliphatic acids or propylene glycol; and if desired, with
conventional additives such as solubilizers, isotonic agents,
suspending agents, emulsifying agents, stabilizers and
preservatives.
[0142] The agents can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0143] Furthermore, the agents can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. An active agent (e.g., an agent that inhibits
a Vif activity; an agent that increases the level of active
APOBEC3G in a cell; an agent that converts a HMW APOBEC3G complex
to a LMW APOBEC3G complex; etc.) can be administered rectally via a
suppository. The suppository can include vehicles such as cocoa
butter, carbowaxes and polyethylene glycols, which melt at body
temperature, yet are solidified at room temperature.
[0144] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more active agents. Similarly, unit dosage forms
for injection or intravenous administration may comprise the active
agent(s) in a composition as a solution in sterile water, normal
saline or another pharmaceutically acceptable carrier.
[0145] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
an active agent, calculated in an amount sufficient to produce the
desired effect in association with a pharmaceutically acceptable
diluent, carrier or vehicle. The specifications for a given active
agent will depend in part on the particular compound employed and
the effect to be achieved, and the pharmacodynamics associated with
each compound in the host.
[0146] Other modes of administration will also find use with the
subject invention. For instance, an active agent (e.g., an agent
that inhibits a Vif activity; an agent that increases the level of
active APOBEC3G in a cell; an agent that converts a HMW APOBEC3G
complex to a LMW APOBEC3G complex; etc.) can be formulated in
suppositories and, in some cases, aerosol and intranasal
compositions. For suppositories, the vehicle composition will
include traditional binders and carriers such as, polyalkylene
glycols, or triglycerides. Such suppositories may be formed from
mixtures containing the active ingredient in the range of about
0.5% to about 10% (w/w), preferably about 1% to about 2%.
[0147] Intranasal formulations will usually include vehicles that
neither cause irritation to the nasal mucosa nor significantly
disturb ciliary function. Diluents such as water, aqueous saline or
other known substances can be employed with the subject invention.
The nasal formulations may also contain preservatives such as, but
not limited to, chlorobutanol and benzalkonium chloride. A
surfactant may be present to enhance absorption of the subject
proteins by the nasal mucosa.
[0148] An active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) can be administered as injectables.
Typically, injectable compositions are prepared as liquid solutions
or suspensions; solid forms suitable for solution in, or suspension
in, liquid vehicles prior to injection may also be prepared. The
preparation may also be emulsified or the active ingredient
encapsulated in liposome vehicles.
[0149] An active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) will in some embodiments be formulated for
vaginal delivery. A subject formulation for intravaginal
administration is formulated as an intravaginal bioadhesive tablet,
intravaginal bioadhesive microparticle, intravaginal cream,
intravaginal lotion, intravaginal foam, intravaginal ointment,
intravaginal paste, intravaginal solution, or intravaginal gel.
[0150] A subject formulation comprising an active agent includes
one or more of an excipient (e.g., sucrose, starch, mannitol,
sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or
calcium carbonate), a binder (e.g., cellulose, methylcellulose,
hydroxymethylcellulose, polypropylpyrrolidone, polyvinylprrolidone,
gelatin, gum arabic, polyethyleneglycol, sucrose or starch), a
disintegrator (e.g., starch, carboxymethylcellulose,
hydroxypropylstarch, low substituted hydroxypropylcellulose, sodium
bicarbonate, calcium phosphate or calcium citrate), a lubricant
(e.g., magnesium stearate, light anhydrous silicic acid, talc or
sodium lauryl sulfate), a flavoring agent (e.g., citric acid,
menthol, glycine or orange powder), a preservative (e.g., sodium
benzoate, sodium bisulfite, methylparaben or propylparaben), a
stabilizer (e.g., citric acid, sodium citrate or acetic acid), a
suspending agent (e.g., methylcellulose, polyvinylpyrrolidone or
aluminum stearate), a dispersing agent (e.g.,
hydroxypropylmethylcellulose), a diluent (e.g., water), and base
wax (e.g., cocoa butter, white petrolatum or polyethylene
glycol).
[0151] Tablets comprising an active agent may be coated with a
suitable film-forming agent, e.g., hydroxypropylmethyl cellulose,
hydroxypropyl cellulose or ethyl cellulose, to which a suitable
excipient may optionally be added, e.g., a softener such as
glycerol, propylene glycol, diethylphthalate, or glycerol
triacetate; a filler such as sucrose, sorbitol, xylitol, glucose,
or lactose; a colorant such as titanium hydroxide; and the
like.
[0152] Suitable excipient vehicles are, for example, water, saline,
dextrose, glycerol, ethanol, or the like, and combinations thereof.
In addition, if desired, the vehicle may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents or pH
buffering agents. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in the art. See, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 17th edition, 1985. The composition or formulation to
be administered will, in any event, contain a quantity of the agent
adequate to achieve the desired state in the subject being
treated.
[0153] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
Dosages
[0154] Although the dosage used will vary depending on the clinical
goals to be achieved, a suitable dosage range is one which provides
up to about 1 .mu.g to about 100 mg, e.g., from about 1 .mu.g to
about 25 .mu.g, from about 25 .mu.g to about 50 .mu.g, from about
50 .mu.g to about 100 .mu.g, from about 100 .mu.g to about 250
.mu.g, from about 250 .mu.g to about 500 .mu.g, from about 500
.mu.g to about 1 mg, from about 1 mg to about 25 mg, from about 25
mg to about 50 mg, or from about 50 mg to about 100 mg, of an
active agent (e.g., an agent that inhibits a Vif activity; an agent
that increases the level of active APOBEC3G in a cell; an agent
that converts a HMW APOBEC3G complex to a LMW APOBEC3G complex;
etc.) can be administered in a single dose. Alternatively, a target
dosage of an active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) can be considered to be about in the range
of about 0.1-1000 .mu.M, about 0.5-500 .mu.M, about 1-100 .mu.M, or
about 5-50 .mu.M in a sample of host blood drawn within the first
24-48 hours after administration of the agent.
[0155] Those of skill will readily appreciate that dose levels can
vary as a function of the specific compound, the severity of the
symptoms and the susceptibility of the subject to side effects.
Preferred dosages for a given compound are readily determinable by
those of skill in the art by a variety of means.
[0156] In some embodiments, a single dose of an active agent is
administered. In other embodiments, multiple doses of an active
agent are administered. Where multiple doses are administered over
a period of time, an active agent is administered twice daily
(qid), daily (qd), every other day (qod), every third day, three
times per week (tiw), or twice per week (biw) over a period of
time. For example, an active agent is administered qid, qd, qod,
tiw, or biw over a period of from one day to about 2 years or more.
For example, an active agent is administered at any of the
aforementioned frequencies for one week, two weeks, one month, two
months, six months, one year, or two years, or more, depending on
various factors.
[0157] As one non-limiting example, where an active agent increases
the level of APOBEC3G, the agent is in some embodiments an anti-CD3
antibody, such as Orthoclone OKT3.RTM. (muromonab-CD3). In these
embodiments, suitable dosage of OKT3 is about 5 mg/day,
administered in a single bolus injection intravenously, for a
period of from about 10 days to about 14 days.
Routes of Administration
[0158] An active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) is administered to an individual using any
available method and route suitable for drug delivery, including in
vivo and ex vivo methods, as well as systemic and localized routes
of administration.
[0159] Conventional and pharmaceutically acceptable routes of
administration include intranasal, intramuscular, intratracheal,
intratumoral, transdermal, subcutaneous, intradermal, topical
application, intravenous, vaginal, nasal, and other parenteral
routes of administration. Suitable routes of administration also
include oral and rectal routes. Routes of administration may be
combined, if desired, or adjusted depending upon the agent and/or
the desired effect. The composition can be administered in a single
dose or in multiple doses.
[0160] An active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) can be administered to a host using any
available conventional methods and routes suitable for delivery of
conventional drugs, including systemic or localized routes. In
general, routes of administration contemplated by the invention
include, but are not necessarily limited to, enteral, parenteral,
or inhalational routes.
[0161] Parenteral routes of administration other than inhalation
administration include, but are not necessarily limited to,
topical, vaginal, transdermal, subcutaneous, intramuscular,
intraorbital, intracapsular, intraspinal, intrasternal, and
intravenous routes, i.e., any route of administration other than
through the alimentary canal. Parenteral administration can be
carried to effect systemic or local delivery of the agent. Where
systemic delivery is desired, administration typically involves
invasive or systemically absorbed topical or mucosal administration
of pharmaceutical preparations.
[0162] The agent can also be delivered to the subject by enteral
administration. Enteral routes of administration include, but are
not necessarily limited to, oral and rectal (e.g., using a
suppository) delivery.
[0163] Methods of administration of the agent through the skin or
mucosa include, but are not necessarily limited to, topical
application of a suitable pharmaceutical preparation, transdermal
transmission, injection and epidermal administration. For
transdermal transmission, absorption promoters or iontophoresis are
suitable methods. Iontophoretic transmission may be accomplished
using commercially available "patches" which deliver their product
continuously via electric pulses through unbroken skin for periods
of several days or more.
[0164] By treatment is meant at least an amelioration of the
symptoms associated with the pathological condition afflicting the
host, where amelioration is used in a broad sense to refer to at
least a reduction in the magnitude of a parameter, e.g. symptom,
associated with the pathological condition being treated, such as
the number of viral particles per unit blood. As such, treatment
also includes situations where the pathological condition, or at
least symptoms associated therewith, are completely inhibited, e.g.
prevented from happening, or stopped, e.g. terminated, such that
the host no longer suffers from the pathological condition, or at
least the symptoms that characterize the pathological
condition.
[0165] A variety of hosts (wherein the term "host" is used
interchangeably herein with the terms "subject" and "patient") are
treatable according to the subject methods. Generally such hosts
are "mammals" or "mammalian," where these terms are used broadly to
describe organisms which are within the class mammalia, and
primates (e.g., humans, chimpanzees, and monkeys), that are
susceptible to lentivirus infection. In many embodiments, the hosts
will be humans.
Combination Therapies
[0166] A subject agent can be administered to an individual in
combination (e.g., in the same formulation or in separate
formulations) with at least a second therapeutic agent
("combination therapy"). The subject agent can be administered in
admixture with a second therapeutic agent or can be administered in
a separate formulation. When administered in separate formulations,
a subject agent and a second therapeutic agent can be administered
substantially simultaneously (e.g., within about 60 minutes, about
50 minutes, about 40 minutes, about 30 minutes, about 20 minutes,
about 10 minutes, about 5 minutes, or about 1 minute of each other)
or separated in time by about 1 hour, about 2 hours, about 4 hours,
about 6 hours, about 10 hours, about 12 hours, about 24 hours,
about 36 hours, or about 72 hours, or more. Effective amounts of a
therapeutic agent are as described above.
[0167] Therapeutic agents that can be administered in combination
therapy, such as anti-inflammatory, anti-viral, anti-fungal,
anti-mycobacterial, antibiotic, amoebicidal, trichomonocidal,
analgesic, anti-neoplastic, anti-hypertensives, anti-microbial
and/or steroid drugs, to treat antiviral infections. In some
embodiments, patients with a viral or bacterial infection are
treated with a combination of one or more subject agents with one
or more of the following; beta-lactam antibiotics, tetracyclines,
chloramphenicol, neomycin, gramicidin, bacitracin, sulfonamides,
nitrofurazone, nalidixic acid, cortisone, hydrocortisone,
betamethasone, dexamethasone, fluocortolone, prednisolone,
triamcinolone, indomethacin, sulindac, acyclovir, amantadine,
rimantadine, recombinant soluble CD4 (rsCD4), anti-receptor
antibodies (e.g., for rhinoviruses), nevirapine, cidofovir
(Vistide.TM.), trisodium phosphonoformate (Foscarnet.TM.),
famcyclovir, pencyclovir, valacyclovir, nucleic acid/replication
inhibitors, interferon, zidovudine (AZT, Retrovir.TM.),
zidovudine/lamivudine (Combivir), didanosine (dideoxyinosine, ddI,
Videx.TM.), stavudine (d4T, Zerit.TM.), zalcitabine
(dideoxycytosine, ddC, Hivid.TM.), nevirapine (Viramune.TM.),
lamivudine (Epivir.TM., 3TC), protease inhibitors, saquinavir
(Invirase.TM., Fortovase.TM.), ritonavir (Norvir.TM.), nelfinavir
(Viracept.TM.), efavirenz (Sustiva.TM.), abacavir (Ziagen.TM.),
amprenavir (Agenerase.TM.) indinavir (Crixivan.TM.), ganciclovir,
AzDU, delavirdine (Rescriptor.TM.), lopinavir/ritonavir (Kaletra),
trizivir, rifampin, clathiromycin, erythropoietin, colony
stimulating factors (G-CSF and GM-CSF), non-nucleoside reverse
transcriptase inhibitors, nucleoside inhibitors, adriamycin,
fluorouracil, methotrexate, asparaginase and combinations thereof.
Anti-HIV agents are those in the preceding list that specifically
target a function of one or more HIV proteins.
[0168] In some embodiments, a subject agent is administered in
combination therapy with two or more anti-HIV agents. For example,
a subject agent can be administered in combination therapy with
one, two, or three nucleoside reverse transcriptase inhibitors
(e.g., Combivir, Epivir, Hivid, Retrovir, Videx, Zerit, Ziagen,
etc.). A subject agent can be administered in combination therapy
with one or two non-nucleoside reverse transcriptase inhibitors
(e.g., Rescriptor, Sustiva, Viramune, etc.). A subject agent can be
administered in combination therapy with one or two protease
inhibitors (e.g., Agenerase, Crixivan, Fortovase, Invirase,
Kaletra, Norvir, Viracept, etc.). A subject agent can be
administered in combination therapy with a protease inhibitor and a
nucleoside reverse transcriptase inhibitor. A subject agent can be
administered in combination therapy with a protease inhibitor, a
nucleoside reverse transcriptase inhibitor, and a non-nucleoside
reverse transcriptase inhibitor. A subject agent can be
administered in combination therapy with a protease inhibitor and a
non-nucleoside reverse transcriptase inhibitor. Other combinations
of a subject inhibitor with one or more of a protease inhibitor, a
nucleoside reverse transcriptase inhibitor, and a non-nucleoside
reverse transcriptase inhibitor are contemplated.
Kits, Containers, Devices, Delivery Systems
[0169] Kits with unit doses of the active agent, e.g. in oral,
vaginal, rectal, transdermal, or injectable doses (e.g., for
intramuscular, intravenous, or subcutaneous injection), are
provided. In such kits, in addition to the containers containing
the unit doses will be an informational package insert describing
the use and attendant benefits of the drugs in treating an HIV
infection. Suitable active agents and unit doses are those
described herein above.
[0170] In many embodiments, a subject kit will further include
instructions for practicing the subject methods or means for
obtaining the same (e.g., a website URL directing the user to a
webpage which provides the instructions), where these instructions
are typically printed on a substrate, which substrate may be one or
more of: a package insert, the packaging, formulation containers,
and the like.
[0171] In some embodiments, a subject kit includes one or more
components or features that increase patient compliance, e.g., a
component or system to aid the patient in remembering to take the
active agent at the appropriate time or interval. Such components
include, but are not limited to, a calendaring system to aid the
patient in remembering to take the active agent at the appropriate
time or interval.
[0172] The present invention provides a delivery system comprising
an active agent that inhibits a Vif activity, an agent that
increases the level of active APOBEC3G in a cell, or an active
agent that converts a HMW APOBEC3G complex to a LMW APOBEC3G
(enzymatically active) complex. In some embodiments, the delivery
system is a delivery system that provides for injection of a
formulation comprising an active agent subcutaneously,
intravenously, or intramuscularly. In other embodiments, the
delivery system is a vaginal delivery system.
[0173] In some embodiments, an active agent is packaged for oral
administration. The present invention provides a packaging unit
comprising daily dosage units of an active agent. For example, the
packaging unit is in some embodiments a conventional blister pack
or any other form that includes tablets, pills, and the like. The
blister pack will contain the appropriate number of unit dosage
forms, in a sealed blister pack with a cardboard, paperboard, foil,
or plastic backing, and enclosed in a suitable cover. Each blister
container may be numbered or otherwise labeled, e.g., starting with
day 1.
[0174] In some embodiments, a subject delivery system comprises an
injection device. Exemplary, non-limiting drug delivery devices
include injections devices, such as pen injectors, and
needle/syringe devices. In some embodiments, the invention provides
an injection delivery device that is pre-loaded with a formulation
comprising an effective amount of an active agent that inhibits a
Vif activity or that increases the level of active APOBEC3G in a
cell or that converts a HMW APOBEC3G complex to a LMW APOBEC3G
complex. For example, a subject delivery device comprises an
injection device pre-loaded with a single dose of an active agent
that inhibits a Vif activity, or that increases the level of active
APOBEC3G, or that converts a HMW APOBEC3G complex to a LMW APOBEC3G
complex. A subject injection device can be re-usable or
disposable.
[0175] Pen injectors are well known in the art. Exemplary devices
which can be adapted for use in the present methods are any of a
variety of pen injectors from Becton Dickinson, e.g., BD.TM. Pen,
BD.TM. Pen II, BD.TM. Auto-Injector; a pen injector from Innoject,
Inc.; any of the medication delivery pen devices discussed in U.S.
Pat. Nos. 5,728,074, 6,096,010, 6,146,361, 6,248,095, 6,277,099,
and 6,221,053; and the like. The medication delivery pen can be
disposable, or reusable and refillable.
[0176] The present invention provides a vaginal delivery system for
vaginal delivery of an active agent (e.g., an agent that inhibits a
Vif activity; an agent that increases the level of active APOBEC3G
in a cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) to the vagina of an individual. The
delivery system comprises a device for insertion into the vagina.
In some embodiments, the delivery system comprises an applicator
for delivery of a formulation into the vagina; and a container that
contains a formulation comprising an active agent (e.g., an agent
that inhibits a Vif activity; an agent that increases the level of
active APOBEC3G in a cell; an agent that converts a HMW APOBEC3G
complex to a LMW APOBEC3G complex; etc.). In these embodiments, the
container (e.g., a tube) is adapted for delivering a formulation
into the applicator. In other embodiments, the delivery system
comprises a device that is inserted into the vagina, which device
includes an active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.). For example, the device is coated with,
impregnated with, or otherwise contains a formulation comprising
the active agent.
[0177] In some embodiments, the vaginal delivery system is a tampon
or tampon-like device that comprises a subject formulation. Drug
delivery tampons are known in the art, and any such tampon can be
used in conjunction with a subject drug delivery system. Drug
delivery tampons are described in, e.g., U.S. Pat. No. 6,086,909 If
a tampon or tampon-like device is used, there are numerous methods
by which an active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) can be incorporated into the device. For
example, the drug can be incorporated into a gel-like bioadhesive
reservoir in the tip of the device. Alternatively, the drug can be
in the form of a powdered material positioned at the tip of the
tampon. The drug can also be absorbed into fibers at the tip of the
tampon, for example, by dissolving the drug in a pharmaceutically
acceptable carrier and absorbing the drug solution into the tampon
fibers. The drug can also be dissolved in a coating material which
is applied to the tip of the tampon. Alternatively, the drug can be
incorporated into an insertable suppository which is placed in
association with the tip of the tampon.
[0178] In other embodiments, the drug delivery device is a vaginal
ring. Vaginal rings usually consist of an inert elastomer ring
coated by another layer of elastomer containing an active agent
(e.g., an agent that inhibits a Vif activity; an agent that
increases the level of active APOBEC3G in a cell; an agent that
converts a HMW APOBEC3G complex to a LMW APOBEC3G complex; etc.) to
be delivered. The rings can be easily inserted, left in place for
the desired period of time (e.g., up to 7 days), then removed by
the user. The ring can optionally include a third, outer,
rate-controlling elastomer layer which contains no drug.
Optionally, the third ring can contain a second drug for a dual
release ring. The drug can be incorporated into polyethylene glycol
throughout the silicone elastomer ring to act as a reservoir for
drug to be delivered.
[0179] In other embodiments, a subject vaginal delivery system is a
vaginal sponge. The active agent is incorporated into a silicone
matrix which is coated onto a cylindrical drug-free polyurethane
vaginal sponge, as described in the literature.
[0180] Pessaries, tablets and suppositories are other examples of
drug delivery systems which can be used in the present invention.
These systems have been described extensively in the
literature.
[0181] Bioadhesive microparticles constitute still another drug
delivery system suitable for use in the present invention. This
system is a multi-phase liquid or semi-solid preparation which does
not seep from the vagina as do many suppository formulations. The
substances cling to the wall of the vagina and release the drug
(e.g., an agent that inhibits a Vif activity; an agent that
increases the level of active APOBEC3G in a cell; an agent that
converts a HMW APOBEC3G complex to a LMW APOBEC3G complex; etc.)
over a period of time. Many of these systems were designed for
nasal use but can be used in the vagina as well (e.g. U.S. Pat. No.
4,756,907). The system may comprise microspheres with an active
agent; and a surfactant for enhancing uptake of the drug. The
microparticles have a diameter of 10-100 .mu.m and can be prepared
from starch, gelatin, albumin, collagen, or dextran.
[0182] Another system is a container comprising a subject
formulation (e.g., a tube) that is adapted for use with an
applicator. The active agent (e.g., an agent that inhibits a Vif
activity; an agent that increases the level of active APOBEC3G in a
cell; an agent that converts a HMW APOBEC3G complex to a LMW
APOBEC3G complex; etc.) is incorporated into creams, lotions,
foams, paste, ointments, and gels which can be applied to the
vagina using an applicator. Processes for preparing pharmaceuticals
in cream, lotion, foam, paste, ointment and gel formats can be
found throughout the literature. An example of a suitable system is
a standard fragrance free lotion formulation containing glycerol,
ceramides, mineral oil, petrolatum, parabens, fragrance and water
such as the product sold under the trademark JERGENS.TM. (Andrew
Jergens Co., Cincinnati, Ohio). Suitable nontoxic pharmaceutically
acceptable systems for use in the compositions of the present
invention will be apparent to those skilled in the art of
pharmaceutical formulations and examples are described in
Remington's Pharmaceutical Sciences, 19th Edition, A. R. Gennaro,
ed., 1995. The choice of suitable carriers will depend on the exact
nature of the particular vaginal dosage form desired, e.g., whether
the active ingredient(s) is/are to be formulated into a cream,
lotion, foam, ointment, paste, solution, or gel, as well as on the
identity of the active ingredient(s). Other suitable delivery
devices are those described in U.S. Pat. No. 6,476,079.
Subject Suitable for Treatment
[0183] The methods of the present invention are suitable for
treating individuals who have a lentiviral infection; who are at
risk of contracting a lentiviral infection; and who were treated
for a lentiviral infection, but who relapsed. Such individuals
include, but are not limited to, individuals with healthy, intact
immune systems, but who are at risk for becoming HIV infected
("at-risk" individuals). At-risk individuals include, but are not
limited to, individuals who have a greater likelihood than the
general population of becoming HIV infected. Individuals at risk
for becoming HIV infected include, but are not limited to,
individuals at risk for HIV infection due to sexual activity with
HIV-infected individuals; intravenous drug users; individuals who
may have been exposed to HIV-infected blood, blood products, or
other HIV-contaminated body fluids; and babies who are being nursed
by HIV-infected mothers. Individuals suitable for treatment include
individuals infected with, or at risk of becoming infected with,
HIV-1 and/or HIV-2 and/or HIV-3, or any variant thereof.
[0184] Individuals suitable for treatment with the methods of the
invention also include individuals who have a lentiviral infection
that is refractory to treatment with other anti-viral
therapies.
EXAMPLES
[0185] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric. Standard abbreviations may be used,
e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or
sec., second(s); min, minute(s); h or hr, hour(s); and the
like.
Example 1
HIV-1 Vif Blocks the Antiviral Activity of APOBEC3G
Experimental Procedures
Plasmids
[0186] The pNL4-3 and pNL4-3 .DELTA.Vif (also known has p-NLND)
expression vectors have been previously described. Adachi et al.
(1991). Arch Virol 117, 45-58; and Sakai et al. (1993) J Virol 67,
1663-1666. pcDNA3.1 APOBEC3G-HA and APOBEC3G expression vectors
were produced by excising an APOBEC3G-HA cassette using the
XhoI/EcoRI restriction sites present in the NG/C 15 retroviral
vector (Sheehy et al. (2002) Nature 418, 646-650) and then cloning
of the fragment into the same restriction enzyme sites in pcDNA3.1.
To prepare the pCMV4-HA-APOBEC3G vector, APOBEC3G cDNA was
amplified by PCR from an H9 cDNA library and then inserted into the
HindIII and XbaI sites of the pCMV4 vector. GST-Vif was prepared by
polymerase chain reaction (PCR) amplification of the Vif coding
region present in a pNL4-3 vector followed by insertion of this
amplicon into the EcoRI and NheI sites of the pBC vector (Chatton
et al. (1995) Biotechniques 18, 142-145). The sequences of each
insert prepared using PCR were confirmed by DNA sequencing.
Cell Lines, Spinoculation, and Preparation of Highly Purified
Virions
[0187] H9, Supt-1, Jurkat, and 293T cells were maintained using
standard tissue culture techniques. Fresh human peripheral blood
lymphocytes (PBLs) were maintained in complete RPMI media (10% FBS)
supplemented with IL-2 (5 .mu.g/L) (Roche Diagnostics). A portion
of PBLs was activated with phytohemagglutinin (PHA) (5 .mu.g/ml)
for 24 hours prior to use. HIV-1 wt and HIV-1 .DELTA.Vif viruses
were pseudotyped with the vesicular stomatitis virus-G (VSV-G)
envelope by calcium phosphate mediated cotransfection of 293T cells
with expression vector DNAs encoding VSV-G and pNL4-3 or pNL4-3
.DELTA.Vif. After 48 hours of culture, virus-containing
supernatants were harvested, clarified by centrifugation at
500.times.g for 5 minutes and filtered through 0.2
micron-pore-sized filters. The virus-containing supernatant was
then incubated with 0.4.times.10.sup.6 H9 cells/well in 48 well
plates and centrifuged at low speed for 90 minutes at 34.degree. C.
The cells were then washed with phosphate-buffered saline (PBS) and
incubated in complete medium for 40 hours at 37.degree. C. The
cells were then pelleted by centrifugation at 500.times.g for 5
min., washed in PBS, and lysed for immunoblotting and
nuclear-cytoplasmic fractionation studies. Highly purified virions
were obtained from the virus-containing supernatant using the
discontinuous step iodixanol gradient purification method
previously described (Dettenhofer and Yu (1999) J Virol 73,
1460-1467. Fractions from the iodixanol gradient were then
subjected to an additional round of ultracentrifugation for 90
minutes at 4.degree. C., and the pellets were resuspended in 30
.mu.l of Laemmli loading buffer prior to analysis by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Antibodies, Immunoblotting Analysis, Intracellular Anti-p24
Immunostaining, and FPLC
[0188] Anti-APOBEC3G antibody was prepared by immunizing and
boosting rabbits with a synthetic peptide corresponding to the C
terminal 16 amino acids of human APOBEC3G [(C)-QDLSGRLRAILQNQEN;
SEQ ID NO:8]. Serum was obtained after 42 days and tested for
immunoreactivity with H9 cellular lysates. As a negative control,
the same lysates were tested for immunoreactivity with pre-immune
serum from the same rabbits (Antibody Solutions). Polyclonal
anti-Vif antiserum (Goncalves et al. (1994) J Virol 68, 704-712).
Rabbit anti-Nef antiserum has been previously described (Bresnahan
et al., 1998). Mouse monoclonal anti-p24 Gag ascites was a generous
gift from BeckmanCoulter. Other antibodies used included rabbit
anti-HA (Santa Cruz); mouse anti-HA (12CA5, Roche); and mouse
anti-ubiquitin (Santa Cruz). Lysis buffers used for immunoblotting
and fast protein liquid chromatography (FPLC) were (1) 100-400 mM
NaCl, 50 mM Hepes, 0.2% NP40, 0-5 mM EDTA, 0.1 mM PMS, plus
complete protease inhibitor cocktail (Roche) (1 tablet per 50 ml
buffer) or (2) 50 mM Hepes, pH 7.2, 135 mM NaCl, 1% Triton X-100,
0.5% deoxycholate, 10% glycerol, 1 mM EDTA, 1.times. protease
inhibitor cocktail (Calbiochem). The FPLC running buffer consisted
of 50 mM Hepes, pH 7.4; 250 mM NaCl; 0.1% NP40, 1 mM DTT; and 10%
glycerol. Intracellular anti-24 immunostaining of H9 cells was
performed as previously described (Eckstein et al. (2001) J. Exp.
Med. 194:1407-1419). Cell lysates for FPLC analysis were
centrifuged twice in a microfuge at maximum speed for 10 minutes
and then applied to a calibrated Superose 6 HR 10/30 or Superose 12
HR 10/30 pre-packed gel filtration column (AKTA, Amersham Pharmacia
Biotech). One ml samples were collected from the column for
subsequent analysis.
Protein Expression Assays
[0189] 293T cells were transfected with the indicated amounts of
expression vector DNA using the calcium phosphate method. Lysis
buffers used for immunoblotting were (1) 100-400 mM NaCl, 50 mM
Hepes, 0.2% NP40, 0-5 mM EDTA, 0.1 mM PMSF, plus complete protease
inhibitor cocktail (Roche) (1 tablet/50 ml buffer) or (2) 50 mM
Hepes, pH 7.2, 135 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 10%
glycerol, 1 mM EDTA, 1.times. protease inhibitor cocktail
(Calbiochem). Samples were analyzed using standard SDS-PAGE
techniques.
[0190] For the proteasome inhibitor experiments in transiently
transfected cells, 293T cells were transiently transfected with 0.1
.mu.g of APOBEC3G-HA DNA, 2 .mu.g of control vector DNA and 2 .mu.g
of either pcDNA3.1 Vif DNA vector or an additional 2 .mu.g of
control vector DNA. Twenty-four hours after transfection, the cells
were treated with the indicated inhibitor and harvested 15 hours
later. For the proteasome inhibitor experiments using infected H9
cells, H9 cells were infected by spinoculation as described above.
Forty-four hours after infection, the cells were treated for 16
hours with either dimethylsulfoxide (DMSO) (0.1%) or 0.25 .mu.M
epoxomicin. All of the inhibitors (ALLN, MG 132, epoxomicin,
Calpain Inhibitor III, and clasto-Lactacystin-.beta.-Lactone) were
purchased from CalBiochem and were dissolved in DMSO.
[0191] Intracellular anti-24 Gag immunostaining of H9 cells was
performed as described (Eckstein et al., 2001, supra). Briefly, H9
cells were harvested, washed and permeabilized by incubation at
room temperature for 30 min in PermeaFix (Ortho Diagnostic Systems,
Raritan, N.J.). After two additional washes, the cells were
incubated with FITC-conjugated anti-p24-Gag antibody (Coulter) for
30 min at room temperature. After additional wash steps, the cells
were then analyzed by flow cytometry.
[0192] To analyze subcellular distribution of the APEBEC3G, H9 or
transfected 293T cells were washed and suspended in swelling buffer
(10 mM HEPES pH 7.9, 1.5 mM MgCl.sub.2, 10 mM KCL, protease
inhibitor cocktail and PMSF). The cells were allowed to swell for
10 min on ice before being vortexed in the presence of 0.4% NP-40.
The lysate was layered on top of 1 ml of 1% sucrose in swelling
buffer and then centrifuged at 3150 RPM in a Beckman GS-6R
centrifuge.
Northern Analysis
[0193] RNA was extracted from 10.sup.7 infected or uninfected H9 or
Supt-1 cells using the Qiagen RNAeasy RNA extraction kit. The RNA
was then separated on a 1% formaldehyde-agarose gel, transferred to
a nitrocellulose membrane and probed with a .sup.32P-labeled
APOBEC3G cDNA. Hybridizing bands on the membrane were then
visualized using autoradiography.
Pulse-Chase and Pulse Radiolabeling Experiments
[0194] HEK 293 cells were co-transfected with 1 .mu.g of
APOBEC3G-HA expression vector and 8 .mu.g of either Vif or control
expression vectors. The transfected HEK 293 cells were
pre-incubated for one hour in labeling media (DMEM without
methionine and cysteine (Cellgro) plus 10% dialyzed FBS). Each
sample was subsequently pulse-labeled for 30 min in labeling media
containing 250 .mu.Ci EasyTag.TM. EXPRESS .sup.35S Protein Labeling
Mix containing radiolabled methionine and cysteine (PerkinElmer).
The initial pulse-labeled (t=0) samples were harvested. The
remaining radiolabeled samples were incubated with chase media
(DMEM plus 10% FBS with 4.02 mM methionine (20.times.) and 3 mM
cysteine (15.times.)) and harvested at various time points. For the
pulse-label experiment, each sample was pulse-labeled for 15 min
and then harvested immediately.
[0195] The harvested cell pellets were lysed in Buffer A (50 mM
HEPES, 135 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 10%
glycerol, 1 mM EDTA, (pH 7.2) plus protease inhibitor cocktail set
I (Calbiochem)) for 15 minutes on ice. The cell lysates were
clarified at 14,000.times.g, at 4.degree. C. for 10 min. Protein
determinations were performed using the BCA Protein Assay Reagents
(Pierce). Immunoprecipitations (IP) were set up at equal protein
concentration/volume in the presence of anti-HA monoclonal
antibodies (262K Mab, Cell Signaling Technology) and incubated on
ice. All subsequent spin/wash steps were performed at 4.degree. C.
The IPs were washed four times with Buffer A and eluted with
1.times.SDS-PAGE sample buffer. The samples were analyzed by
SDS-PAGE and the gels fixed for at least 1 hour before treatment
with Amplify fluorographic reagent (Amersham Biosciences) and gel
drying. The dried gels were subjected to autoradiography and the
scanned images were quantified using Scion Image software (version
1.62).
In Vitro Translation
[0196] Vif or control GST protein was initially transcribed and
translated for 90 min using the Promega TNT T7-coupled Reticulocyte
Lysate system according to the manufacturer's instructions. The
pre-synthesized Vif or GST was then added to a new
transcription/translation mix programmed with APOBEC3G DNA on ice.
The reactions were then transferred to a 30.degree. C. water bath
and allowed to proceed for 15 or 30 min. The proteins were
separated by SDS-PAGE, and the gel was analyzed by
autoradiography.
Results
Native APOBEC3G Protein is Selectively Expressed in Nonpermissive T
Cells and Principally Localizes in the Cytoplasm
[0197] To address the question of how Vif overcomes the antiviral
activity of APOBEC3G, a rabbit polyclonal antibody was developed,
which was directed to the C terminal 16 amino acids of the
.about.46 kD APOBEC3G protein. Although a previous study
demonstrated that APOBEC3G mRNA is expressed in nonpermissive but
not in permissive cells (Sheehy et al., 2002, supra), it remained
unknown whether APOBEC3G protein was actually expressed in
nonpermissive cells. Accordingly, the expression pattern of
APOBEC3G protein was assessed by immunoblotting lysates from
permissive and nonpermissive cell types with the anti-APOBEC3G
antibody. The results are depicted in FIG. 1A.
[0198] The rabbit anti-APOBEC3G antibody specifically reacted with
a .about.46 kD protein that was present in nonpermissive
PHA-stimulated primary peripheral blood lymphocytes (PBLs) and H9
cells (FIG. 1A, lanes 2 and 3). In contrast, this 46 kD protein was
not detected in lysates prepared from permissive Supt-1, Jurkat, or
293T cells (FIG. 1A, lanes 4-6), nor was it detected in
nonpermissive cell types when pre-immune serum from the same rabbit
was used. The levels of .alpha.-tubulin expression were similar
between the nonpermissive and permissive cell lysates, confirming
equal loading. Of note, PHA stimulation of the PBLs (designated by
+ symbol) resulted in increased expression of endogenous APOBEC3G
(compare lanes 1 and 2). Although .alpha.-tubulin was expressed at
lower levels in these unstimulated PBL lysates, p56.sup.lck was
nearly equally expressed in the unstimulated versus PHA-activated
PBL lysates, suggesting that APOBEC3G is inducibly expressed in
response to mitogen activation of primary PBLs.
[0199] To further confirm the specificity of the anti-APOBEC3G
antibody, cellular lysates from 293T cells transfected with either
control vector DNA (mock) or HA-APOBEC3G expression vector were
immunoblotted. The anti-APOBEC3G antibody reacted with a .about.50
kD protein present in the lysate from the HA-APOBEC3G transfected
cells while lysates from the mock-transfected 293T cells did not
exhibit a comparable protein band (FIG. 1A, lanes 6 and 7).
Together, these findings demonstrate that the rabbit anti-APOBEC3G
antibody specifically reacts with endogenous human APOBEC3G,
confirm that endogenous APOBEC3G protein is expressed in
nonpermissive cells, and reveal that APOBEC3G protein expression is
upregulated following PHA stimulation of primary PBLs.
[0200] Next, the intracellular localization of APOEC3G was analyzed
by immunoblotting nuclear and cytoplasmic fractions isolated from
nonpermissive H9 cells and permissive SupT1 cells. The results are
shown in FIG. 1B. Tubulin and histone H3 were employed as
cytoplasmic and nuclear markers, respectively, to monitor the
purity of these subcellular fractions. The endogenous APOBEC3G
protein was not detectably expressed in either the nucleus or the
cytoplasm of permissive SupT1 cells. Conversely, in nonpermissive
H9 cells, APOBEC3G was readily detected and was principally
localized to the cytoplasm. However, small amounts of APOBEC3G were
present in the nucleus. Since the expression of tubulin and histone
H3 shows that the nuclear and cytoplasmic fractions were not cross
contaminated, these findings raise the possibility that the
APOBEC3G protein may in fact shuttle into and out of the nucleus.
Nevertheless, APOBEC3G is predominantly expressed in the
cytoplasmic compartment.
[0201] FIGS. 1A and 1B. Characterization of the expression and
localization of APOBEC3G. (A) Immunoblot analysis of APOBEC3G
expression in nonpermissive and permissive cells. The indicated
cell types were lysed and analyzed by SDS-PAGE followed by
immunoblotting with anti-APOBEC3G antiserum, anti-tubulin and
anti-p56.sup.lck. To confirm specificity of the antiserum, 293T
cells were transfected with a HA-APOBEC3G expression vector. PBL,
peripheral blood lymphocytes. (B) Subcellular localization of
APOBEC3G. Nuclear and cytoplasmic fractions were prepared from H9
(nonpermissive) or Supt-1 (permissive) cells and analyzed by
immunoblotting with antiserum specific for APOBEC3G. Immunoblotting
with anti-.alpha.-tubulin and anti-Histone H3 confirmed the
integrity of the cytoplasmic and nuclear fractions. Note that
APOBEC3G is predominantly localized in the cytoplasm although small
amounts of this protein are also present in the nucleus.
Vif Prevents Intravirion Incorporation of Endogenous APOBEC3G
Protein
[0202] Recent studies have demonstrated that APOBEC3G inhibits the
infectivity of HIV-1 .DELTA.Vif virions produced by nonpermissive
cells by causing extensive hypermutation of newly-synthesized HIV-1
DNA transcripts formed during reverse transcription in the
subsequent target cell (Harris et al., 2003; Lecossier et al.,
2003; Mangeat et al., 2003; Zhang et al., 2003). These findings
support the conclusion that an antiviral factor is incorporated
into the .DELTA.Vif virions and transported to the next target cell
(Harris et al., 2003; Lecossier et al., 2003; Mangeat et al., 2003;
Zhang et al., 2003, supra). In this regard, APOBEC3G-HA has been
detected in virions produced by transfected 293T cells in both the
presence and absence of Vif (Sheehy et al., 2002, supra). However,
given the unnaturally high levels of APOBEC3G protein that are
expressed in transfected 293T cells, this system may not faithfully
recapitulate the actual biological events. It was hypothesized that
Vif may prevent endogenous APOBEC3G from being incorporated into
the virion and tested this possibility in a more physiologically
relevant cellular setting. H9 cells were infected with vesicular
stomatitis protein-G (VSV-G) pseudotyped HIV-1.sub.NL4-3 wt or
VSV-G-HIV-1.sub.NL4-3-.DELTA.Vif viruses. To isolate highly
purified virions, supernatants from infected H9 cells were
collected and applied to discontinuous iodixanol gradients.
Sequential fractions from these gradients were analyzed for p24Gag
content as a marker of HIV-1 wt and .DELTA.vif virions. Probing of
these fractions revealed the presence of endogenous APOBEC3G in the
HIV-1 .DELTA.Vif virions (FIG. 2). Conversely, virtually no
full-length APOBEC3G protein was present in the HIV-1 wt virions.
These findings indicate that Vif expression in the producer cell
markedly impairs the incorporation of APOBEC3G protein into newly
formed virions.
[0203] FIG. 2. Vif prevents incorporation of full-length endogenous
APOBEC3G into virions. HIV-1 and HIV-1 .DELTA.Vif virions were
prepared by spinoculation infection of H9 cells with VSV-G
pseudyotyped-HIV-1 or HIV-1 .DELTA.Vif viruses. Forty eight hours
post-infection, virus-containing supernatants were collected and
virions were purified on discontinous step iodixanol gradients.
Gradient fractions were immunoblotted with anti-p24 Gag antibody to
identify virions and anti-APOBEC3G antiserum to evaluate virion
incorporation of this antiviral factor. Note that APOBEC3G is
markedly diminished in HIV-1 wt virions compared with HIV-1
.DELTA.Vif virions.
Vif Expression is Associated with the Disappearance of Endogenous
APOBEC3G Protein in HIV-1-infected H9 T Cells
[0204] To further elucidate the mechanism by which HIV-1 Vif
prevents the incorporation of APOBEC3G into virions, intracellular
levels of endogenous APOBEC3G in H9 cells were analyzed 2 days
after infection with HIV-1 wt or HIV-1 .DELTA.Vif viruses. For
these studies H9 cells were employed that displayed comparable
levels of HIV-1 infection (76.4% versus 73.9%) as assessed by
intracellular anti-p24-Gag immunostaining (FIG. 3A, top) and
equivalent viability as assessed by forward and side light
scattering properties (FIG. 3A, bottom).
[0205] Next, cellular lysates were prepared from uninfected H9
cells, or H9 cells equivalently infected with HIV-1 wt or HIV-1
.DELTA.Vif and analyzed expression of endogenous APOBEC3G protein
by immunoblotting with the anti-APOBEC3G antibody (FIG. 3B). A
marked reduction was observed in the expression of endogenous
APOBEC3G protein in H9 cells infected with HIV-1 wt. Conversely,
APOBEC3G expression was readily detected in HIV-1 .DELTA.Vif
infected H9 cell cultures. This level was very similar to that
found in uninfected H9 cells suggesting that HIV-1 infection does
not induce APOBEC3G expression. In contrast, .alpha.-tubulin levels
did not decline in the HIV-1 wt infected sample in comparison to
uninfected or HIV-1 .DELTA.Vif infected samples (FIG. 3B). Vif was
expressed in the HIV-1 wt infected sample, and similar levels of
Nef were detected in the two virally infected samples (FIG. 3B).
These findings demonstrate that infection of nonpermissive cells
with HIV-1 expressing Vif leads to a marked loss of native APOBEC3G
protein expression.
[0206] FIG. 3. Vif induces an almost complete disappearance of
endogenous APOBEC3G in H9 cells infected with HIV-1-wt but not in
cells infected with HIV-1 .DELTA.Vif (A). Infection of H9 cells. H9
cells were infected with VSV-G pseudotyped HIV-1-wt or HIV-1
.DELTA.Vif virus by spinoculation and analyzed by flow cytometry
using intracellular Gag expression to monitor the extent of viral
infection and by forward and side scattering of light (FSC/SSC) to
identify live cells. Note that H9 cells infected with HIV-1 wt and
HIV-1 .DELTA.Vif were comparably infected and displayed equivalent
numbers of cells in the live cell gate. (B) Analysis of endogenous
APOBEC3G expression in uninfected H9 cells or H9 cells infected
with HIV-1 wt and HIV-1 .DELTA.Vif viruses. Cellular lysates from
these cultures were immunoblotted with anti-C-terminal APOBEC3G
antiserum and with anti-.alpha.-tubulin, anti-Vif or anti-Nef
antibodies. Note the sharp decline in intracellular APOBEC3G levels
in cells infected with HIV-1 wt viruses.
APOBEC3G mRNA Levels and Integrity Do Not Change in the Presence of
HIV-1 Vif.
[0207] To further dissect the mechanism underlying the apparent
disappearance of APOBEC3G protein expression in HIV-1 wt infected
H9 cells, the possibility that Vif alters the expression or
integrity of APOBEC3G mRNA was considered. RNA and protein were
simultaneously prepared from uninfected and infected H9 cells and
levels of APOBEC3G mRNA and protein were assessed. As expected,
APOBEC3G protein expression was decreased in the H9 cells infected
with HIV-1 wt. However, there was no apparent difference in the
level of expression of APOBEC3G mRNA among the three samples (FIG.
4). Equivalent RNA loading in these samples was confirmed by
hybridization with a DNA probe specific for GAPDH (FIG. 4). These
findings show that Vif does not impair APOBEC3G protein expression
by interfering with the production or stability of APOBEC3G
mRNA.
[0208] FIG. 4. Vif does not alter APOBEC3G mRNA expression or
integrity. Total RNA was extracted from lysates of H9 cells that
were either uninfected or infected with VSV-G pseudotyped HIV-1 or
HIV-1 .DELTA.Vif viruses and then subjected to Northern blot
analysis using a P-labeled APOBEC3G probe. Note that while APOBEC3G
protein expression is decreased in H9 cells infected with HIV-1 wt,
the level of APOBEC3G mRNA in these same cells is equivalent to
that present in cells infected with HIV-1 .DELTA.Vif. Radiolabeled
GAPDH probes were employed as a control for mRNA loading.
Vif Alone is Sufficient to Reduce Intracellular Levels of
APOBEC3G
[0209] The question of whether Vif alone could recapitulate the
decline in endogenous APOBEC3G levels observed when H9 cells were
infected with virus containing the full complement of HIV-1
proteins was examined. For these studies, 293T cells were
transiently transfected with increasing doses of untagged APOBEC3G
expression plasmid (0.1-1.0 ug) in the presence of a fixed amount
of Vif expression vector (FIG. 5A). The expression of Vif alone
reduced the intracellular levels of APOBEC3G protein, although this
effect was proportionately less at high APOBEC3G input levels. The
levels of Vif expression were comparable in each of the cell
lysates from cells transfected with Vif expression vector DNA; and
the cellular lysates contained similar amounts of .alpha.-tubulin,
indicating comparable loading. The Vif-induced decrease of APOBEC3G
expression was also not caused by translocation of APOBEC3G into
the nucleus since analysis of whole cell lysates revealed the same
loss of APOBEC3G. These results demonstrate that Vif is able to act
in the absence of other viral components to promote an
intracellular decline in APOBEC3G protein expression.
[0210] FIG. 5. Intracellular depletion of APOBEC3G by Vif does not
require the participation of other viral proteins and Vif
physically interacts with APOBEC3G. (A) Expression of Vif in 293T
cells is sufficient to promote a decline in intracellular APOBEC3G
expression in the absence of other HIV-1 proteins. 293T cells were
transfected with graded amounts of APOBEC3G expression vector DNA
in the presence or absence of a constant amount of Vif expression
vector. Cellular lysates from these cultures were immunoblotted
with anti-APOBEC3G antiserum to assess intracellular levels of this
protein. Note that Vif alone is sufficient to induce a decline in
APOBEC3G expression over the range of APOBEC3G inputs although the
effect is most pronounced at the lower levels of APOBEC3G
expression. Blotting for .alpha.-tubulin expression confirmed
loading of comparable amounts of these cellular lysates. (B)
APOBEC3G interacts with GST-Vif. Lysates from 293T cells
transfected with HA-APOBEC3G expression vector DNA and either GST
or GST-Vif mammalian expression vectors were incubated with
glutathione-sepharose beads. After washing, the beads were boiled
in sample buffer and analyzed by SDS-PAGE followed by
immunoblotting. Note that GST-Vif but not GST binds HA-APOBEC3G.
(C) GST-Vif is biologically active. Immunoblotting analysis of
lysates from 293T cells co-transfected with APOBEC3G-HA expression
plasmid and either GST or GST-Vif expression vector. Note that
expression of GST-Vif but not GST induced a decline in
intracellular APOBEC3G levels.
Vif Physically Assembles with APOBEC3G
[0211] A potential physical interaction of HIV-1 Vif with APOBEC3G
was evaluated by using a mammalian GST expression system.
HA-APOBEC3G and GST-Vif or control GST expression plasmids were
co-transfected into 293T cells (FIG. 5B). Cell lysates were
prepared and incubated with glutathione sepharose beads. Subsequent
immunoblotting analysis revealed that HA-APOBEC3G was precipitated
by GST-Vif but not by GST alone (FIG. 5B). We also confirmed that
GST-Vif retained biological activity by monitoring APOBEC3G-HA
levels in the presence and absence of GST-Vif. APOBEC3G-HA levels
were reduced in the presence of GST-Vif when compared to GST alone
(FIG. 5C). These findings indicate that Vif either directly or
indirectly assembles with the APOBEC3G protein in living cells and
that the chimeric GST-Vif protein retains the ability to impair
APOBEC3G expression.
APOBEC3G is Present in a High-molecular Weight Ribonucleoprotein
Complex and Vif Co-Fractionates with this Complex.
[0212] To further discern the mechanism employed by Vif, we
investigated whether APOBEC3G resides in a larger complex and, if
so, whether Vif could associate with this complex. First, using
size-exclusion fast performance liquid chromatography (FPLC) of
lysates from transfected 293T cells, it was found that APOBEC3G-HA
is present in a high molecular weight complex (>700 kD) (FIG.
6A). Interestingly, pre-treating the cellular lysates with RNAse
promoted disruption of this high molecular weight APOBEC3G complex
(FIG. 6A). Furthermore, size-exclusion FPLC analysis of infected H9
cell lysates revealed that a portion of Vif expressed in these
cells co-fractionates with the endogenous high molecular weight
APOBEC3G complex. These findings indicate that APOBEC3G assembles
into a high molecular weight complex that includes cellular RNAs
and that the RNA maintains the integrity of the complex.
Furthermore, a portion of Vif present in HIV-1 wt infected H9 cells
co-fractionates with the high molecular weight APOBEC3G complex
(FIG. 6B), which is consistent with our prior finding indicating
that Vif binds directly or indirectly to APOBEC3G (FIG. 5B).
[0213] FIG. 6. APOBEC3G assembles in an RNAse-sensitive high
molecular weight complex and a portion of Vif co-fractionates with
this APOBEC3G complex in infected H9 cells. (A) APOBEC3G assembles
in a high-molecular weight RNAse-sensitive complex. Lysates from
293T cells transfected with APOBEC3G-HA were either treated with
diluent control or 0.01 mg/ml RNAse on ice for 30 minutes. The
lysates were then applied to a Superose 6 column and subjected to
FPLC size-exclusion fractionation followed by SDS-PAGE and
immunoblotting with anti-HA antibodies. Note that APOBEC3G-HA
predominantly resides in a >700 kD complex and that RNAse
treatment results in a shift of most of the APOBEC3G to a lower
molecular weight complex. (B) Vif and APOBEC3G co-fractionate in
infected H9 cells. H9 cells were infected with VSV-G pseudotyped
HIV-1 wt virus followed by the preparation of cellular lysates and
analysis of lysate proteins on a Superose 12 FPLC column. Fractions
were collected and analyzed by immunoblotting with anti-APOBEC3G or
anti-Vif antibodies. Note that a portion of Vif co-fractionates
with native APOBEC3G in a >700 kD complex.
Vif Induces Proteolytic Degradation of APOBEC3G
[0214] It was next determined whether Vif induces APOBEC3G
degradation or, alternatively, whether it simply induces
proteolytic clipping of APOBEC3G leading to loss of
immunoreactivity with the rabbit anti-APOBEC3G antibody. Since this
antibody reacts specifically with the C-terminus of APOBEC3G,
whether a similar loss of APOBEC3G immunoreactivity occurred when
an antibody directed to the N-terminus was employed was examined.
For these studies, graded doses of either HA-APOBEC3G or
APOBEC3G-HA expression vector DNA together with Vif expression
plasmid DNA were co-transfected into 293T cells and tested the
cellular lysates for APOBEC3G expression using an anti-HA antibody.
Both N- and C-terminally tagged versions of APOBEC3G were expressed
at reduced levels in the presence of Vif (FIG. 7 A&B). These
findings support the conclusion that Vif induces degradation of
APOBEC3G or extensive proteolysis of APOBEC3G involving both the N
and C-termini of the protein.
APOBEC3G is Polyubiquitylated and Vif Promotes Degradation of
(Ub).sub.n-APOBEC3G
[0215] To obtain further insight into the mechanism of Vif-induced
degradation of APOBEC3G, it was investigated whether APOBEC3G is
modified by ubiquitylation. APOBEC3G and His-ubiquitin expression
vectors (Treier et al., 1994) were cotransfected into 293T cells in
the presence or absence of Vif expression vector. After 48 hours in
culture, cellular lysates were prepared. Subsequent immunoblotting
of these proteins with anti-HA antibodies revealed a continuum of
polyubiquitylated forms of APOBEC3G-HA (FIG. 7C, lanes 2 and 4).
Immunoblotting of lysates prepared in the absence of APOBEC3G-HA
confirmed that anti-HA antibodies selectively reacted with modified
forms of APOBEC3G (FIG. 7C, lane 10). Coexpression of Vif was
associated with a complete loss of these polyubiquitylated APOBEC3G
species as well as diminished expression of unmodified APOBEC3G
(FIG. 7C, lanes 6 and 8). To investigate whether the 26S proteasome
mediated Vif-induced degradation of these polyubiquitylated
APOBEC3G protein species, parallel experiments were performed in
the presence of the proteasome inhibitor, MG132. This proteasome
inhibitor did not block the Vif-dependent disappearance of
polyubiquitylated APOBEC3G or increase levels of the unmodified
protein (FIG. 7C, lanes 5 and 7). However, MG132 promoted increased
accumulation of many polyubiquitylated proteins within these cells,
indicating that the inhibitor was biologically active. These
findings suggest that Vif-induced degradation of polyubiquitylated
APOBEC3G occurs through a mechanism that is not dependent on the
function of the 26S proteasome. However, it was also observed in
multiple experiments that one of the many APOBEC3G degradation
products appearing in Vif-expressing cells increased after the
addition of MG132 (FIG. 7C, compare lanes 7 and 8). These findings
raise the possibility that Vif action may involve multiple pathways
including the targeting of select proteolytic fragments of APOBEC3G
for degradation by the 26S proteasome.
[0216] FIG. 7. HIV-1 Vif induces degradation of APOBEC3G, and
polyubiquitylated forms of APOBEC3G form a major target for Vif
induced degradation. (A and B) Vif promotes degradation or
extensive proteolysis of APOBEC3G as reflected by the loss of both
N- and C-terminal immunoreactive epitopes. Graded amounts of
expression vector DNA encoding HA-APOBEC3G or APOBEC3G-HA were
co-transfected with and without Vif expression vector DNA into 293T
cells. Cellular lysates prepared from these cultures were analyzed
by immunoblotting with anti-HA antibodies. Immunoblotting for
.alpha.-tubulin expression confirmed comparable loading of these
cellular lysates. Note that Vif expression induces a loss of
anti-HA immunoreactivity regardless of whether the HA epitope is
positioned at the N- or C-terminus of APOBEC3G. (C) APOBEC3G is
polyubiquitylated and Vif induces degradation of (Ub)n-APOBEC3G
apparently by a proteasome-independent mechanism. 293T cells were
cotransfected with APOBEC3G-HA and his-ubiquitin expression vectors
in the presence or absence of Vif expression vector. 48 hours
later, cellular lysates were prepared. As indicated, select
cultures were supplemented with the proteasome inhibitor, MG132,
for the final 6 hours of culture. Lysates were analyzed by
immunoblotting with anti-HA antibodies. Note that coexpression of
Vif resulted in marked loss of polyubiquitylated APOBEC3G-HA
proteins and that the disappearance of these modified forms of
APOBEC3G-HA was not blocked by addition of the MG132 proteasome
inhibitor to the cell cultures. In addition to the
polyubiquitylated forms of APOBEC3G-HA, degradation products of
APOBEC3G-HA were apparent both in the presence and absence of Vif
expression. One of these multiple degradation products of
APOBEC3G-HA present in Vif expressing cells increased in quantity
in the presence of MG132 (*) suggesting that the degradation likely
involved an action of the proteasome.
Vif Induced Depletion of APOBEC3G Involves Disappearance of the
Entire Protein
[0217] It was investigated whether the apparent loss of APOBEC3G in
Vif expressing cells could be explained by proteolytic clipping of
the C-terminus, which would result in a loss of the epitope used
for immunodetection. When 293T cells were co-transfected with
graded doses of HA-APOBEC3G or APOBEC3G-HA expression vector DNA in
the presence or absence of Vif expression plasmid DNA, both the N-
and C-terminally tagged versions of the APOBEC3G enzyme were
diminished in the presence of Vif. These findings indicate that Vif
targets the entire APOBEC3G protein for elimination rather than
inducing proteolysis only at the C-terminus.
Vif Physically Assembles with APOBEC3G
[0218] To evaluate whether HIV-1 Vif physically interacts with
APOBEC3G, 293T cells were contransfected with HA-APOBEC3G and
GST-Vif or control GST expression plasmid DNA. Cellular lysates
were prepared and incubated with glutathione-Sepharose beads.
Immunoblotting of proteins bound to these beads demonstrated
recovery of HA-APOBEC3G by GST-Vif but not by GST. To assess
whether the GST-Vif protein retained biological activity,
HA-APOBEC3G levels were monitored in the presence and absence of
GST-Vif and observed lower levels of this enzyme in the presence of
GST-Vif (FIG. 8D). These findings indicate that Vif is able to
assemble with the APOBEC3G protein in living cells and further that
the chimeric GST-Vif protein retains the ability to impair
intracellular APOBEC3G expression.
Vif Shortens the In Vivo Half-Life of APOBEC3G.
[0219] To investigate whether Vif depletes the intracellular levels
of APOBEC3G by accelerating its degradation,
[.sup.35S]-methionine/cysteine pulse-chase radiolabeling studies
were performed in HEK 293 cells co-transfected with APOBEC3G-HA and
HIV-1 Vif or control expression vector DNA. Thirty-six hours after
transfection, the cells were incubated in methionine and
cysteine-deficient medium for 60 minutes followed by pulse labeling
with .sup.35S-methionine/cysteine for 30 minutes at 37.degree. C.
Subsequently, the cultures were chased for up to 12 hours at
37.degree. C. in media containing excess unlabeled methionine and
cysteine. APOBEC3G-HA was then immunoprecipitated from lysates of
cells harvested at different intervals (FIG. 8A). In the absence of
Vif, the radiolabeled APOBEC3G protein decayed with a T1/2 of
approximately 10 hrs. However, in the presence of Vif, the
radiolabeled APOBEC3G disappeared more rapidly, displaying a T1/2
of approximately 1.9 hrs (FIG. 8A). These findings demonstrate that
Vif shortens the half-life of APOBEC3G.
Proteasome Inhibitors Partially Block Vif-Mediated Depletion of
APOBEC3G
[0220] To investigate whether the 26S proteasome might mediate the
Vif-induced degradation of APOBEC3G, H9 cells were infected with
HIV-1 wt or were mock-infected. Forty-four hours after infection,
the cells were treated for 16 hours with either DMSO (0.1%) or 0.25
.mu.M epoxomicin, which specifically inhibits three proteolytic
activities in the proteasome. Whole cell lysates were subsequently
prepared by boiling the cells in Laemmli lysis buffer, followed by
protein separation by SDS-PAGE and immunoblotting. As expected,
APOBEC3G levels were much lower in the infected sample when
compared to the uninfected sample (FIG. 8B, lanes 1 and 3).
However, APOBEC3G levels were elevated in infected cells treated
with epoxomicin when compared to DMSO-treated cells (FIG. 8B, lanes
1 and 2), while epoxomicin did not appear to cause a similar
increase in the level of APOBEC3G in the uninfected cultures (FIG.
8B, lanes 3 and 4). Interestingly, epoxomicin also caused an
increase in Vif levels.
[0221] To confirm this finding in a different system, a similar
experiment was performed using 293T cells transiently transfected
with an APOBEC3G-HA expression vector and either a Vif expression
vector or a control vector. Twenty-four hours after transfection
the cells were treated with a panel of different proteasome
inhibitors for a 15-hour period. It was observed that the broadly
acting proteasome inhibitors MG132 (12.5 .mu.M) and ALLN (25 .mu.M)
as well as the more specific proteasome inhibitor
clasto-Lactacystin-.beta.-Lactone (10 .mu.M, 20 .mu.M) enhanced the
levels of APOBEC3G-HA in the presence of Vif when compared to the
diluent control sample (0.1% DMSO) (FIG. 8C, lanes 7 through 12).
In contrast, Calpain Inhibitor III (50 .mu.M), which inhibits a
separate proteolytic pathway (FIG. 8C, lane 12), did not cause an
increase in APOBEC3G levels. Taken together, our results indicate
that Vif's effect on APOBEC3G is at least partially mediated by the
proteasome.
[0222] FIG. 8A-C. Vif shortens the half-life of APOBEC3G, and
proteasome inhibitors partially block the intracellular depletion
of APOBEC3G-HA. (A) Pulse-Chase Radiolabeling. HEK 293 cells
transiently transfected with APOBEC3G-HA and Vif or control vector
DNA were pulse-labeled for 30 min. The initial pulse-labeled (t=0)
samples were harvested, and the remaining samples were incubated
with chase media for the indicated time points. After
immunoprecipitation with anti-HA antibody, the samples were
analyzed by SDS-PAGE and the gels were subjected to
autoradiography. The relative intensity of the bands were
quantified and the Vif (+) and Vif (-) samples were normalized to 1
and graphed (FIG. 8, right side). The T.sub.1/2 of APOBEC3G was 1.9
hours in the presence of Vif and 10 hours in the absence of Vif.
(B) Proteasome inhibitor studies using infected H9 cells. Mock or
HIV-1 wt-infected H9 cells were treated with the broadly active
proteasome inhibitor epoxomicin (0.25 .mu.M) or DMSO (0.1%) 44
hours after infection. Sixteen hours following treatment, the cells
were harvested; and whole cell lysates were prepared by boiling the
cells in Laemmli lysis buffer. Note that Epoxomicin caused an
increase in endogenous APOBEC3G levels in the infected sample
(compare lane 2 with lane 1). (C) Proteasome inhibitor studies
using transiently-transfected 293T cells. Twenty-four hours
following transfection of 293T cells with 0.1 .mu.g APOBEC3G and 2
.mu.g Vif or control vector DNA, the cells were treated with the
indicated concentration of inhibitor for 15 hours. APOBEC3G-HA
levels were elevated in the presence of the proteasome inhibitors
MG132, ALLN, and clasto-lactacystin-.beta.-lactone (lanes 8-11),
but not in the presence of calpain inhibitor III (lane 12).
Vif Also Impairs Translation of APOBEC3G mRNA
[0223] In the course of the in vivo pulse-chase radiolabeling
experiments, it was observed that 15-40% less radiolabeled APOBEC3G
was recovered after the pulse labeling phase in cells expressing
Vif as compared to cells not expressing Vif. These results raised
the possibility that Vif might inhibit APOBEC3G expression by
impairing the translation of APOBEC3G mRNA. To further investigate
this possibility, shorter pulse-labeling experiments (15 minutes)
were performed in the presence of increasing amounts of Vif (0-8
.mu.g) (FIG. 9A, left side). These studies revealed that Vif
impaired 35S-methionine/cysteine radiolabeling of APOBEC3G-HA in
vivo in a dose-related manner, consistently causing a 30-50%
decline in APOBEC3G translation during the pulse-labeling period.
Immunoblotting analysis of the same samples revealed an 80% decline
in the steady-state levels of APOBEC3G-HA (FIG. 9A, right side).
Since a 30-50% decline in translation does not fully explain the
80% decline in the steady-state levels of APOBEC3G-HA, Vif must
function both by partially blocking the synthesis of the APOBEC3G
protein and by inducing APOBEC3G degradation after translation.
[0224] To further examine the effects of Vif on APOBEC3G mRNA
translation, an in vitro coupled transcription/translation system
was employed. Rabbit reticulocyte lysates were programmed with
APOBEC3G mRNA in the presence of pre-translated Vif or GST control
protein (FIG. 9B). In this in vitro system, Vif impaired APOBEC3G
translation by approximately 70-75%. These findings indicate that
Vif partially blocks the in vitro translation of APOBEC3G mRNA.
[0225] Together, these results demonstrate that Vif depletes
intracellular APOBEC3G in a bimodal manner by inhibiting
translation of APOBEC3G mRNA and by accelerating
proteasome-mediated degradation of the APOBEC3G enzyme. In
combination, these two mechanisms effectively eliminate the
APOBEC3G enzyme in infected T cells.
[0226] FIGS. 9A and 9B. Vif impairs the translation of APOBEC3G-HA
mRNA. (A) In vivo pulse label studies. HEK 293 cells transiently
transfected with 1 .mu.g of APOBEC3G-HA expression vector DNA and 0
to 8 .mu.g Vif expression vector DNA were pulsed with radiolabeled
methionine and cysteine for 15 min and then immediately harvested.
After immunoprecipitation with anti-HA antibody, the samples were
either (1) subjected to SDS-PAGE and analyzed by autoradiography
(left panel) or (2) subjected to SDS-PAGE and analyzed by
immunoblotting (right panel). Higher concentrations of Vif caused a
significant (36% -50%) impairment in the in vivo translation of
APOBEC3G-HA (left panel). Vif caused an even greater (80%)
reduction in the steady-state levels of APOBEC3G-HA (right panel).
(B) In vitro Transcription/Translation Studies. Vif or control GST
protein was initially transcribed and translated for 90 min using
the Promega TNT T7-coupled Reticulocyte Lysate system according to
the manufacturer's instructions. The pre-synthesized Vif or GST was
then added to a new transcription/translation mix programmed with
APOBEC3G DNA on ice. The reactions were then transferred to a
30.degree. C. water bath and allowed to proceed for 15 or 30 min.
The proteins were then separated by SDS-PAGE, and the gel was
analyzed by autoradiography. Note that Vif produced a .about.70-75%
impairment in the in vitro synthesis of APOBEC3G.
Example 2
Characterization of High- and Low-Molecular Weight APOBEC3G
Complexes
[0227] Human peripheral blood lymphocytes (PBLs) were isolated from
blood and then incubated with anti-CD3 antibody (.alpha.-CD3),
anti-CD28 antibody (.alpha.-CD28), IL-9, IL-12, IL-13, IL-15, or
IFN-.gamma. for 48 hours. Cells were lysed, the lysates clarified,
and the protein concentration of the lysates was quantitated. Equal
amounts of proteins in the lysates were separated by SDS-PAGE.
After separation of the proteins by SDS-PAGE, the proteins were
transferred to a membrane, and antibody to APOBEC3G was used to
detect APOBEC3G protein on the membrane. The results are depicted
in FIGS. 11-13.
[0228] FIG. 11 depicts the effect of PHA, anti-CD3 antibody,
anti-CD28 antibody, and IL-15 on the level of APOBEC3G in
peripheral blood lymphocytes. PBLs treated with PHA, anti-CD3
antibody, anti-CD28 antibody, or IL15 display increased expression
of the APOBECG enzyme. Western blot of cell lysates deriving from
stimulated and unstimulated peripheral blood lymphocytes. PBLs were
isolated from blood and then incubated with the indicated stimulant
for 48 hours. Following separation of the proteins by SDS-PAGE,
antibody to APOBEC3G was used to detect APOBEC3G protein. As a
loading control, anti-.beta.-actin was used to detect
.beta.-actin.
[0229] FIG. 12 depicts the effect of IL-2 on APOBEC3G levels in
PBL. Treatment with IL-2 results in upregulation of the APOBEC3G
enzyme in peripheral blood lymphocytes. PBLs were treated with the
indicated cytokine and then analyzed as in FIG. 11.
[0230] FIG. 13 depicts the effect of PMA, PHA, anti-CD3 antibody,
anti-CD28 antibody, and prostratin on the level of APOBEC3G in PBL.
PMA, PHA, anti-CD3, anti-CD28, and prostratin all cause APOBEC3G to
be upregulated in PBLs. PBLs were treated with the indicated
mitogen or antibody and then analyzed as in FIGS. 11 and 12.
[0231] The assembly of APOBEC3G into low molecular weight or high
molecular weight complexes was analyzed in various cells. The
enzymatic activity of low molecular weight (LMW) APOBEC3G complexes
and high molecular weigh (HMW) APOBEC3G complexes was analyzed.
[0232] H9 cells and 293T cells transiently transfected with
HA-APOBEC3G DNA were cultured in standard conditions prior to use.
CD4.sup.+ T cells were isolated from fresh human peripheral blood
lymphocytes (PBLs) by MACS.RTM. microbeads (Miltenyi Biotec, Inc.)
sorting and were maintained in complete RPMI media (10% FBS). A
portion of the CD4.sup.+ T cells was activated with
phytohemagglutinin (PHA) (5 .mu.g/ml) for 36 hours and then IL-2 (5
.mu.g/L) for another 24 hours prior to use. Monocytes were isolated
from buffy-coat peripheral blood mononuclear cells (PBMC). Immature
dendritic cells were obtained by incubating monocytes in complete
RPMI medium supplemented with GM-CSF (50 ng/mL) and IL-4 (25 ng/mL)
for 6 days. Mature dendritic cells were derived from immature
dendritic cells treated with TNF-alpha (5 ng/mL) and Poly IC (25
.mu.g/mL) for 24 hours. Clarified lysates were made from these
cells. Total protein in the clarified lysates was quantified and
subjected to FPLC analysis with or without prior RNase treatment.
Each FPLC fraction was subjected to SDS-PAGE and immunoblotting
analysis. HA-APOBEC3G in the high and low molecular weight
fractions was immunoprecipitated and subjected to the in vitro
cytidine deaminase assay.
[0233] FIG. 14 depicts results indicating that endogenous APOBEC3G
is assembled into a HMW, RNase-sensitive complex in non-permissive
H9 cells.
[0234] FIG. 15 depicts results showing that transfection of
HA-APOBEC3G DNA into permissive cells leads to HMW APOBEC3G complex
formation. This HMW fraction is RNase-sensitive as a LMW APOBEC3G
complex results from RNase treatment of the HMW fraction.
[0235] FIG. 16 depicts results indicating that the LMW APOBEC3G
complex, but not the HMW APOBEC3G complex, displays cytidine
deaminase activity. Thus, APOBEC3G is recruited into a HMW
ribonucleoprotein complex that lacks cytidine deaminase activity.
Treatment of the HMW APOBEC3G complex with RNase produces a LMW
APOBEC3G complex that exhibits high cytidine deaminase
activity.
[0236] FIG. 17 depicts results showing that APOBEC3G in resting PBL
is present in a LMW complex.
[0237] FIG. 18 depicts results showing that PHA/IL-2 stimulation of
CD4.sup.+ PBL promotes assembly of HMW APOBEC3G complexes.
[0238] FIG. 19 depicts results showing that APOBEC3G in monocytes
is present in a LMW complex.
[0239] FIG. 20 depicts results showing that differentiation of
monocytes promotes assembly of HMW APOBEC3G complexes.
[0240] In summary, it was shown that:
[0241] 1) APOBEC3G is recruited into a HMW ribonucleoprotein
complex which lacks cytidine deaminase activity;
[0242] 2) Treatment of the HMW APOBEC3G complex with RNase produces
a LMW complex that exhibits a high level of cytidine deaminase
activity.
[0243] 3) Resting peripheral blood T cells and monocytes display
LMW APOBEC3G complexes. Activation and/or differentiation of
resting peripheral blood T cells and monocytes promotes the
formation of HMW APOBEC3G complexes.
Example 3
The Effect of an N-Terminal Fragment of APOBEC3G on Vif-Induced
Degradation of APOBEC3G
[0244] HEK 293 cells were co-transfected with an HA-APOBEC3G
expression vector or with increasing amounts (2 .mu.g or 4 .mu.g)
of an HA-(1-104)APOBEC3G expression vector; and either an HIV-1 Vif
or a control expression vector. The HA-APOBEC3G expression vector
encodes wild-type (e.g., full-length) APOBEC3G fused in-frame to
the hemagglutinin-epitope tag at the N-terminus. The
HA-(1-104)APOBEC3G expression vector encodes amino acids 1-104 of
APOBEC3G fused in-frame to the hemagglutinin-epitope tag. Cell
lysates were clarified by centrifugation, and equal amounts of
protein separated by SDS-PAGE. The separated proteins were
transferred to nitrocellulose membranes, and both HA-APOBEC3G and
HA-(1-104)APOBEC3G on the membranes were detected by anti-HA
antibody. In cells transfected with the HA-APOBEC3G expression
vector (and not co-transfected with the Vif expression vector),
HA-APOBEC3G was produced that migrated at about 50 kD. In cells
co-transfected with the Vif expression vector and the HA-APOBEC3G
expression vector (and not co-transfected with the
HA-(1-104)APOBEC3G expression vector), most of the HA-APOBEC3G was
degraded. However, in cells co-transfected with the Vif expression
vector, the HA-APOBEC3G expression vector, and either 2 .mu.g or 4
.mu.g of the HA-(1-104)APOBEC3G expression vector, the steady-state
production of the .about.50 kD HA-APOBEC3G fusion protein was
significantly restored, in the presence of the HA-(1-104)APOBEC3G
protein, to the level found in the absence of Vif co-expression.
The results indicate that over-expression of HA-(1-104)APOBEC3G
inhibits HIV-1 Vif-mediated degradation of HA-APOBEC3G.
Example 4
Construction of a Vif/luc-APOBEC3G Cell Line
[0245] Several cell lines were constructed that could serve as the
basis for a high-throughput assay for inhibitors of HIV-1 Vif
action. The cell lines produce Vif and an APOBEC3G fusion protein
composed of APOBEC3G fused to a heterologous protein that provides
a detectable signal. One such cell line was constructed as follows.
HeLa cells were co-transfected with both a Vif expression vector
and a vector expressing APOBEC3G tagged with renilla-luciferase
(luc-APOBEC3G), to generate a Vif/luc-APOBEC3G cell line. The Vif
expression vector contains a selectable marker that provides for
resistance to histidinol. The luc-APOBEC3G expression vector
contains a selectable marker that protects transfected cells from
neomycin or G418.
[0246] The Vif/luc-APOBEC3G cell line was treated with the
proteasome inhibitor MG132, an agent capable of blocking
Vif-mediated degradation of APOBEC3G, and the cells were
subsequently lysed. The cell lysates were clarified, and subjected
to a conventional luciferase assay. The level of luc-APOBEC3G was
expressed as relative light units (RLU). The RLU emitted from the
cell lysate sample of the Vif/luc-APOBEC3G cells treated with MG132
was 203333. However, in the absence of the proteasome inhibitor,
the RLU from lysates of the Vif/luc-APOBEC3G cells was 10180,
representing about a 20-fold reduction. From these results, it was
concluded that the Vif present in the cell line caused an
approximately 20-fold reduction in the level of the luc-APOBEC3G
protein, and that this activity of Vif was reversed by MG132.
[0247] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Sequence CWU 1
1
8 1 10 PRT Artificial Sequence synthetic protein created in a
laboratory 1 Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 10 2 8 PRT
Artificial Sequence synthetic protein created in a laboratory 2 Asp
Tyr Lys Asp Asp Asp Asp Lys 1 5 3 11 PRT Artificial Sequence
synthetic protein created in a laboratory 3 Cys Glu Gln Lys Leu Ile
Ser Glu Glu Asp Leu 1 5 10 4 384 PRT Homo sapiens 4 Met Lys Pro His
Phe Arg Asn Thr Val Glu Arg Met Tyr Arg Asp Thr 1 5 10 15 Phe Ser
Tyr Asn Phe Tyr Asn Arg Pro Ile Leu Ser Arg Arg Asn Thr 20 25 30
Val Trp Leu Cys Tyr Glu Val Lys Thr Lys Gly Pro Ser Arg Pro Pro 35
40 45 Leu Asp Ala Lys Ile Phe Arg Gly Gln Val Tyr Ser Glu Leu Lys
Tyr 50 55 60 His Pro Glu Met Arg Phe Phe His Trp Phe Ser Lys Trp
Arg Lys Leu 65 70 75 80 His Arg Asp Gln Glu Tyr Glu Val Thr Trp Tyr
Ile Ser Trp Ser Pro 85 90 95 Cys Thr Lys Cys Thr Arg Asp Met Ala
Thr Phe Leu Ala Glu Asp Pro 100 105 110 Lys Val Thr Leu Thr Ile Phe
Val Ala Arg Leu Tyr Tyr Phe Trp Asp 115 120 125 Pro Asp Tyr Gln Glu
Ala Leu Arg Ser Leu Cys Gln Lys Arg Asp Gly 130 135 140 Pro Arg Ala
Thr Met Lys Ile Met Asn Tyr Asp Glu Phe Gln His Cys 145 150 155 160
Trp Ser Lys Phe Val Tyr Ser Gln Arg Glu Leu Phe Glu Pro Trp Asn 165
170 175 Asn Leu Pro Lys Tyr Tyr Ile Leu Leu His Ile Met Leu Gly Glu
Ile 180 185 190 Leu Arg His Ser Met Asp Pro Pro Thr Phe Thr Phe Asn
Phe Asn Asn 195 200 205 Glu Pro Trp Val Arg Gly Arg His Glu Thr Tyr
Leu Cys Tyr Glu Val 210 215 220 Glu Arg Met His Asn Asp Thr Trp Val
Leu Leu Asn Gln Arg Arg Gly 225 230 235 240 Phe Leu Cys Asn Gln Ala
Pro His Lys His Gly Phe Leu Glu Gly Arg 245 250 255 His Ala Glu Leu
Cys Phe Leu Asp Val Ile Pro Phe Trp Lys Leu Asp 260 265 270 Leu Asp
Gln Asp Tyr Arg Val Thr Cys Phe Thr Ser Trp Ser Pro Cys 275 280 285
Phe Ser Cys Ala Gln Glu Met Ala Lys Phe Ile Ser Lys Asn Lys His 290
295 300 Val Ser Leu Cys Ile Phe Thr Ala Arg Ile Tyr Asp Asp Gln Gly
Arg 305 310 315 320 Cys Gln Glu Gly Leu Arg Thr Leu Ala Glu Ala Gly
Ala Lys Ile Ser 325 330 335 Ile Met Thr Tyr Ser Glu Phe Lys His Cys
Trp Asp Thr Phe Val Asp 340 345 350 His Gln Gly Cys Pro Phe Gln Pro
Trp Asp Gly Leu Asp Glu His Ser 355 360 365 Gln Asp Leu Ser Gly Arg
Leu Arg Ala Ile Leu Gln Asn Gln Glu Asn 370 375 380 5 40 PRT
Artificial Sequence synthetic protein created in a laboratory 5 Met
Lys Pro His Phe Arg Asn Thr Val Glu Arg Met Tyr Arg Asp Thr 1 5 10
15 Phe Ser Tyr Asn Phe Tyr Asn Arg Pro Ile Leu Ser Arg Arg Asn Thr
20 25 30 Val Trp Leu Cys Tyr Glu Val Lys 35 40 6 29 PRT Artificial
Sequence synthetic protein created in a laboratory 6 Cys Pro Phe
Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Asp Leu 1 5 10 15 Ser
Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Glu Asn 20 25 7 104 PRT
Artificial Sequence synthetic protein created in a laboratory 7 Met
Lys Pro His Phe Arg Asn Thr Val Glu Arg Met Tyr Arg Asp Thr 1 5 10
15 Phe Ser Tyr Asn Phe Tyr Asn Arg Pro Ile Leu Ser Arg Arg Asn Thr
20 25 30 Val Trp Leu Cys Tyr Glu Val Lys Thr Lys Gly Pro Ser Arg
Pro Pro 35 40 45 Leu Asp Ala Lys Ile Phe Arg Gly Gln Val Tyr Ser
Glu Leu Lys Tyr 50 55 60 His Pro Glu Met Arg Phe Phe His Trp Phe
Ser Lys Trp Arg Lys Leu 65 70 75 80 His Arg Asp Gln Glu Tyr Glu Val
Thr Trp Tyr Ile Ser Trp Ser Pro 85 90 95 Cys Thr Lys Cys Thr Arg
Asp Met 100 8 16 PRT Artificial Sequence synthetic protein created
in a laboratory 8 Gln Asp Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln
Asn Gln Glu Asn 1 5 10 15
* * * * *