U.S. patent application number 13/035891 was filed with the patent office on 2011-09-22 for fusion protein delivery system and uses thereof.
This patent application is currently assigned to THE UAB RESEARCH FOUNDATION. Invention is credited to John C. Kappes, Xiaoyun Wu.
Application Number | 20110229964 13/035891 |
Document ID | / |
Family ID | 23829157 |
Filed Date | 2011-09-22 |
United States Patent
Application |
20110229964 |
Kind Code |
A1 |
Kappes; John C. ; et
al. |
September 22, 2011 |
Fusion Protein Delivery System and Uses Thereof
Abstract
The present invention provides a composition of matter,
comprising: DNA encoding a viral Vpx protein fused to DNA encoding
a protein. In another embodiment of the present invention, there is
provided a composition of matter comprising: DNA encoding a viral
Vpr protein fused to DNA encoding a protein. The present invention
further provides DNA, vectors and methods for expressing a
lentiviral pol gene in trans, independent of the lentiviral
gag-pol. A gene transduction element is optionally delivered to a
lentiviral vector according to the present invention. Also provided
are various methods of delivering a virus inhibitory molecule to a
target in an animal. Further provided is a pharmaceutical
composition.
Inventors: |
Kappes; John C.;
(Birmingham, AL) ; Wu; Xiaoyun; (Birmingham,
AL) |
Assignee: |
THE UAB RESEARCH FOUNDATION
Birmingham
AL
|
Family ID: |
23829157 |
Appl. No.: |
13/035891 |
Filed: |
February 25, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11894224 |
Aug 20, 2007 |
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13035891 |
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10202457 |
Jul 24, 2002 |
7622300 |
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11894224 |
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09709751 |
Nov 10, 2000 |
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10202457 |
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09460548 |
Dec 14, 1999 |
6555342 |
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09709751 |
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09089900 |
Jun 3, 1998 |
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09460548 |
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Current U.S.
Class: |
435/348 ;
435/252.3; 435/320.1; 435/325; 536/23.2 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2740/10022 20130101; C12N 7/00 20130101; C07K 2319/23
20130101; C07K 2319/735 20130101; C12N 2740/16023 20130101; C12N
15/62 20130101; C12N 2740/16043 20130101; C07K 2319/00 20130101;
C07K 2319/40 20130101; C12N 15/86 20130101; C12N 2740/16052
20130101; C12N 2710/16122 20130101 |
Class at
Publication: |
435/348 ;
435/320.1; 435/325; 536/23.2; 435/252.3 |
International
Class: |
C12N 5/10 20060101
C12N005/10; C12N 15/63 20060101 C12N015/63; C07H 21/00 20060101
C07H021/00; C12N 1/21 20060101 C12N001/21 |
Claims
1. A nucleic acid molecule comprising a first nucleotide sequence
expressing a Vpr polypeptide fused in frame to a second nucleotide
sequence expressing a full length Integrase polypeptide, wherein
said first nucleotide sequence expresses a full length Vpr
polypeptide capable of association with an HIV viron, an SIV viron,
or a virus like particle.
2. A nucleic acid molecule comprising a first nucleotide sequence
expressing a Vpr polypeptide fused in frame to a second nucleotide
sequence expressing a full length Reverse Transcriptase
polypeptide, wherein said first nucleotide sequence expresses a
full length Vpr polypeptide capable of association with an HIV
viron, an SIV viron, or a virus like particle.
3. The nucleic acid molecule of claim 1, wherein said first
nucleotide sequence is from a Human Immunodeficiency Virus or a
Simian Immunodeficiency Virus.
4. An expression plasmid comprising a nucleic acid molecule of
claim 1.
5. An expression plasmid comprising a nucleic acid molecule of
claim 2.
6. The expression plasmid of claim 4, wherein said nucleic acid
molecule is operably linked to a regulatory element necessary for
expression of said nucleic acid molecule in a cell.
7. The expression plasmid of claim 6, wherein said regulatory
element is selected from the group consisting of an HIV-2 RRE and
an HIV-2 LTR.
8. The expression plasmid of claim 6, wherein said regulatory
element comprises a promoter capable of driving expression in a
cell.
9. A cell having the nucleic acid molecule of claim 1.
10. A cell having the nucleic acid molecule of claim 2.
11. The cell of claim 9, wherein said cell is selected from the
group consisting of a bacterial cell, a mammalian cell, and an
insect cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The patent application is a continuation of U.S. patent
application Ser. No. 11/894,223, filed on Aug. 20, 2007, which is
currently pending. U.S. patent application Ser. No. 11/894,223 is a
continuation of U.S. patent application Ser. No. 10/245,475, filed
Sep. 17, 2002 which is a continuation of U.S. patent application
Ser. No. 09/460,548, filed Dec. 14, 1999 which is a
continuation-in-part of U.S. patent application Ser. No.
09/089,900, filed Jun. 3, 1998, all of which are incorporated
herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the fields of
molecular virology and protein chemistry. More specifically, the
present invention relates to the use of Human and Simian
Immunodeficiency Virus (HIV/SIV) Gag proteins, or amino acid
residues that mediate their packaging, as vehicles for delivery of
proteins/peptides to virions or virus-like particles and uses
thereof.
DESCRIPTION OF THE RELATED ART
[0003] Unlike simple retroviruses, human and simian
immunodeficiency viruses (HIV/SIV) encode proteins in addition to
Gag, Pol, and Env that are packaged into virus particles. These
include the Vpr protein, present in all primate lentiviruses, and
the Vpx protein, which is unique to the
HIV-2/SIV.sub.SM/SIV.sub.MAC group of viruses. Since Vpr and Vpx
are present in infectious virions, they have long been thought to
play important roles early in the virus life cycle. Indeed, recent
studies of HIV-1 have shown that Vpr has nucleophilic properties
and that it facilitates, together with the matrix protein, nuclear
transport of the viral preintegration complex in nondividing cells,
such as the macrophage. Similarly, Vpx-deficient HIV-2 has been
shown to exhibit delayed replication kinetics and to require 2-3
orders of magnitude more virus to produce and maintain a productive
infection in peripheral blood mononuclear cells. Thus, both
accessory proteins appear to be important for efficient replication
and spread of HIV/SIV in primary target cells.
[0004] Incorporation of foreign proteins into retrovirus particles
has previously been reported by fusion with Gag. The yeast
retrotransposon Tyl was tested as a retrovirus assembly model to
interfere with viral replication (Natsoulis et al. (1991) Nature
352:632-5). More recently, the expression of a murine retrovirus
capsid-staphylococcal nuclease fusion protein was found to inhibit
murine leukemia virus replication in tissue culture cells. The
expression of Gag-staphylococcal nuclease reduces viral titer and
diminishes viral infectivity to promote an anti-HIV strategy
(Schumann et al. (1996) J. Virol. 70:432937).
[0005] Lentiviral vectors, specifically those based on HIV-1, HIV-2
and SIV, have utility in gene therapy, due to their attractive
property of stable integration into nondividing cell types (Naldini
et al. (1996) Science 272:263-267; Stewart et al. (1997) J. Virol.
71:5579-5592; Zhang et al. (1993) Science 259:234-238). The utility
of lentiviral-based vector use for human therapy requires the
development of a safe lentiviral-based vector. HIV virion
associated accessory proteins (Vpr and Vpx) have been shown to be
useful as vehicles to deliver protein of both viral and non-viral
origin into HIV particles (Liu et al. (1995) J. Virol.
69:7630-7638; Liu et al. (1997) J. Virol. 71:7704-7710; Wu et al.
(1994) J. Virol. 68:6161-6169; Wu et al. (1997) EMBO Journal
16:5113-5122; Wu et al. (1996) J. Virol. 70:3378-3384). We recently
demonstrated that trans-RT and IN mimic cis-RT and IN (derived from
Gag-Pol). The trans-RT and IN proteins effectively rescue the
infectivity and replication of virions derived from RT-IN minus
provirus through the complete life cycle (Liu et al. (1997) J.
Virol. 71:7704-7710 and Wu et al. (1994) J. Virol. 68:6161-6169).
Moreover, these findings demonstrate that truncated Gag-Pol
precursor polyprotein (Gag-Pro) support the formation of infectious
particles when the functions of RT and IN are provided in trans.
This finding demonstrated for the first time for a lentivirus that
the full length Gag-Pol precursor is not required for the formation
of infectious particles. Our data also show that trans Vpr-RT-IN,
or Vpr-RT together with Vpr-IN are fully functional and support
virus infectivity, integration of the proviral DNA, and replication
(through one cycle) of RT defective, IN defective and RT-IN
defective viruses (Liu et al. (1997) J. Virol. 71:7704-7710 and Wu
et al. (1994) J. Virol. 68:6161-6169). It should also be noted that
our data demonstrate that enzymatically active RT does not require
Vpr for incorporation into virions (FIGS. 19A and B). RT can be
incorporated into HIV-1 virions when expressed in trans even
without its expression as a fusion partner of Vpr. These data
demonstrate that the functions of these critical enzymes can be
provided in trans, independent of their normal mechanism for
expression and virion incorporation as components of the Gag-Pol
precursor protein.
[0006] The prior art is deficient in the lack of effective means of
delivering or targeting foreign, e.g., toxic proteins to virions.
The present invention fulfills this longstanding need and desire in
the art.
SUMMARY OF THE INVENTION
[0007] The present invention shows that Vpr and Vpx can be used as
vehicles to target foreign proteins to HIV/SIV virons. Vpr1 and
Vpx2 gene fusions were constructed with bacterial staphylococcal
nuclease (SN) and chloramphenicol acetyl transferase (CAT) genes.
Unlike Gag or Pol proteins, Vpr and Vpx are dispensable for viral
replication in immortalized T-cell lines. Thus, structural
alteration of these accessory proteins may be more readily
tolerated than similar changes in Gag or Gag/Pol. Fusion proteins
containing a Vpx or Vpr moiety should be packaged into HIV
particles by expression in trans, since their incorporation should
be mediated by the same interactions with Gag that facilitates
wild-type Vpr and Vpx protein packaging.
[0008] Vpr and Vpx fusion proteins were constructed and their
abilities to package into HIV particles were demonstrated. Fusion
partners selected for demonstration were: staphylococcal nuclease
because of its potential to degrade viral nucleic acid upon
packaging and chloramphenicol acetyl transferase because of its
utility as a functional marker. To control for cytotoxicity, an
enzymatically inactive nuclease mutant (SN*), derived from SN by
site-directed mutagenesis was also used. This SN* mutant differs
from wild-type SN by two amino acid substitutions; Glu was changed
to Ser (position 43) and Arg was changed to Gly (position 87). SN*
folds normally, but has a specific activity that is 10.sup.6-fold
lower than wild-type SN. Using transient expression systems and in
trans complementation approaches, fusion protein stability,
function and packaging requirements was shown. The present
invention shows that Vpr1 and Vpx2 fusion proteins were expressed
in mammalian cells and were incorporated into HIV particles even in
the presence of wild-type Vpr and/or Vpx proteins. More
importantly, however, the present invention shows that virion
incorporated Vpr and Vpx fusion remains enzymatically active. Thus,
targeting heterologous Vpr and Vpx fusion proteins, including
deleterious enzymes, to virions represents a new avenue toward
anit-HIV drug discovery.
[0009] In one embodiment of the present invention, there is
provided a composition of matter, comprising: DNA encoding a viral
Vpx protein fused to DNA encoding a virus inhibitory protein.
[0010] In another embodiment of the present invention, there is
provided a composition of matter, comprising: DNA encoding a viral
Vpr protein fused to DNA encoding a virus inhibitory protein.
[0011] The present invention shows that Gag and/or Gag variants can
be used as vehicles to target proteins of viral and non-viral
origin into HIV/SIV virions. Gag gene fusions were constructed with
bacterial staphylococcal nuclease (SN), chloramphenicol acetyl
transferase (CAT) genes, green fluorescence protein (GFP), reverse
transcriptase (RT), integrase (IN) and combinations thereof. Fusion
proteins containing a Gag moiety should be packaged into HIV
particles by expression in trans, to the native viral genome.
[0012] Gag fusion proteins were constructed and their abilities to
package into HIV particles were demonstrated. The present invention
shows that Gag fusion proteins were expressed in mammalian cells
and were incorporated into HIV particles even in the presence of
wild-type Gag proteins. The present invention further shows that
virion incorporated Gag fusions remain infective in contrast to the
prior art (Schuman et al. (1996) J. Virol. 70:4379-37). Thus,
targeting heterologous Gag fusion proteins, including deleterious
enzymes, to virions represents a new avenue toward anti-HIV drug
discovery and gene therapy.
[0013] The invention shows that Gag proteins and variants thereof
are operative as vehicles to deliver fully functional RT and IN in
trans into lentiviral and retroviral particles, independently of
their normal expression as components of the Gag-Pol precursor
protein. Therefore this invention generates a novel packaging
component (Gag-Pro), and a novel trans-enzymatic element that
provides enzyme function for retroviral-based vectors. According to
the present invention, the generation of potentially
infectious/replicating retroviral forms (LTR-gag-pol-LTR) is
decreased, since according to the present invention this requires
recombination of at least three separate RNAs derived from the
different plasmids: vector plasmid, packaging plasmid, a
trans-enzyme expression plasmid and envelope plasmid, and as such
is unlikely to occur. Virion Gag proteins are utilized in the
present invention as vehicles to deliver the RT and IN proteins
into lentiviral vectors, independently of Gag-Pol. As such, a
"trans-lentiviral" or "transretroviral" vector is utilized for gene
delivery, and gene therapy.
[0014] In one embodiment of the present invention, there is
provided a composition of matter, comprising: DNA encoding a viral
Gag protein fused to DNA encoding a virus inhibitory protein.
[0015] In another embodiment of the present invention, there is
provided a composition of matter, comprising: DNA encoding a viral
Gag protein truncate fused to DNA encoding a virus inhibitory
protein.
[0016] In yet another embodiment of the present invention, there is
provided a method of delivering a virus inhibitory molecule to a
target in an animal, comprising the step of administering to said
animal an effective amount of the composition of the present
invention.
[0017] In still yet another embodiment of the present invention,
there is provided a pharmaceutical composition, comprising a
composition of the present invention and a pharmaceutically
acceptable carrier.
[0018] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention given for
the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions of the invention briefly summarized
above may be had by reference to certain embodiments thereof which
are illustrated in the appended drawings. These drawings forth a
part of the specification. It is to be noted, however, that the
appended drawings illustrate preferred embodiments of the invention
and therefore are not to be considered limiting in their scope.
[0020] FIG. 1 shows the construction of vpr1, vpr1SN/SN*, vpx2 and
vpx2SN/SN* expression plasmids. FIG. 1A shows the illustration of
the pTM-vpr1 expression plasmid. The HIV-1.sub.YU2 vpr coding
region was amplified by PCR and ligated into pTM1 at the NcoI and
BamHI restriction sites. FIG. 1B shows the illustration of the
pTM-vpx2 expression plasmid. The HIV-2.sub.ST vpx coding region was
amplified by PCR and ligated into pTM1 at the NcoI and Bg1 II/SmaI
sites. FIG. 1C shows the illustration of the fusion junctions of
the pTM-vpr1 SN/SN* expression plasmids (SEQ ID NO: 11 and 12).
SmaI/XhoI DNA fragments containing SN and SN* were ligated into
HpaI/XhoI cut pTM-vpr1. Blunt-end ligation at HpaI and SmaI sites
changes the vpr translational stop codon (TAA) to Trp and
Substituted the C terminal Ser with a Cys residue. FIG. 1D shows
the illustration of the fusion junctions of the pTM-vpx2SN/SN*
expression plasmids (SEQ ID NO:13 and 14). BamHI/XhoI DNA fragments
containing SN and SN* were ligated into BamHI/XhoI cut pTM-vpx2. In
the construction of these plasmids, the Vpx C terminal Arg codon
was changed to a Val codon and a Ser residue was introduced in
place of the Vpx translational stop codon (TAA). Fusion of vpx and
SN/SN* at the BamHI sites left a short amino acid sequence of the
pTM1 polylinker (double underlined) between the two coding
regions.
[0021] FIG. 2 shows the expression of Vpr1- and Vpx2-SN and SN*
fusion proteins in mammalian cells. FIG. 2A shows the pTM1,
pTM-vpr1, pTM-vpr1SN and pTM-vpr1 SN* were transfected into HeLa
cells one hour after infection with rVT7 (MOI=10). Twenty-four
hours later cell lysates were prepared and examined by immunoblot
analysis. Replica blots were probed with anti-Vpr1 (left) and
anti-SN (right) antibodies. FIG. 2B shows that replica blots,
prepared from rVT7 infected HeLa cells transfected with pTM1,
pTM-vpx2, pTM-vpx2SN and pTM-vpx2SN*, were probed with anti-Vpx2
(left) and anti-SN (right) antibodies. Bound antibodies were
detected by ECL (Amersham) methods as described by the
manufacturer.
[0022] FIG. 3 shows the incorporation of Vpr1- and Vpx2-SN and SN*
fusion proteins into virus-like particles (VLP). FIG. 3A
transfection of T7 expressing (rVT7 infected) HeLa cells with
pTM-vpr1, pTM-vpr1SN, and pTM-vpr1 SN* alone and in combination
with pTM-gag1. pTM1 was also transfected for control. Culture
supernatant were collected twenty-four hours after transfection,
clarified by centrifugation (1000.times.g, 10 min.) and
ultracentrifuged (125,000.times.g, 2 hrs.) over cushions of 20%
sucrose. Pellets (VLPs, middle and bottom panels) and cells (top
panel) were solubilized in loading buffer and examined by
immunoblot analysis using anti-Vpr1 (top and middle) and anti-Gag
(bottom) antibodies as probes. FIG. 3B transfection of T7
expressing HeLa cells pTM-vpx2, pTM-vpx2SN and pTM-vpx2SN* alone
and in combination with pTM-gag2. Pellets (VLPs, middle and bottom
panels) and cells (top panel) were lysed, proteins were separated
by SDS-PAGE and electroblotted to nitrocellulose as described
above. Replica blots were probed with anti-Vpx2 (top and middle
panels) and anti-Gag (bottom panel) antibodies. Bound antibodies
were detected using ECL methods.
[0023] FIG. 4 shows that virus-specific signals mediate
incorporation of Vpr- and Vpx-SN into VLPs. FIG. 4A shows that
HIV-1 Gag mediates packaging of Vpr1SN. rVT7 infected (T7
expressing) HeLa cells were transfected with pTM-vpr1 SN alone and
in combination with pTM-gag2 and pTM-gag1. Pellets (VLPs, middle
and bottom panels) and cells (top panel) were prepared 24 hours
after transfection and examined by immunoblot analysis using
anti-Vpr1 (top and middle) and anti-Gag (bottom) antibodies for
probes. (B) HIV-2 Gag mediates packaging of Vpx2SN. T7 expressing
HeLa cells were transfected with pTM-vpx2SN alone and in
combination with pTM-gag1 and pTM-gag2. Pellets (VLPs, middle and
bottom panels) and cells (top panel) were prepared 24 hours after
transfection and examined by immunoblot analysis using anti-Vpx2
(top and middle) and anti-Gag (bottom) antibodies for probes.
[0024] FIG. 5 shows a competition analysis of Vpr1SN and Vpx2SN for
incorporation into VLPs. FIG. 5A shows transfection of T7
expressing HeLa cells with different amounts of pTM-vpr1 (2.5, 5
and 10 .mu.g) and pTM-vpr1 SN (2.5, 5 and 10 .mu.g), either
individually or together in combination with pTMgag1 (10 .mu.g).
FIG. 5B shows that HeLa cells were transfected with different
amounts of pTM-vpx2 (2.5, 5 and 10 .mu.g) and pTM-vpx2SN (2.5, 5
and 10 .mu.g), either individually or together with pTM-gag2 (10
.mu.g). Twenty hours after transfection, particles were
concentrated by ultracentrifugation through sucrose cushions and
analyzed by immunoblotting using anti-Vpr1 (A) or anti-Vpx2 (B)
antibodies.
[0025] FIG. 6 shows the nuclease activity of VLP-associated Vpr1SN
and Vpx2SN proteins. Virus-like particles were concentrated from
culture supernatants of T7 expressing HeLa cells cotransfected with
pTM-gag1/pTM-vpr1SN, pTM-gag1/pTM-vpr1 SN*, pTM-gag2/pTM-vpx2SN and
pTMgag2/pTM-vpx2SN* by ultracentrifugation (125,000.times.g, 2 hrs)
through 20% cushions of sucrose. Pellets containing Vpr1-SN and SN*
(B) and Vpx2-SN and SN* (C) were resuspended in PBS. Tenfold
dilutions were made in nuclease reaction cocktail buffer (100 mM
Tris-HCl pH 8.8, 10 mM CaCl.sub.2, 0.1% NP40) and boiled for 1
minute. 5 .mu.l of each dilution was added to 14 ul of reaction
cocktail buffer containing 500 ng of lambda phage DNA (HindIII
fragments) and incubated at 37.degree. C. for 2 hours. Reaction
products were electrophoresed on 0.8% agarose gels and DNA was
visualized by ethidium bromide staining. Standards (A) were
prepared by dilution of purified staphylococcal nuclease (provided
by A. Mildvan) into cocktail buffer and assayed.
[0026] FIG. 7 shows the incorporation of Vpx2SN into HIV-2 by trans
complementation. FIG. 7A shows the construction of the
pLR2P-vpx2SN/SN* expression plasmids. To facilitate efficient
expression of HIV genes, the HIV-2 LTR and RRE were engineered into
the polylinker of pTZ19U, generating pLR2P. The organization of
these elements within the pTZ19U polylinker is illustrated.
NcoI/XhoI vpx2SN and vpx2SN* (vpxSN/SN*) containing DNA fragments
were ligated into pLR2P, generating pLR2P-vpx2SN and pLR2P-vpx2SN*
(pLR2P-vpxSN/SN*). FIG. 7B shows the association of Vpx2SN with
HIV-2 virions. Monolayer cultures of HLtat cells were transfected
with HIV-2.sub.ST proviral DNA (pSXB1) and cotransfected with
pSXB1/pTM-vpx2SN and pSXB1/pTM-vpx2SN*. Extracellular virus was
concentrated from culture supernatants forty-eight hours after
transfection by ultracentrifugation (125,000.times.g, 2 hrs.)
through cushions of 20% sucrose. Duplicate Western blots of viral
pellets were prepared and probed independently with anti-Vpx2
(left) anti-SN (middle) and anti-Gag (right) antibodies. FIG. 7C
shows a sucrose gradient analysis. Pellets of supernatant-virus
prepared from pSXB1/pTM-vpx2SN cotransfected HLtat cells were
resuspended in PBS, layered over a 20-60% linear gradient of
sucrose and centrifuged for 18 hours at 125,000.times.g. Fractions
(0.5 ml) were collected from the bottom of the tube, diluted 1:3 in
PBS, reprecipitated and solubilized in electrophoresis buffer for
immunoblot analysis. Replica blots were probed with anti-SN (top)
and anti-Gag (bottom) antibodies. Fraction 1 represents the first
collection from the bottom of the gradient and fraction 19
represents the last collection. Only alternate fractions are shown,
except at the peak of protein detection. FIG. 7D shows the
incorporation of Vpx2SN into HIV-2.sub.7312A Vpr and Vpx competent
virus. Virus concentrated from supernatants of HLtat cells
transfected with HIV-2.sub.7312A proviral DNA (pJK) or
cotransfected with pJK/pLR2P-vpx2SN or pJK/pLR2P-vpx2SN* was
prepared for immunoblot analysis as described above. Included for
control were virions derived by pSXB1/pLR2P-vpx2SN* cotransfection.
Duplicate blots were probed with anti-Vpx (left) and anti-Gag
(right) antibodies.
[0027] FIG. 8 shows the incorporation of Vpr1 SN into HIV-1 virions
by trans complementation. Culture supernatant virus from HLtat
cells transfected with pNL4-3 (HIV-1) and pNL4-3R (HIV-1 vpr
mutant) or cotransfected with pNL4-3/pLR2P-vpr1SN and
pNL4-3R/pLR2P-vpr1SN was prepared for immunoblot analysis as
described above. Blots were probed with anti-SN (FIG. 8A),
anti-Vpr1 (FIG. 8B) and anti-Gag (FIG. 8C) antibodies.
[0028] FIG. 9 shows the inhibition of Vpr1/Vpx2-SN processing by an
HIV protease inhibitor. HIV-1 (pSG3) and HIV-2 (pSXB1) proviral
DNAs were cotransfected separately into replica cultures of HLtat
cells with pLR2P-vpr1SN and pLR2P-vpx2SN, respectively. One culture
of each transfection contained medium supplemented with 1 .mu.M of
the HIV protease inhibitor L-699-502. Virions were concentrated
from culture supernatants by ultracentrifugation through cushions
of 20% sucrose and examined by immunoblot analysis using anti-Gag
(FIG. 9A) and anti-SN (FIG. 9B) antibodies.
[0029] FIG. 10 shows the incorporation of enzymatically active
Vpr1- and Vpx2-CAT fusion proteins into HIV virions. FIG. 10A shows
an illustration of the fusion junctions of the pLR2P-vpr1CAT and
pLR2P-vpx2CAT expression plasmids (SEQ ID NOS:15, 16, 17 and 18).
PCR amplified BamHI/XhoI DNA fragments containing CAT were ligated
into Bg1 II/XhoI cut pLR2P-vpr1SN and pLR2P-vpx2SAN, replacing SN
(see FIG. 1). This construction introduced two additional amino
acid residues (Asp and Leu, above blackened bar) between the
vpr1/vpx2CAT coding regions. FIG. 10B shows the incorporation of
Vpr1CAT into HIV-1 virions. Virus produced from HLtat cells
transfected with pNL4-3 (HIV-1) and pNL4-3R (HIV-1-R), or
cotransfected with pNL4-3/pLR2P-vpr1 CAT and pNL4-3R/pLR2P-vpr1 CAT
was prepared as described above and examined by immunoblot
analysis. Replica blots were probed with anti-Vpr1 (left) and
anti-Gag (right) antibodies. FIG. 10C shows the incorporation of
Vpx2CAT into HIV-2 virions. Virus produced from HLtat cells
transfected with pSXB1 (HIV-2) or cotransfected with pSXB
1/pLR2P-vpx2CAT was prepared as described above and examined by
immunoblot analysis. Replica blots were probed with anti-Vpx2
(left) and anti-Gag (right) antibodies. FIG. 10D shows that virion
incorporated Vpr1- and Vpx2-CAT fusion proteins possess enzymatic
activity. Viruses pelleted from HLtat cells transfected with pSXB1
(HIV-2) or cotransfected with pSXBI/pLR2P-vpx2CAT and
pNL4-3/pLR2P-vpr1 CAT were lysed and analyzed for CAT activity.
HIV-2 was included as a negative control.
[0030] FIG. 11 shows virion association of enzymatically active CAT
and SN fusion proteins. FIG. 11A shows that HIV-2 virions collected
from the culture supernatant of HLtat cells cotransfected with
pSXB1 and pLR2P-vpx2 were sedimented in linear gradients of 20-60%
sucrose. 0.7 ml fractions were collected and analyzed by immunoblot
analysis using Gag monoclonal antibodies as a probe. FIG. 11B shows
CAT enzyme activity was determined in each fraction by standard
methods. The positions of nonacetylated [.sup.14C]chloramphenicol
(Cm) and acetylated chloramphenicol (Ac--Cm) are indicated. FIG.
11C shows HIV-1 virions derived from HLtat cells cotransfected with
pSG3 and pLR2P-vpr1SN and cultured in the presence of L-689,502
were sedimented in linear gradients of 20-60% sucrose. Fractions
were collected and analyzed for virus content by immunoblot
analysis using Gag monoclonal antibodies. FIG. 11D shows that SN
activity was determined in each fraction as described in FIG.
6.
[0031] FIG. 12 shows the HIV-1 genome, the construction of
p.DELTA.8.2, pCMV-VSV-G, pHR-CMV-.beta.-gal, pCR-gag-pro,
pLR2P-vpr-RT-IN, pCMV-VSV-G and pHR-CMV-.beta.-gal plasmids. FIG.
12A shows an illustration of the HIV-1 genome. FIG. 12B shows the
lentivirus vector plasmid expression system. FIG. 12C shows the
illustration of a trans-lentiviral vector expression system, where
RT and IN are contiguous as Vpr fusion partners.
[0032] FIG. 13 shows positive gene transduction with a
trans-lentiviral vector of the instant invention as determined by
fluorescence microscopy.
[0033] FIG. 14 shows positive gene transduction with a lentiviral
vector as a control as determined by fluorescence microscopy.
[0034] FIG. 15 shows the construction of a pHR-CFTR
trans-lentiviral vector of the present invention.
[0035] FIG. 16 shows the expression of CFTR on HeLa cells using the
trans-lentiviral vector, and the lentiviral vector as a control.
Transduced cells were probed with polyclonal antibodies in
immunofluorescence microscopy.
[0036] FIG. 17 shows the expression of CFTR on HeLa cells using
monoclonal antibodies in immunofluorescence microscopy.
[0037] FIG. 18 shows the restoration of CFTR function in
trans-lentiviral transduced HeLa cells as measured by a halide
sensitive fluorophore.
[0038] FIGS. 19A and B show the presence in progeny virions of RT
in trans without Vpr-dependent incorporation.
[0039] FIG. 20 shows that both Vpr-RT and RT support vector
transduction when provided in trans.
[0040] FIG. 21 shows component constructs of a trans-retroviral
vector according to the present invention. FIG. 21 A shows a pCMV,
Gag-Pro packaging plasmid. FIG. 21 B shows a pCMV, GagNC-RT-IN
trans-enzyme expression plasmid. FIG. 21C shows a vector plasmid.
FIG. 21D shows an envelope plasmid construct operative in the
present invention.
[0041] FIG. 22 shows component constructs of a retroviral vector
according to the present invention. FIG. 22A shows a pCMV,
Gag-Pro-RT-IN retroviral packaging plasmid. FIGS. 22B and C are the
vector plasmid and envelope plasmids of FIGS. 21C and D,
respectively.
[0042] FIG. 23 shows component constructs of a lentiviral vector
according to the present invention. FIG. 23A shows a pTRE,
Gag-Pro-RT-IN packaging plasmid. FIG. 23B shows a pHR-CTS, CMV,
GFP, WPRE lentiviral vector plasmid. FIG. 23C is the envelope
plasmid at FIG. 21D.
[0043] FIGS. 24(A)-(C) show representative trans-lentiviral
trans-enzyme plasmids according to the present invention indicating
Pro cleavage sites and zinc finger locations.
DETAILED DESCRIPTION OF THE INVENTION
[0044] As used herein, the term "fusion protein" refers to either
the entire native protein amino acid sequence of Vpx (of any HIV-2
and SW) or Vpr (of any HIV-1 and SIV) or retroviral Gag or any
subfraction of their sequences that have been joined through
recombinant DNA technology and are capable of association with
either native HIV/SIV virions or a retrovirus-like particle.
[0045] As used herein, the term "virion" refers to HIV-1, HIV-2 and
SIV virus particles.
[0046] As used herein, the term "retrovirus-like particle" refers
to any composition of HIV-1, HIV-2, SIV or retrovirus proteins
other than which exists naturally in naturally infected hosts that
are capable of assembly and release from either natural or
immortalized cells that express these proteins.
[0047] As used herein, the term "variant" refers to a polypeptide
or nucleotide sequence having at least 30% sequence identity with
the native sequence including fragments thereof as calculated by
Fast DB as per "Current Methods in Sequence Comparison and
Analysis," Macromolecule Sequencing and Synthesis, Selected Methods
and Applications, pp. 127-149.
[0048] As used herein, the term "transfect" refers to the
introduction of nucleic acids (either DNA or RNA) into eukaryotic
or prokaryotic cells or organisms.
[0049] As used herein, the term "gene transduction element" refers
to the minimal required genetic information to transduce a cell
with a gene.
[0050] As used herein, the term "virus-inhibitory protein" refers
to any sequence of amino acids that have been fused with Vpx or Vpr
or Gag sequences that may alter in any way the ability of a
retrovirus to multiply and spread in either individual cells
(prokaryotic and eukaryotic) or in higher organisms. Such
inhibitory molecules may include: HIV/SIV proteins or sequences,
including those that may possess enzymatic activity (examples may
include the HIV/SIV protease, integrase, reverse transcriptase, Vif
and Nef proteins) HIV/SIV proteins or proteins/peptide sequences
that have been modified by genetic engineering technologies in
order to alter in any way their normal function or enzymatic
activity and/or specificity (examples may include mutations of the
HIV/SIV protease, integrase, reverse transcriptase, Vif and Nef
proteins), or any other non-viral protein that, when expressed as a
fusion protein with Vpr or Vpx or Gag, alter virus multiplication
and spread in vitro or in vivo.
[0051] In the present invention, the HIV Vpr and Vpx proteins were
packaged into virions through virus type-specific interactions with
the Gag polyprotein precursor. HIV-1 Vpr (Vpr1) and HIV-2 Vpx
(Vpx2) are utilized to target foreign proteins to the HIV particle
as their open reading frames were fused in-frame with genes
encoding the bacterial staphylococcal nuclease (SN), an
enzymatically inactive mutant of SN(SN*), and the chloramphenicol
acetyl transferase (CAT). Transient expression in a T7-based
vaccinia virus system demonstrated the synthesis of appropriately
sized Vpr1SN/SN* and Vpx2SN/SN* fusion proteins which, when
co-expressed with their cognate p55.sup.Gag protein, were
efficiently incorporated into virus-like particles (VLPs).
Packaging of the fusion proteins was dependent on virus
type-specific determinants, as previously seen with wild-type Vpr
and Vpx proteins. Particle associated Vpr1SN and Vpx2SN fusion
proteins were enzymatically active as determined by in vitro
digestion of lambda phage DNA. To demonstrate that functional Vpr1
and Vpx2 fusion proteins were targeted to HIV particles, the
gene-fusions were cloned into an HIV-2 LTR/RRE regulated expression
vector and co-transfected with wild-type HIV-1 and HIV-2
proviruses. Western blot analysis of sucrose gradient purified
virions revealed that both Vpr1 and Vpx2 fusion proteins were
efficiently packaged regardless of whether SN, SN* or CAT were used
as C terminal fusion partners. Moreover, the fusion proteins
remained enzymatically active and were packaged in the presence of
wild-type Vpr and Vpx proteins. Interestingly, virions also
contained smaller sized proteins that reacted with antibodies
specific for the accessory proteins as well as SN and CAT fusion
partners. Since similar proteins were absent from Gag-derived VLPs
as well as in virions propagated in the presence of an HIV protease
inhibitor, they must represent cleavage products produced by the
viral protease. Taken together, these results demonstrate that Vpr
and Vpx can be used to target functional proteins, including
potentially deleterious enzymes, to the HIV/SIV particle. These
properties are useful for the development of novel antiviral
strategies.
[0052] In the present invention, a gene cassette is coupled to a
retrovirus Gag variant within a trans-enzyme plasmid to induce
fusion protein expression of the gene. Through selection of the
gene and modification of the Gag nucleotide sequence, the vectors
of the present invention are operative as antiviral therapeutics
and/or gene delivery vectors when transfected into host cells in
conjunction with genes or variants thereof coding packaging, vector
and envelope polypeptides. While the present invention is detailed
herein with plasmids each encoding different vector functions, it
is appreciated that such functions are readily combined into a
lesser number of plasmids including one, two and three plasmids
which are cotransfected into a host cell. Preferably, a multiple
plasmid gene delivery system is utilized according to the present
invention.
[0053] A Gag based trans-lentiviral vector was produced by
transfecting 293T cells with the pCMV-gag-pro (packaging plasmid),
a different trans-enzyme plasmid based on Gag, the pPCMV-eGFP
(transfer vector), and the pMD-G (env plasmid). The Gag based
trans-lentiviral vector of the present invention demonstrates that
the Gag precursor protein is able to deliver function fusion
proteins to a host cell. The fusion proteins illustratively
including RT, IN, RT-IN, GFP, CAT, CFTR and the like. As a control,
trans-lentiviral vector based Vpr was produced by transfecting 293T
cells with the pCMV-gag-pro (packaging plasmid), the pLR2P-Vpr-RTIN
(trans-enzyme plasmid), the pPCMV-eGFP (transfer vector), and the
pMD-G (env plasmid) (Wu et al. (1997) EMBO Journal 16:5113-5122).
Using fluorescence microscopy to monitor GFP expression, the
infectivity of the trans-lentiviral vector particles was monitored
on monolayer cultures of HeLa cells. As shown in Table 1, the titer
of the trans-lentiviral vector based on Gag ranged from 0.4 to
4.times.10.sup.5/ml, while that of the trans-lentiviral vector
based on Vpr ranged from 5 to 9.times.10.sup.5/ml. The Gag
precursor protein according to the present invention is capable of
delivering functional proteins into the vector particles.
Reproducibly, the titer of the trans-lentiviral vector based Gag
was approximately 2-5 fold less than that of the trans-lentiviral
vector based Vpr for RT-IN.
TABLE-US-00001 TABLE 1 Titers of Trans-Lentiviral GagRTIN Vectors
Con- Viral Control structs Trans-Lentiviral Delivery Vectors Titer
Viral Titer* A pTRE, GagNC-RT-IN, RRE-1 3.5 .times. 10.sup.4 5
.times. 10.sup.5 B pPLR2P-GagNC(1ZF)-RT-IN, 3.75 .times. 10.sup.5
6.5 .times. 10.sup.5 RRE-2** C pLR2P-GagCA-RT-IN, RRE-2*** 1.25
.times. 10.sup.5 8.75 .times. 10.sup.5 D pLR2P-GagNC-RT-IN, RRE-2
2.5 .times. 10.sup.5 5 .times. 10.sup.5 E
pLR2P-GagNC(.DELTA.PC)-RT-IN, 1.25 .times. 10.sup.5 5 .times.
10.sup.5 RRE-2**** *pLR2P-Vpr-RT-IN plasmid was used as positive
control. **The 3' Zinc Finger domain was deleted in the NC domain
of this construct. ***The whole NC domain was deleted in this
construct. ***Only the Pro-RT protease cleavage site exists between
the Gag and RT domains of this construct.
[0054] The ability of trans-RT-IN to support virus infectivity of
the lentivirus particles (virions derived from RT-IN minus proviral
DNA of HIV-1) or a lentivirus-based vector, indicates that
trans-RT-IN fusion protein is readily substituted for cis acting
RT-IN. To determine whether the trans-RT-IN (derived from the
Gag-RT-IN fusion protein) of a simple retrovirus, like the
lentivirus, also cis acting RT-TN derived from the native Gag-Pol
structure (GAG-PR-RT-IN) is replaced by trans-RT-IN derived from
Gag-RT and Gag-IN or a triple fusion of Gag-RT-IN in a retrovirus
such as a lentivirus. Thus, a trans-retroviral vector based Gag was
produced by transfecting 293T cells with the 5 .mu.g of packaging
construct (pCMV-ATG/gag-pro), 2 .mu.g of the trans-enzyme plasmid
(pCMV-ATG/gag-RT-IN), 5 .mu.g of the transfer vector
(pRTCMV-eGFP-WPRE) and the pMD-G (env plasmid). As a control, the
retrovirus vector was produced by transfecting 293T cells with the
pCMV-ATG/gag-pol (packaging plasmid), 5 .mu.g of the transfer
vector (pRTCMV-eGFP-WPRE) and the pMD-G (env plasmid). Using
fluorescence microscopy to monitor GFP expression, the infectivity
of the trans-lentiviral vector particles was monitored on monolayer
cultures of HeLa cells. As shown in Table 2, the titer of the
trans-retrovirus vector ranged from 0.6 to 1.8.times.10.sup.7/ml.
Retrovirus vector titers ranged from 0.8 to 2.5.times.10.sup.7/ml.
This result demonstrates that the simple Gag precursor protein of a
retrovirus also can deliver the functional proteins into a vector
particle in trans. Thus, according to the present invention gene
delivery to a host cell occurs with a Gag precursor gene as a
fusion partner to a protein of interest, thereby making a variety
of retroviruses operative as gene delivery vector systems.
TABLE-US-00002 TABLE 2 Titers of the Trans-Retroviral and
Retroviral Vectors Trans-enzyme Packaging Envelope Plasmid Plasmid
Vector Plasmid Plasmid Viral Titer NA* pCMV, Gag-Pro-RT-IN pRT-CMV,
GFP, WPRE pMD-G 7.18 .times. 10.sup.6 NT** pCMV, Gag-Pro pRT-CMV,
GFP, WPRE pMD-G 6 .times. 10.sup.3 pCMV, GagNC- NT** pRT-CMV, GFP,
WPRE pMD-G 0 RT-IN pCMV, GagNC- pCMV, Gag-Pro pRT-CMV,GFP, WPRE
pMD-G 1.78 .times. 10.sup.7 RT-IN *NA not applicable **NT Not
transfected
[0055] Gag fusions are operative here from retroviruses and
lentiviruses including Moloney Leukemia Virus (MLV), Abelson murine
leukemia virus, AKR (endogenous) murine leukemia virus, Avian
carcinoma, Mill Hill virus 2, Avian leukosis virus-RSA, Avian
myeloblastosis virus, Avian myelocytomatosis virus 29, Bovine
syncytial virus, Caprine arthritis encephalitis virus, Chick
syncytial virus, Equine infectious anemia virus, Feline leukemia
virus, Feline syncytial virus, Finkel-Biskis-Jinkins murine sarcoma
virus, Friend murine leukemia virus, Fujinami sarcoma virus,
Gardner-Arnstein feline sarcoma virus, Gibbon ape leukemia virus,
Guinea pig type C oncovirus, Hardy-Zuckerman feline sarcoma virus,
Harvey murine sarcoma virus, Human foamy virus, Human spumavirus,
Human T-lymphotropic virus 1, Human T-lymphotropic virus 2,
Jaagsiekte virus, Kirsten murine sarcoma virus, Langur virus,
Mason-Pfizer monkey virus, Moloney murine sarcoma virus, Mouse
mammary tumor virus, Ovine pulmonary adenocarcinoma virus, Porcine
type C oncovirus, Reticuloendotheliosis virus, Rous sarcoma virus,
Simian foamy virus, Simian sarcoma virus, Simian T-lymphotropic
virus, Simian type D virus 1, Snyder-Theilen feline sarcoma virus,
Squirrel monkey retrovirus, Trager duck spleen necrosis virus, UR2
sarcoma virus, Viper retrovirus, Visna/maedi virus, Woolly monkey
sarcoma virus, and Y73 sarcoma virus human-, simian-, feline-, and
bovine immunodeficiency viruses (HIV, SIV, FIV, BIV). While RT and
IN fusions with Gag are operative herein, it is appreciated that a
variety of therapeutic and diagnostic fusion proteins are similarly
deliverable to a target cell according to the methodologies and
vectors disclosed herein.
[0056] Gag-based trans-lentiviral vectors are disclosed based on
non-primate lentiviruses and simple retroviruses encoding
retrovirus Gag precursor proteins which have functions akin to
those of primate lentiviral Vpr or Vpx proteins. Contrary to the
prior art, the present invention maintains viral infectivity in
non-dividing primary cells. The WPRE sequence encoded within a
vector of the present invention is necessary therefor. Infectivity
of the vectors of the present invention is further enhanced through
the use of a gene transfer vector containing the
post-transcriptional regulatory element of woodchuck hepatitis
virus (WPRE). While the inclusion of a WPRE gene or gene fragment
capable of regulating post-transcription increases trans-lentiviral
titer alone, the inclusion of additional PPT-CTS sequences creates
a cumulative enhancement in viral infectivity. WPRE has been shown
to increase luciferase or GFP production in similar virus-based
vectors (Zufferey et al. (1999) J. Virol. 73:2886-2892.
Alternatively, the central terminator sequence (CTS) and central
polypurine tract (PPT) are introduced into the gene transfer vector
to independently increase titer, as detailed in U.S. Provisional
Application 60/164,626 filed Nov. 10, 1999. PPT and CTS have been
implicated in HIV-1 reverse transcription. Charneau et al. (1994) 1
Mol. Biol. 241:651-662. It is appreciated that the other control
sequences capable of stabilizing messenger RNA and thereby
facilitating protein expression are operative in place of WPRE,
PPT, and CTS within the present invention.
[0057] The present invention provides for a delivery of a
trans-protein or gene to a viral vector through coupling to either
a viral protein or gene delivery, respectively; wherein the viral
protein is Vpr or Vpx or Gag and the gene encodes either Vpr or Vpx
or Gag. Certain truncation variants of these trans-proteins or
genes perform the regulatory or enzymatic functions of the full
sequence protein or gene. For example, the nucleic acid sequences
coding for Protease, Integrase, Reverse Transcriptase, Vif, Nef,
Gag, and CFTR can be altered by substitutions, additions, deletions
or multimeric expression that provide for functionally equivalent
proteins or genes. Due to the degeneracy of nucleic acid coding
sequences, other sequences which encode substantially the same
amino acid sequences as those of the naturally occurring proteins
may be used in the practice of the present invention. These
include, but are not limited to, nucleic acid sequences comprising
all or portions of the nucleic acid sequences encoding the above
proteins, which are altered by the substitution of different codons
that encode a functionally equivalent amino acid residue within the
sequence, thus producing a silent change. For example, one or more
amino acid residues within a sequence can be substituted by another
amino acid of a similar polarity which acts as a functional
equivalent, resulting in a silent alteration. Substitutes for an
amino acid within the sequence may be selected from other members
of the class to which the amino acid belongs. For example, the
nonpolar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan and
methionine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The
positively charged (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. Also included within the scope of
the present invention are proteins or fragments or derivatives
thereof which are differentially modified during or after
translation, e.g., by glycosolation, protolytic cleavage, linkage
to an antibody molecule or other cellular ligands, etc. In
addition, the recombinant ligand encoding nucleic acid sequences of
the present invention may be engineered so as to modify processing
or expression of a ligand. For example, a signal sequence may be
inserted upstream of a ligand encoding sequence to permit secretion
of the ligand and thereby facilitate apoptosis.
[0058] Additionally, a ligand encoding nucleic acid sequence can be
mutated in vitro or in vivo to create and/or destroy translation,
initiation, and/or termination sequences or to create variations in
coding regions and/or form new restriction endonuclease sites or
destroy pre-existing ones, to facilitate further in vitro
modification. Any technique for mutagenesis known in the art can be
used, including but not limited to in vitro site directed
mutagenesis, J. Biol. Chem. 253:6551, use of Tab linkers
(Pharmacea), etc.
[0059] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
EXPERIMENTAL
Example 1
Cells and Viruses
[0060] HeLa, HeLa-tat (HLtat), 293T and CV-1 cells were maintained
in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal
bovine serum (FBS), 100 U penicillin and 0.1 mg/ml streptomycin.
HLtat cells constitutively express the first exon of HIV-1 tat and
were provided by Drs. B. Felber and G. Pavlakis. A recombinant
vaccinia virus (rVT7) containing the bacteriophage T7 RNA
polymerase gene was used to facilitate expression of viral genes
placed under the control of a T7 promoter. Stocks of rVT7 were
prepared and titrated in CV-1 cells as described previously by Wu
et al. (1992) J Virol. 66:7104-7112. HIV-1.sub.YU2, HIV-1 pNL 4-3-R
and pNL 4-3, HIV-1.sub.HXB2, H1V-2.sub.ST, and HIV-2.sub.7312A
proviral clones were used for the construction of recombinant
expression plasmids and the generation of transfection derived
viruses.
Example 2
Antibodies
[0061] To generate HIV-1 Vpr specific antibodies, the
HIV-1.sub.YU-2 vpr open reading frame was amplified by polymerase
chain reaction (PCR) using primers (sense:
5'-GCCACCTTTGTCGACTGTTAAAAAACT-3' (SEQ ID NO:1) and antisense:
5'-GTCCTAGGCAAGCTTCCTGGATGC-3' (SEQ ID NO:2)) containing Sa1I and
HindIII sites and ligated into the prokaryotic expression vector,
pGEX, generating pGEX-vpr1. This construct allowed expression of
Vpr1 as a C terminal fusion protein and Glutathione S-Transferase
(GST), thus allowing protein purification using affinity
chromatography. E. coli (DH5d) were transformed with pGEX-vpr1 and
protein expression was induced with isopropyl (.beta.-D
thiogalactopyranoside (IPTG)). Expression of the GST-Vpr1 fusion
protein was confirmed by SDS-PAGE. Soluble GST-Vpr1 protein was
purified and Vpr1 was released by thrombin cleavage using
previously described procedures of Smith et al. (1988) Gene
67:31-40. New Zealand White rabbits were immunized with 0.4 mg of
purified Vpr1 protein emulsified 1:1 in Freunds complete adjuvant,
boosted three times at two week intervals with 0.25 mg of Vpr1
mixed 1:1 in Freunds' incomplete adjuvant and bled eight and ten
weeks after the first immunization to collect antisera. Additional
antibodies used included monoclonal antibodies to HIV-1 Gag (ACT1,
and HIV-2 Gag (6D2.6), polyclonal rabbit antibodies raised against
the HIV-2 Vpx protein and anti-SN antiserum raised against purified
bacterially expressed SN protein.
Example 3
Construction of T7-Based Expression Plasmids
[0062] A DNA fragment encompassing .sup.HIV-1HXB2D.sup.gag
(nucleotides 335-1837) was amplified by PCR using primers (sense:
5'-AAGGAGAG CCATGGGTGCGAGAGCG-3' (SEQ ID NO:3) and anti-sense:
5-GGGGATCC CTTTATTG TGACGAGGGG-3' (SEQ ID NO:4)) containing NcoI
and BamHI restriction sites (underlined). The PCR product was
digested with NcoI and BamHI, purified and ligated into the
polylinker of the pTM1 vector, generating pTM-gag1. Similarly, a
DNA fragment containing the Gag coding region of HIV-2.sub.ST,
(nucleotides 547-2113) was amplified by PCR using sense and
anti-sense primers 5'-ATTGTGGGCCATGGGCGCGAGAAAC-3' (SEQ ID NO:5)
and 5'-GGGGGG CCCCTACTGGTCTTTTCC-3 (SEQ ID NO:6), respectively. The
reaction product was cut with NcoI and SmaI (underlined), purified
and ligated into the polylinker of pTM1, generating pTM-gag2.
[0063] For expression of Vpr1 under the control of the T7 promoter,
a DNA fragment containing the HIV-1.sub.YU2 vpr coding region
(nucleotides 5107-5400) was amplified by PCR using primers (sense:
5'-GAAGATCTACCATGG AAGCCCCAGAAGA-3' (SEQ ID NO:7) and anti-sense:
5'-CGCGGATCCGTTAACATCT ACTGGCTCCATTTCTTGCTC-3' (SEQ ID NO:8))
containing NcoI and HpaI/BamHI sites, respectively (underlined).
The reaction product was cut with NcoI and BamHI and ligated into
pTM1, generating a pTM-vpr1 (FIG. 12A). In order to fuse SN and SN*
in-frame with vpr1, their coding regions were excised from
pGN1561.1 and pGN1709.3, respectively and through a series of
subcloning steps, ligated into the SmaI/XhoI sites of pTM-vpr1,
generating pTM-vpr1SN and pTM-vpr1SN*. This approach changed the
translational stop codon of Vpr1 to a Trp codon and the C terminal
Ser residue to a Cys. The resulting junctions between vpr1 and
SN/SN* are depicted in FIG. 12C.
[0064] For expression of Vpx2 under T7 control, a DNA fragment
containing the HIV-2.sub.ST vpx coding sequence (nucleotides
5343-5691) was amplified by PCR using primers (sense:
5'-GTGCAACACCATGGCAGGCCCCAGA-3' (SEQ ID NO: 9) and anti-sense:
5'-TGCACTGCAGGAAGATCTTAGACCTGGAGGGGGAG GAGG-3' (SEQ ID NO: 10))
containing NcoI and Bg1 II sites, respectively (underlined). After
cleavage with BgLII and Klenow fill-in, the PCR product was cleaved
with NcoI, purified and ligated into the NcoI and SmaI sites of
pTM1, generating pTM-vpx2 (FIG. 12B). To construct in-frame fusions
with vpx2, BamHI/XhoI, SN- and SN*-containing DNA fragments were
excised from pTM-vpr1SN and pTM-vpr1SN* and ligated into pTM-vpx2,
generating pTM-vpx2SN and pTM-vpx2SN*, respectively. This approach
introduced one amino acid substitution at the C terminus of Vpx
(Val to Arg), changed the translational stop codon of vpx to Ser
and left five amino acids residues of the pTM1 plasmid polylinker.
The resulting junctions between vpx2 and SN/SN* are depicted in
FIG. 1D.
Example 4
Construction of HIV LTR-Based Vpr or Vpx Expression Plasmids
[0065] For efficient expression of Vpr and Vpx fusion proteins in
the presence of HIV, a eukaryotic expression vector (termed pLR2P)
was constructed which contains both an HIV-2 LTR (HIV-2.sub.ST,
coordinates -544 to 466) and an HIV-2 RRE (HIV-2.sub.ROD,
coordinates 7320 to 7972) element (FIG. 7A). These HIV-2 LTR and
RRE elements were chosen because they respond to both HIV-1 and
HIV-2 Tat and Rev proteins. The vpr1, vpr1SN,vpx2 and vpx2SN coding
regions were excised from their respective pTM expression plasmids
(see FIG. 1) with NcoI and XhoI restriction enzymes and ligated
into pLR2P, generating pLR2P-vpr1, pLR2P-vpr1SN, pLR2P-vpx2 and
pLR2P-vpx2SN, respectively (FIG. 7A). For construction and
expression of vpr- and vpx-CAT gene fusions, the SN containing
regions (BamHI/XhoI fragments) of pLR2P-vpr1SN and pLR2P-vpx2SN
were removed and substituted with a PCR amplified Bgl II/XhoI DNA
fragment containing CAT, generating pLR2P-vpr1CAT and
pLR2P-vpx2CAT, respectively (FIG. 9A).
Example 5
Construction of Lentiviral Plasmids Involving Gag Fusions
[0066] The pHRCMV-eGFP plasmid was derived by modifying pHRCMV-LacZ
which has been described (Naldini et al. (1996) Science
272:263-267). The pHRCMV-eGFP plasmid was constructed by ligating a
BamHI/XhoI DNA fragment containing eGFP (derived from pEGFP-C1;
CLONTECH Laboratories, Palo Alto, Calif.) into the pHRCMV-lacz
plasmid after removing lacz by digestion with BamHI and XhoI. To
construct the pPCMV-eGFP, a 150 bp sequence (with coordinates
4327-4483) and containing the central PPT and central terminal site
(CTS) was amplified from the SG3 molecular clone by PCR and ligated
into pHRCMV-eGFP that was cut with ClaI. To construct the
Tet-inducible expression plasmids, 430 bps of TRE-inducible
promoter derived from pTRE; CLONTECH Laboratories, Palo Alto,
Calif., was cut by XhoI filled to blunt ends and BamHI. The CMV
promoter of pcDNA3.1(+) plasmid (Invitrogen, CA) was replaced using
SpeI (filled to blunt ends) and BamHI, generating pTRE-neo. The 6.7
kb fragment containing the HIV-based packaging components derived
from pCMVgag-pol was cloned into pTRE-neo using EcoRI and XhoI,
generating pTRE-gag-pol which contains functional vif, tat, rev,
gag and pal genes. To construct the RT-IN minus plasmid shown in
FIG. 23A, the region (from 1975 to 5337) of pTREgag-pol were
substituted with an RT-IN containing BcII/Sa1I DNA fragment (from
1975 to 5337) of pSG3S-RT was ligated into the Bc1I and Sa1I sites
of the pTREgag-pol plasmid, generating pTREgag-pro. The RT-IN
sequence contained translational stop codons (TAA) at the first
amino acid position of the RT and IN coding regions and was under
control of CMV promoter. A 39-base pair internal deletion in the 4
sequence was introduced, the internal region (1357 bp) of envelope
gene was deleted from 5827 to 7184, generating pCMVgag-pol. To
construct the RT-IN minus plasmid, the region (from 1975 to 5337)
of pCMVgag-pol was substituted with an RT-IN containing Bc1I/Sa1I
DNA fragment (from 1975 to 5337) of pSG3S-RT was ligated into the
Bc1I and Sa1I sites of the pCMVgag-pol plasmid, generating
pCMVgag-pro shown in Table 2. The RT-IN sequence contained
translational stop colons (TAA) at the first amino acid position of
the RT and IN coding regions. To construct the series of
trans-enzyme plasmids shown in FIG. 24A-C and Table 2, different
fragments of HIV-1 gag genes were amplified by PCR and were cloned
into pLR2Pvpr-RT-IN using Nco1 1 and Bg1 11, generating a series of
gag-RTIN fusion expression plasmids. pLR2gagNC-RTIN shown in Table
1 as construct D contained the Gag gene with the p6 portion
deleted. pLR2PgagNC(1ZF)-RTIN shown in FIG. 24B and contains the
Gag gene with the second Zing finger of NC and p1-p6 fragment
deleted. pLR2PgagCA-RTIN shown as FIG. 24C and in Table 1 as
construct C contains the Gag gene which deleted the NC-p1-p6
fragment. pLR2PVprRTIN construction is described in FIG. 12. pMD-G
is constructed according to existing techniques (Wu et al. (1997)
EMBO Journal 16:5113-5122).
Example 6
Construction of Retroviral Plasmids Involving Gag Fusions
[0067] A RT-IN minus packaging construct was formed based on
Moloney murine leukemia virus. pCMV-ATG/gag-pol was cut by SalI and
filled with Klenow, generating pCMV-ATG/gag-pro, with the RT gene
being mutated by the reading frame shift at amino acid position 366
as shown in FIG. 21A. To generate a trans-enzyme plasmid, the 312
bp fragment which contains the protease region was deleted from the
gag-pol of pCMV-ATG/gag-pol, generating pCMV-ATG/gag-RT-IN as shown
in FIG. 21B. The GFP transfer vector based on Moloney murine
leukemia virus, GFP-WPRE which was obtained from pPCMV-eGFP-WPRE
(Finer et al. (1994) Blood 83:43-50) and cloned into pRTCMV using
BamHI and ApoI, generating pRTCMV-eGFP-WPRE as shown in FIG.
21C.
Example 7
Preparation of Vector Stocks and Infection
[0068] Trans-lentiviral vector stocks were produced by transfecting
the 5 .mu.g of packaging construct (pTREgag-pol), the 2 .mu.g of
VSV-G construct (pMD-G), and 5 .mu.g of the transfer vector
(pPCMV-eGFP WPRE) and 1 .mu.g of pTet-off (CLONTECH Laboratories,
Palo Alto, Calif.) and different trans-enzyme plasmids into the
subcontinent 293T cell by the calcium phosphate precipitation
method. Trans-retroviral vector stocks were produced by
transfecting the 5 .mu.g of packaging construct (pCMV-ATG/gag-pro),
the 2 .mu.g of VSV-G construct (pMD-G), and 5 .mu.g of the transfer
vector (pRTCMV-eGFP-WPRE) and 2 .mu.g of the trans-enzyme plasmid
(pCMV-ATG/gag-RT-IN). Approximately 1.times.10.sup.6 cells were
seeded into six-well plates 24 hr prior to transfection. The vector
stocks were harvested 60 hr posttransfection. Supernatants of the
transfected cultures were clarified by low speed centrifugation
(1000 g, 10 min), and filtered through a 0.45-.mu.g-pore-size
filter, aliquoted and subsequently frozen at -80.degree. C. The
target cells were infected in the DMEM-1% FBS containing 10
.mu.g/ml of DEAETestron for 4 hr at 37.degree. C. The medium was
subsequently replaced with fresh DMEM-10% FBS or preconditional
medium. To determine the titer of eGFP vector, the supernatant
stock of 1.0, 0.2, 0.04, and 0.008 .mu.l were used to infect the
culture of HeLa cell. 2-3 days later, positive (green) cell
colonies were counted using a fluorescence microscope.
Example 8
Transfections
[0069] Transfections of proviral clones were performed in HLtat
cells using calcium phosphate DNA precipitation methods as
described by the manufacturer (Strategene). T7-based (pTM1)
expression constructs were transfected using Lipofectin (BioRad)
into rVT7 infected HeLa cells as described previously by Wu et al.
(1994) J. Virol. 68:6161-6169. These methods were those recommended
by the manufacturer of the Lipofectin reagent.
Example 9
Western Immunoblot Analysis
[0070] Virions and virus-like particles (VLPs) were concentrated
from the supernatants of transfected or infected cells by
ultracentrifugation through 20% cushions of sucrose
(125,000.times.g, 2 hrs., 4.degree. C.). Pellets and
infected/transfected cells were solubilized in loading buffer (62.5
mM Tris-HCl (pH 6.8) 0.2% sodium dodecyl sulfate (SDS), 5%
2-mercaptoethanol, 10% glycerol), loaded and separated on 12.5%
polyacrylamide gels containing SDS. Following electrophoresis,
proteins were transferred to nitrocellulose (0.2 .mu.m; Schleicher
34 & Schnell) by electroblotting, incubated for one hour at
room temperature in blocking buffer (5% nonfat dry milk in
phosphate buffered saline [PBS]) and then for two hours with the
appropriate antibodies diluted in blocking buffer. Protein bound
antibodies were detected with HRP-conjugated specific secondary
antibodies using ECL methods according to the manufacturer's
instructions (Amersham).
Example 10
SN Nuclease Activity Assay
[0071] Cells and viral pellets were resuspended in nuclease lysis
buffer (40 mM Tris-HCl, pH 6.8, 100 mM NaCl, 0.1% SDS, 1% Triton
X-100) and clarified by low speed centrifugation (1000.times.g, 10
min.). Tenfold dilutions were made in nuclease reaction cocktail
buffer (100 mM Tris-HCl, pH 8.8, 10 mM CaCl.sub.2, 0.1% NP40) and
boiled for 1 minute. 5 .mu.l of each dilution was added to 14 .mu.l
of reaction cocktail buffer containing 500 ng of lambda phage DNA
(HindIII fragments) and incubated at 37.degree. C. for 2 hours.
Reaction products were electrophoresed on 0.8% agarose gels and DNA
was visualized by ethidium bromide staining.
Example 11
Expression of Vpr1- and Vpx2-SN and SN* Fusion Proteins in
Mammalian Cells
[0072] Expression of Vpr1- and Vpx2-SN/SN* fusion proteins in
mammalian cells was assessed using the recombinant vaccinia
virus-T7 system (rVT7). HeLa cells were grown to 75-80% confluency
and transfected with the recombinant plasmids pTM-vpr, pTM-vpx,
pTM-vpr1 SN/SN*, and pTMvpx2SN/SN* (FIG. 1). Twenty-four hours
after transfection, cells were washed twice with PBS and lysed.
Soluble proteins were separated by SDS-PAGE and subjected to
immunoblot blot analysis. The results are shown in FIG. 2.
Transfection of pTM-vpr1SN and pTM-vpr1SN* resulted in the
expression of a 34 kDa fusion protein that was detectable using
both anti-Vpr and anti-SN antibodies (A). Similarly, transfection
of pTM-vpx2SN and pTM-vpx2SN* resulted in the expression of a 35
kDa fusion protein which was detected using anti-Vpx and anti-SN
antibodies (B). Both fusion proteins were found to migrate slightly
slower than expected, based on the combined molecular weights of
Vpr1 (14.5 kDa) and SN (16 kDa) and Vpx2 (15 kDa) and SN,
respectively. Transfection of pTM-vpr1 and pTM-vpx2 alone yielded
appropriately sized wild-type Vpr and Vpx proteins. Anti-Vpr,
anti-Vpx and anti-SN antibodies were not reactive with lysates of
pTM1 transfected cells included as controls. Thus, both SN and SN*
fusion proteins can be expressed in mammalian cells.
Example 12
Incorporation of Vpr1- and Vpr2-SN/SN* Fusion Proteins into
Virus-Like Particles
[0073] In vaccinia and baculovirus systems, the expression of HIV
Gag is sufficient for assembly and extracellular release of VLPs.
Vpr1 and Vpx2 can be efficiently incorporated into Gag particles
without the expression of other viral gene products. To demonstrate
that the Vpr1 and Vpx2 fusion proteins could be packaged into VLPs,
recombinant plasmids were coexpressed with HIV-1 and HIV-2 Gag
proteins in the rVT7 system. pTM-vpr1, pTM-vpr1SN and pTM-vpr1SN*
were transfected into HeLa cells alone and in combination with the
HIV-1 Gag expression plasmid, pTM-gag1. Twenty-four hours after
transfection, cell and VLP extracts were prepared and analyzed by
immunoblot analysis (FIG. 3A). Anti-Vpr antibody detected Vpr1,
Vpr1SN and Vpr1SN* in cell lysates (top panel) and in pelleted VLPs
derived by coexpression with pTM-gag1 (middle panel). In the
absence of HIV-1Gag expression, Vpr1 and Vpr1SN were not detected
in pellets of culture supernatants (middle panel). As expected VLPs
also contained p55 Gag (bottom panel). Thus, Vpr1SN/SN* fusion
proteins were successfully packaged into VLPs.
[0074] To demonstrate that Vpx2SN was similarly capable of
packaging into HIV-2 VLPs, pTM-vpx2, pTM-vpx2SN and pTM-vpx2SN*
were transfected into HeLa cells alone and in combination with the
HIV-2 Gag expression plasmid, pTM-gag2. Western blots were prepared
with lysates of cells and VLPs concentrated from culture
supernatants by ultracentrifugation (FIG. 3B). Anti-Vpx antibody
detected Vpx2, Vpx2SN and Vpx2SN* in cell lysates (top panel) and
in VLPs derived by coexpression with pTM-gag2 (middle panel).
Anti-Gag antibody detected p55 Gag in VLP pellets (bottom panel).
Comparison of the relative protein signal intensities suggested
that the Vpr1 and Vpx2-SN and SN* fusion proteins were packaged
into VLPs in amounts similar to wild-type Vpr1 and Vpx2 proteins.
Sucrose gradient analysis of VLPs containing Vpr1 SN and Vpx2SN
demonstrated co-sedimentation of these fusion proteins with VLPs
(data not shown).
[0075] The Gag C terminal region is required for incorporation of
Vpr1 and Vpx2 into virions. However, packaging was found to be
virus type-specific, that is, when expressed in trans, Vpx2 was
only efficiently incorporated into HIV-2 virions and HIV-2 VLPs.
Similarly, HIV-1 Vpr required interaction with the HIV-1 Gag
precursor for incorporation into HIV-1 VLPs. To show that the
association of Vpr1SN and Vpx2SN with VIPs was not mediated by the
SN moiety, but was due to the Vpr and Vpx specific packaging
signals, pTM-vpr1SN and pTM-vpx2SN were cotransfected individually
with either pTM-gag1 or pTM-gag2. For control, pTM-vpr1 and
pTM-vpx2 were also transfected alone. Twenty-four hours later,
lysates of cells and pelleted VLPs were examined by immunoblotting
(FIG. 4). While Vpr1SN was expressed in all cells (FIG. 4A, top
panel), it was only associated with VLPs derived from cells
transfected with pTM-gag1. Similarly, Vpx2SN was detected in all
pTM-vpx2 transfected cells (FIG. 4B, top panel), but was only
associated with VLPs derived by cotransfection with pTM-gag2 (FIG.
4B, middle panel). HIV-1 and HIV-2 Gag monoclonal antibodies
confirmed the presence of Gag precursor protein in each VLP pellet
(FIG. 4B, bottom panels). Thus, incorporation of Vpr1SN and Vpx2SN
into VLPs requires interaction of the cognate Gag precursor
protein, just like native Vpr1 and Vpx2.
[0076] While Vpr1SN and Vpx2SN fusion proteins clearly associated
with VLPs (FIG. 3), the question remained whether they would
continue to do so in the presence of the native accessory proteins.
The efficiency of Vpr1SN and Vpx2SN packaging was compared by
competition analysis (FIG. 5). pTM-vpr1SN and pTM-vpx2SN were
cotransfected with pTM-gag1/pTM-vpr1 and pTMgag2/pTM-vpx2,
respectively, using ratios that ranged from 1:4 to 4:1 (FIG. 5A and
FIG. 5B, left panels). For comparison, pTM-vpr1 SN and pTM-vpr1
were transfected individually with pTM-gag1 (FIG. 5A, middle and
right panels respectively) and pTM-vpx2SN and pTM-vpx2 were
transfected with pTM-gag2 (FIG. 5B, middle and right panels
respectively). VLPs were pelleted through sucrose cushions, lysed,
separated by PAGE, blotted onto nitrocellulose and probed with
anti-SN antibody. The results revealed the presence of both Vpr1
and Vpr1SN in VLPs when cotransfected into the same cells (FIG. 5A,
left panel). Similarly, coexpressed Vpx2 and Vpx2SN were also
copackaged (FIG. 5B, left panel). Comparison of the relative
amounts of VLP-associated Vpr1SN and Vpx2SN when expressed in the
presence and absence of the native protein, indicated that there
were no significant packaging differences. Thus, Vpr1/Vpx2 fusion
proteins can efficiently compete with wild-type proteins for virion
incorporation.
Example 13
Vpr1 SN and Vpx2SN Fusion Proteins Possess Nuclease Activity
[0077] To demonstrate that virion associated SN fusion proteins
were enzymatically active, VLPs concentrated by ultracentrifugation
from culture supernatants of HeLa cells transfected with
pTM-gag1/pTM-vpr1SN and pTMgag2/pTM-vpx2SN were analyzed for
nuclease activity using an in vitro DNA digestion assay. Prior to
this analysis, immunoblotting confirmed the association of Vpr1 SN
and Vpx2SN with VLPs (data not shown). FIG. 6 shows lambda phage
DNA fragments in 0.8% agarose gels after incubation with dilutions
of VLPs lysates that contained Vpr1- or Vpx2-SN fusion proteins.
VLPs containing Vpr1 SN* and Vpx2SN* were included as negative
controls and dilutions of purified SN served as reference standards
(FIG. 6A). Both virion associated Vpr1SN (FIG. 6B) and Vpx2SN (FIG.
6C) fusion proteins exhibited nuclease activity as demonstrated by
degradation of lambda phage DNA. Cell-associated Vpr1SN and Vpx2SN
fusion proteins also possessed nuclease activity when analyzed in
this system (data not shown). To control for SN specificity, this
analysis was also conducted in buffers devoid of Ca.sup.++ and
under these conditions no SN activity was detected (data not
shown). Thus, SN remains enzymatically active when expressed as a
fusion protein and packaged into VLPs.
Example 14
Incorporation of Vpx2SN Fusion Protein into HIV-2 Virions
[0078] Vpx is incorporated into HIV-2 virions when expressed in
trans. To show that Vpx2 fusion proteins were similarly capable of
packaging into wild-type HIV-2 virions, an expression plasmid
(pLR2P) was constructed placing the vpx2SN and vpx2SN* coding
regions under control of HIV-2 LTR and RRE elements. The HIV-2 RRE
was positioned downstream of the fusion genes to ensure mRNA
stability and efficient translation (FIG. 7A). To show that the
fusion proteins could package when expressed in trans,
HIV-2.sub.ST, proviral DNA (pSXBI) was transfected alone and in
combination with pLR2P-vpx2SN and pLR2P-vpx2SN*. Forty-eight hours
later, extracellular virus as pelleted from culture supernatants by
ultracentrifugation through cushions of 20% sucrose and examined by
immunoblot analysis (FIG. 7B). Duplicate blots were probed using
anti-Vpx (left), anti-SN (middle) and anti-Gag (right) antibodies.
Anti-Vpx antibody detected the 15 kDa Vpx2 protein in all viral
pellets. In virions derived by cotransfection of HIV-2.sub.ST with
pLR2P-vpx2SN and pLR2P-vpx2SN*, additional proteins of
approximately 35 and 32 kDa were clearly visible. The same two
proteins were also apparent on a duplicate blot probed with anti-SN
antibodies, indicating that they represented Vpx2SN fusion proteins
(FIG. 7B, middle panel). The predicted molecular weight of
full-length Vpx2SN fusion protein is 33 kDa. As native Vpx and SN
run slightly slower than predicted, it is likely that the 35 kDa
species represents the full-length Vpx2SN fusion protein. Anti-SN
antibodies detected additional proteins of approximately 21 and 17
kDa (these proteins were more apparent after longer exposure).
Since only the 35 kDa protein was detected in Gag derived VLPs,
which lack Pol proteins (FIG. 2), the smaller proteins represented
cleavage products of Vpx2SN and Vpx2SN* generated by the viral
protease. Anti-Gag antibodies confirmed the analysis of
approximately equivalent amounts of virions from each
transfection.
[0079] To show packaging of Vpx2SN into HIV-2 virions, sucrose
gradient analysis was performed. Extracellular virus collected from
culture supernatants of HLtat cells forty-eight hours after
cotransfection with pLR2P-vpx2SN and HIV-2.sub.ST was pelleted
through cushions of 20% sucrose. Pellets were resuspended in PBS
and then centrifuged for 18 hours over linear gradients of 20-60%
sucrose. Fractions were collected and analyzed by immunoblotting
(FIG. 7C). Duplicate blots were probed separately with anti-SN
(top) and anti-Gag (bottom) antibodies. Peak concentrations of both
Vpx2SN and Gag were detected in fractions 8-11, demonstrating
direct association and packaging of Vpx2SN into HIV-2 virions.
These same sucrose factions (8-11) were found to have densities
between 1.16 and 1.17 g/ml, as determined by refractometric
analysis (data not shown). Again, both the 35 kDa and 32 kDa forms
of Vpx2SN were detected, providing further evidence for protease
cleavage following packaging into virus particles.
[0080] Since HIV-2.sub.ST is defective in Vpr, this may have
affected the packaging of the Vpx2SN fusion protein. A second
strain of HIV-2, termed HIV-2.sub.7312A, was analyzed which was
cloned from short-term PBMC culture and contains open reading
frames for all genes, including intact vpr and vpx genes
(unpublished). A plasmid clone of HIV-2.sub.7312A proviral DNA
(pJK) was transfected alone and in combination with pLR2P-vpx2SN
into HLtat cells. For comparison, HIV-2.sub.ST was also
co-transfected with pLR2P-vpx2SN. Progeny virus was concentrated by
ultracentrifugation through sucrose cushions and examined by
immunoblot analysis (FIG. 7D). Duplicate blots were probed with
anti-Vpx (left) and anti-Gag (right) antibodies. The results
revealed comparable levels of Vpx2SN incorporation into vpr
competent virus (HIV-2.sub.7312A) compared with vpr-defective virus
(HIV-2.sub.ST). Moreover, the 35 kDa and 32 kDa proteins were again
detected in HIV-2.sub.7312A virions. Thus, efficient incorporation
of the Vpx2SN protein into replication-competent wild-type HIV-2
was demonstrated, even in the presence of native Vpr and Vpx
proteins.
Example 15
Incorporation of Vpr1SN into HIV-1 Virions
[0081] Using the same LTR/RRE-based expression plasmid, it was also
shown that Vpr1SN could package into HIV-1 virions by co-expression
with HIV-1 provirus (as discussed above, the HIV-2 LTR can be
transactivated by HIV-1 Tat and the HIV-2 RRE is sensitive to the
HIV-1 Rev protein). Virions released into the culture medium 48
hours after transfection of HLtat cells with pNL4-3 (HIV-1) and
pNL4-3-R.sup.- (HIV-1-R.sup.-) alone and in combination with
pLR2P-vpr1SN were concentrated by ultracentrifugation and examined
by immunoblot analysis (FIG. 8). As observed in cotransfection
experiments with HIV-2, anti-SN antibodies identified two major
Vpr1SN fusion proteins of approximately 34 to 31 kDa. These
proteins were not detected in virions produced by transfection of
pNL4-3 and pNL4-e-R.sup.- alone. From expression in the rVT7
system, the full-length Vpr1SN fusion protein was expected to
migrate at 34 kDa. Therefore, the 31 kDa protein likely represents
a cleavage product. Anti-SN antibodies also detected a protein
migrating at 17 kDa. Anti-Vpr antibody detected the 34 and 31 kDa
proteins in virions derived from cotransfected cells. It is
noteworthy that both the anti-Vpr and anti-SN antibodies detected
the 31 kDa protein most strongly, and that anti-Vpr antibody did
not detect the 17 kDa protein recognized by anti-SN antibody. These
results also show that even in virions in which native Vpr protein
was packaged, Vpr1SN was also incorporated in abundance. Gag
monoclonal antibody detected similar amounts of Gag protein in all
viral pellets and demonstrated processing of the p55.sup.Gag
precursor (FIG. 8C).
[0082] To demonstrate more directly that cleavage of the Vpr1- and
Vpx2-SN fusion proteins was mediated by the HIV protease, virus was
concentrated from pNL4-3-R.sup.-/pLR2P-vpr1SN and
pSXB1/pLR2P-vpx2SN transfected cells that were culture in the
presence of 1 .mu.M of the HIV protease inhibitor L-689,502
(provided by Dr. E. Emini, Merck & Co. Inc.). As expected,
immunoblot analysis of virions demonstrated substantially less
processing of p55.sup.Gag (FIG. 9A). Similarly, virions produced in
the presence of L-689,502 also contained greater amounts of the
uncleaved species of Vpr1SN and Vpx2SN fusion proteins (FIG. 9B).
Taken together, these results demonstrate that Vpr1- and Vpx2-SN
fusion proteins are subject to protease cleavage during or
subsequent to virus assembly.
Example 16
Vpr1-CAT and Vpr2-CAT Fusion Protein Incorporation into HIV
Virions
[0083] To show that Vpx2 and Vpr1 could target additional proteins
to the HIV particle, the entire 740 bp cat gene was substituted for
sn in the pLR2P-vpx2SN and pLR2P-vpr1SN vectors, generating
pLR2P-vpr1 CAT and pLR2P-vpx2CAT (FIG. 10A). pNL4-3/pLR2P-vpr1 CAT,
pn14-3-R.sup.-/pLR2P-vpr1 CAT and pSXB1/pLR2P-vpx2CAT were
co-transfected into HLtat cells. As controls, pNL4-3,
pNL4-3-R.sup.- and pSXB1 were transfected alone. Progeny virions,
concentrated from culture supernatants, were analyzed by
immunoblotting (FIGS. 10B and 10C). Using anti-Vpr antibodies, 40
kDa fusion proteins were detected in viral pellets derived by
co-transfection of pRL2P-vpr1 CAT with both pNL4-3 and
pNL4-3-R.sup.- (FIG. 10B). This size is consistent with the
predicted molecular weight of the full-length Vpr1 CAT fusion
protein. In addition, anti-Vpr antibodies also detected a 17 kDa
protein which did not correspond to the molecular weight of native
Vpr1 protein (14.5 kDa in virions derived from cells transfected
with pNL4-3). The same protein was recognized weakly with anti-CAT
antibodies, suggesting a fusion protein cleavage product containing
most Vpr sequence. Very similar results were obtained with virions
derived from HLtat cells co-transfected with HIV-2.sub.ST and
pRL2P-vpx2CAT, in which anti-Vpx antibody detected 41 and 15 kDa
proteins (FIG. 10C). These results demonstrate that Vpr1 CAT and
Vpx2CAT fusion proteins are packaged into virions. However, like in
the case of SN fusion proteins, CAT fusion proteins were also
cleaved by the HIV protease (the Vpx2CAT cleavage product is not
visible because of co-migration with the native Vpx protein. CAT
cleavage appeared less extensive, based on the intensity of the
full-length CAT fusion protein on immunoblots.
[0084] Lysates of HIV-1 and HIV-2 viral particles were diluted 1:50
in 20 mM Tris-base and analyzed for CAT activity by the method of
Allon et al. (1979) Nature 282:864-869. FIG. 10D indicates that
virions which contained Vpr1CAT and Vpx2CAT proteins possessed CAT
activity. These results show the packaging of active Vpr1- and
Vpx2-CAT fusion proteins.
Example 17
Virion Incorporated SN and CAT Fusion Proteins are Enzymatically
Active
[0085] The ability of Vpr1 and Vpx 2 to deliver functionally active
proteins to the virus particle was further confirmed by sucrose
gradient analysis. Virions derived from HLtat cells co-transfected
with HIV-2.sub.ST and pLR2P-vpx2 were sedimented in linear
gradients of 20-60% sucrose as described above. Fractions were
collected and analyzed for viral Gag protein (FIG. 11) and
corresponding CAT activity (FIG. 11B). Peak amounts of Gag protein
were detected in fractions 6 and 7 (density 1.16 and 1.17,
respectively). Similarly, peak amounts of acetylated
chloramphenicol (Ac-cm) were also detected in fractions 6 and
7.
[0086] Whether virion associated SN fusion protein retained
nuclease activity was also shown. HIV-1.sub.SG3 virions containing
Vpr1SN were analyzed after sedimentation in linear gradients of
sucrose (FIG. 11). Since the present invention demonstrated that
protease cleavage of SN fusion proteins (FIGS. 7, 8 and 9) markedly
reduced Vpr1SN nuclease activity (data not shown), these
experiments were performed by culturing pSG3/pLR2P-vpr1SN
co-transfected cells in the presence of L-689,502 as described
above. Immunoblot analysis of sedimented virions revealed peak
concentrations of Gag in fractions 6 and 7 and substantially
reduced p55 processing (FIG. 11C). Peak SN activity was associated
with the fractions that contained the highest concentrations of
virus (FIG. 11D). These results thus document that virion
incorporation per se does not abrogate the enzymatic activity of
Vpr/Vpx fusion proteins, although cleavage by the viral protease
may inactivate the fusion partners.
Example 18
Construction and Design of a Gag-Pro (RT-IN Minus) Packaging
Plasmid
[0087] Several different strategies have been used to express
Gag-Pro. Placing Gag and Pro in the same reading frame leads to
overexpression of Pro and marked cell toxicity. It is known that
deletions within the RT and IN coding regions, including smaller
deletion mutations, may cause marked defects in the expression
levels of the Gag-Pro and Gag-Pol proteins, respectively
(Ansari-Lari et al. (1995) Virology 211:332-335; Ansari-Lari et al.
(1996) J. Virol. 70:3870-3875; Bukovsky et al. (1996) J. Virol.
70:6820-6725; Engelman et al. (1995) J. Virol. 69:2729-2736;
Schnell et al. (1997) Cell Press 90:849-857). Importantly, the
viral particles produced under these circumstances are defective in
proteolytic processing and are not infectious, even if RT and IN
are provided in trans (Wu et al. (1994) J. Virol. 68:6161-6169).
The reduced levels of expression and virion associated Gag-Pol
protein is apparently due to an effect on the frequency of Gag-Pol
frame-shifting. Gag-Pol frame-shifting is not markedly affected
when the translation of RT and IN is abrogated, which is distinct
from deletions of viral DNA fragment. Virions which assembly
Gag-Pro, when RT and IN protein synthesis is abrogated by a
translational stop codon, mature and are infectious when RT and IN
are provided in trans (Wu et al. (1994) J. Virol., 68:6161-6169).
Therefore, a Gag-Pro packaging plasmid of the present invention is
preferably constructed by abrogating translation of sequence
downstream of Pro (RT-IN). Other mutations in Gag and Pol would
also function as part(s) of the trans-lentiviral packaging system
if they did not cause major defects in particle assembly and
infectivity. In addition to introducing a translational stop codon
(TAA) at the first amino acid residue of RT, at least one addition
"fatal" mutation is positioned within RT and IN (FIG. 12B). This
mutation further decreases the likelihood of reestablishing a
complete Gag-Pol coding region by genetic recombination between
packaging (gag-pro) and enzymatic (vpr-RT-IN) plasmids. It is
appreciated that the stop codon can be inserted within the gene
sequence in a position other than at the first codon for the first
amino acid residue of a protein and still be an effective measure
to prevent infectivity. A stop codon generally inserted with the
front half of the amino acid encoding nucleic acid residues is
effective, although the stop codon is preferentially at the
beginning of the translational sequence. A fatal mutation as used
herein refers to a mutation within the gene sequence that render
the coded polypeptide sequence functionally ineffectual in
performing the biological role of the wild protein.
[0088] The Gag-Pro expression plasmid (pCR-gag-pro) includes the
CMV promoter and the HIV-2 Rev responsive element (RRE) (FIG. 12C).
The RRE allows for the efficient expression of HIV proteins
(including Gag, PR, RT, IN) that contain mRNA inhibitory sequences.
RT and IN are provided by trans-expression with the pLR2P-vpr-RT-IN
expression plasmid (FIG. 12C). This vector expresses the Vpr-RT-IN
fusion protein which is incorporated into HIV-1 virions/vector in
trans, and is proteolytically processed by the viral protease to
generate functional forms of RT (p51 and p66) and IN (Wu et al.
(1994) J. Virol. 68:6161-6169). This earlier work shows that
functional RT and IN can be provided separately (Vpr-RT and Vpr-IN)
(Liu et al. (1997) J. Virol. 71:7704-7710 and Wu et al. (1994) J.
Virol. 68:6161-6169). Preferably, the Vpr component of the fusion
protein contains a His71Arg substitution which knocks out the Vpr
cell cycle arrest function.
Example 19
Production of the Trans-Lentiviral Vector
[0089] 4 .mu.g each of pCR-gag-pro, pLR2P-vpr-RT-IN (enzymatic
plasmid), pHR-CMV-.beta.-gal (marker gene transduction plasmid) and
pCMV-VSV-G (env plasmid were transfected into 293T cell line. 293T
cells were used since they produce high titered stocks of HIV
particles/vector and are exquisitely sensitive to transfection,
including multiple plasmid transfections. As a control, in
side-by-side experiments, the p.DELTA.8.2 packaging plasmid was
also transfected with pHR-CMV-.beta.-gal and pCMV-VSV-G (FIG. 12B).
The p.DELTA.8.2 plasmid is a lentivirus packaging vector obtained
from Dr. D. Trono. The p.DELTA.8.2 produces high titered vector
stocks upon transfection with pHR-CMV-.beta.-gal and pCMV-VSV-G
(Naldini et al. (1996) Science 272:263-267 and Zhang et al. (1993)
Science 259:234-238), (approximately 1-5.times.10.sup.5 infectious
particles/ml supernatant, with a p24 antigen concentration of
150-800 ng/ml). Approximately 72 hours after transfection, the
culture supernatants were harvested, clarified by low-speed
centrifugation, filtered through a 0.45 micron filter, and analyzed
for p24 antigen concentration by ELISA. To examine the titer of the
trans-lentiviral vector, supernatant stocks of 25, 5, 1, and 0.2
.mu.l were used to infect cultures of HeLa cells and IB3 cells. Two
days later, the cells are stained with X-gal, and positive (blue)
cells are counted using a light microscope. Table 3 shows the titer
of trans-lentiviral vector. These results show that the
trans-lentiviral vector can achieve titers as high as
2.times.10.sup.5/ml, although its titer is consistently lower than
that of lentiviral vector (2-5 folds less). For direct examination
of transduction in living cells the transduction plasmid was also
constructed to contain the GFP gene/marker (FIGS. 12B and 12C).
Stocks of trans-lentiviral and lentiviral vector were produced as
described above and used to infect HeLa cells. Two days later the
cells were examined by fluorescence microscopy. FIGS. 13 and 14
show positive gene transduction with the trans-lenti and lentiviral
vectors respectively.
TABLE-US-00003 TABLE 3 Generation of Trans-Lentiviral Vector Titer
(inf. units/ml .times. 10.sup.-5) Packaging Plasmid RT-IN Plasmid
HeLA IB3 pCMV.DELTA.R9 -- 2.5 (+/-5.1) 1.2 (+/-2.7)
pCMV.DELTA.R9-S.sup.RT-IN -- 0 0 pCMV.DELTA.R9-S.sup.RT-IN
Vpr-RT-IN 1.1 (+/-3.1) 0.8 (+/-2.5)
Example 20
Concentration of Trans-Lentiviral Vector by Ultracentrifugation
[0090] To examine whether the trans-lentiviral vector was stable
during the concentration by ultracentrifugation, the
supernatant-trans-lentiviral vector was concentrated by
ultracentrifugation (SW28, 23,000 rpm, 90 min., 4.degree. C.). As a
control supernatant-lentiviral vector was concentrated in parallel.
The titers for both were determined both before and after
concentration. Table 4 shows our results and indicates that the
trans-lentiviral vector is stable during concentration by
ultracentrifugation.
TABLE-US-00004 TABLE 4 Concentration of Trans-Lentiviral Vector
Titer (inf. units/ml .times. 10.sup.-5) Packaging Plasmid RT-IN
Plasmid HeLA IB3 pCMV.DELTA.R9 -- 54 31 pCMV.DELTA.R9-S.sup.RT-IN
Vpr-RT-IN 28 19
Example 21
Trans-Lentiviral Vector for CFTR Gene Transduction
[0091] Lentiviral-based vectors are attractive for use in the lung
due to their ability to transduce non-divided cells. This unique
characteristic may represent an important advantage of lentiviral
vectors for gene therapy of CF. A translentiviral vector was used
to deliver the CFTR gene into HeLa cells. The CFTR gene was cloned
into the pHR transduction plasmid, using SmaI and XhoI sites (FIG.
15). Trans-lentiviral and lentiviral (as control) vectors were
generated by transduction as described above, and used to transduce
HeLa cells grown on cover slips. Two days later the cells were
examined by immunofluorescence microscopy, using both polyclonal
(FIG. 16) and monoclonal antibodies (FIG. 17). The results show
CFTR expression and localization of CFTR on the cell surface.
Furthermore, the transduced HeLa cells examined by SPQ
(halide-sensitive fluorophore) showed restored CFTR function (FIG.
18).
[0092] The present invention demonstrated the capability of HIV-1
Vpr and HIV-2 Vpx to direct the packaging of foreign proteins into
HIV virions when expressed as heterologous fusion molecules. The
trans complementation experiments with HIV proviral DNA revealed
that Vpr1 and Vpx2 fusion proteins were also incorporated into
replication-competent viruses. Moreover, packaging of the fusion
proteins in the presence of wild-type Vpx and/or Vpr proteins
(FIGS. 16 and 17) indicated that the viral signals mediating their
packaging were not obstructed by the foreign components of the
fission molecules. Likewise, virion-associated SN and CAT fusion
proteins remained enzymatically active.
[0093] Based on the immunoblot analysis of VLPs and virions, the
present invention illustrates that both virion associated CAT and
SN/SN* are susceptible to cleave by the viral protease. There
appears to be at least one cleavage site in CAT and two cleavage
sites in the SN/SN* proteins. Based on calculated molecular weights
of the major SN/SN* cleavage products, it appears that SN and SN*
are cleaved one near their C termini and once near the fusion
protein junctions. Since the fusion protein junctions of Vpr1SN and
Vpx2SN are not identical it is also possible that these regions
differ with respect to their susceptibility to the viral protease.
Although Vpx2SN/SN* were processed to a lesser extent than Vpr1SN
(FIGS. 7 and 8), the major cleavage sites appear to be conserved.
There is no doubt that both the HIV-1 and HIV-2 proteases recognize
processing sites in the fusion partners and that there is
sufficient physical contact to enable cleavage. This is evidenced
both by the reduction of cleavage product intensities on
immunoblots as well as by an increased enzymatic activity in the
presence of an HIV protease inhibitor.
[0094] The demonstration that Vpr1 and Vpx2 fusion proteins are
capable of associating with both VLPs and virions facilitates
studies on these accessory proteins and on HIV assembly in general.
The approach of generating deletion mutants to study protein
structure/function relationships is often of limited value since
this can reduce protein stability or change the three-dimensional
structure of the protein. In the case of Vpr, a single amino acid
substitution at residue 76 has been shown to destabilize its
expression in infected cells. Studies have indicated that deletion
mutations in vpr and vpx result in premature degradation of the
proteins following expression. Fusion of Vpr and Vpx mutant
proteins with, e.g., SN or CAT as demonstrated by the present
invention, increase stability.
[0095] The successful packaging of Vpr1/Vpx2SN fusion proteins into
virions indicates their use for accessory protein targeted viral
inactivation. The present invention demonstrates that Vpr and Vpx
may serve as vehicles for specific targeting of virus inhibitory
molecules, including SN. In contrast to HIV Gag, Vpr and Vpx are
small proteins that can be manipulated relatively easily without
altering virus replication and thus may represent vehicles with
considerable versatility for application to such an antiviral
strategy.
Example 22
Incorporation of RT in trans into a Lentivirus Independent of HIV
Accessory Proteins
[0096] The HIV accessory proteins, Vpr and Vpx, are incorporated
into virions through specific interactions with the p6 portion of
the Pr55.sup.Gag precursor protein (Kappes et al. 1993; Kondo et
al. (1995) J. Virol. 69:2759-2764; Lu et al. (1995) J. Virol.
69:6873-6879; Paxton et al. (1993) J. Virol. 67:7229-7237; Wu et
al. (1994) J. Virol. 68:6161-6169). Similarly, it has been
demonstrated that Vpr and Vpx fusion proteins (Vpr- and Vpx-SN and
CAT) are incorporated into virions through interactions with
p6.sup.Gag, similar to that of the wild-type Vpr and Vpx proteins
(Wu et al. (1995) J. Virol. 69:3389-3398). To analyze the
contribution of Vpr for incorporation of the Vpr-RT fusion protein
into virions, an HIV-1 proviral clone mutated in p6.sup.Gag and PR
(designated pNL43-.DELTA. p6.sup.Gag, provided by Dr. Mingjun
Huang) was cotransfected with pLR2P-vprRT into 293T cells. This
mutant contains a TAA translational stop colon at the first amino
acid residue position of p6.sup.Gag. This abrogated the Gag
sequences that are required for Vpr virion incorporation. The
pNL43-.DELTA. p6.sup.Gag clone also contains a mutation (D25N) in
the active site of PR, which enhances the release of the p6.sup.Gag
mutant virus from the cell surface membrane (Gottlinger et al.
1991; Huang et al. 1995). As a control, the HIV-1 PR mutant PM3
(Kohl et al. 1988), derived from the same pNL4-3 parental clone,
was also included for analysis. Progeny virions, purified from
pNL43-.DELTA. p6.sup.Gag transfected cell cultures, contained
detectable amounts of RT protein (labeled as Vpr-p66), albeit in
lesser amounts compared with virions derived from PM3 (FIG. 19).
Analysis of cell lysates confirmed expression, and compared with
PM3, the accumulation of Vpr-RT in pLR2P-vprRT/pNL43-.DELTA.
p6.sup.Gag cotransfected cells. Vpr.sup.S-RT was included as an
additional control and was shown to incorporate Vpr efficiently
into PM3 virions but not into those derived by coexpression with
pNL43-.DELTA. p6.sup.Gag. Wild-type Vpr protein was also absent
from .DELTA. p6.sup.Gag virions. Approximately equal amounts of Gag
protein was detected in the different virus pellets, confirming
that similar amounts of the different virions were compared in the
quantitation. These results show that RT protein can be
incorporated into virions independently of Vpr-p6 mediated
interaction. These data also indicate that expression of RT (and IN
by inference) in trans, independently of Gag-Pol, is sufficient for
its incorporation and function.
Example 23
Expression of RT in trans in a Lentivirus Vector Independent of HIV
Accessory
[0097] It has been demonstrated that functional RT can be
incorporated into HIV-1 virions by its expression in trans, even
without fusion to Vpr (Example 19). To determine if RT expressed in
trans can package into lentiviral vector and support the
transduction of a marker gene RT was ligated into the pLR2P
expression plasmid under control of the HIV LTR and RRE, generating
the pLR2P-RT expression plasmid. The pLR2P-RT, pHR-CMV-VSV-G,
pHR-CMV-.beta.-gal, and p.DELTA.8.2-RT.sup.D185N was transfected
together into 293T cells. The p.DELTA.8.2-RT.sup.D185N plasmid
contains a point mutation in RT at amino acid residue position 185
(D185N), which abolishes polymerase activity and destroys its
ability to support gene transduction. As a control Vpr-RT
(pLR2P-vpr-RT) was substituted for pLR2P-RT in a parallel
experiment. As another control neither RT or Vpr-RT were provided.
Virions generated by transfection were used to infect HeLa cells.
Two days later, transduction positive cells were counted. FIG. 20
shows that both Vpr-RT and RT support vector transduction when
provided in trans. The vector titer was reduced by about 10-fold
when RT was provided without fusion with Vpr. These results
demonstrate that enzymatic function (RT and IN) can be provided in
trans, independently of Gag-Pol.
[0098] The present invention demonstrated that Vpr and Vpx can
serve as vehicles to deliver functionally active enzymes to the HIV
virion, including those that may exert an antiviral activity such
as SN. The present invention has demonstrated that the concept of
accessory protein targeted virus inactivation is feasible.
[0099] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. These patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0100] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The present examples along with the methods, procedures,
treatments, molecules, and specific compounds described herein are
presently representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention as
defined by the scope of the claims
Sequence CWU 1
1
18127DNAArtificial Sequenceoligonucleotide primers 1gccacctttg
tcgactgtta aaaaact 27224DNAArtificial Sequenceoligonucleotide
primers 2gtcctaggca agcttcctgg atgc 24325DNAArtificial
Sequenceoligonucleotide primers 3aaggagagcc atgggtgcga gagcg
25426DNAArtificial Sequenceoligonucleotide primers 4ggggatccct
ttattgtgac gagggg 26525DNAArtificial Sequenceoligonucleotide
primers 5attgtgggcc atgggcgcga gaaac 25624DNAArtificial
Sequenceoligonucleotide primers 6ggggggcccc tactggtctt ttcc
24728DNAArtificial Sequenceoligonucleotide primers 7gaagatctac
catggaagcc ccagaaga 28839DNAArtificial Sequenceoligonucleotide
primers 8cgcggatccg ttaacatcta ctggctccat ttcttgctc
39925DNAArtificial Sequenceoligonucleotide primers 9gtgcaacacc
atggcaggcc ccaga 251039DNAArtificial Sequenceoligonucleotide
primers 10tgcactgcag gaagatctta gacctggagg gggaggagg
391117DNAArtificial Sequencefusion junctions of the pTM-vpr1SN/SN*
plasmid 11agtagatgtt gggatcc 17125PRTArtificial Sequencefusion
junctions of the pTM-vpr1SN/SN* plasmid 12Ser Arg Cys Trp Asp1
51329DNAArtificial Sequencefusion junctions of the pTM-vpx2SN/SN*
plasmid 13ctaagatcgg ggagctcact agtggatcc 29149PRTArtificial
Sequencefusion junctions of the pTM-vpx2SN/SN* plasmid 14Leu Arg
Ser Gly Ser Ser Leu Val Asp1 51521DNAArtificial Sequencefusion
junctions of the pLR2P-vpr1CAT plasmid 15agtagatgtt gggatctaat g
21167PRTArtificial Sequencefusion junctions of the pLR2P-vpr1CAT
plasmid 16Ser Arg Cys Trp Asp Leu Met1 51733DNAArtificial
Sequencejunctions of the pLR2P-vpx2CAT plasmid 17ctaagatcgg
ggagctcact agtggatcta atg 331811PRTArtificial Sequencefusion
junctions of the pLR2P-vpx2CAT plasmid 18Leu Arg Ser Gly Ser Ser
Leu Val Asp Leu Met1 5 10
* * * * *