U.S. patent application number 10/392503 was filed with the patent office on 2003-09-04 for fusion protein delivery system and uses thereof.
This patent application is currently assigned to University of Alabama Research Foundation. Invention is credited to Kappes, John C., Wu, Xiaoyun.
Application Number | 20030166563 10/392503 |
Document ID | / |
Family ID | 23672889 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166563 |
Kind Code |
A1 |
Kappes, John C. ; et
al. |
September 4, 2003 |
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) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
University of Alabama Research
Foundation
|
Family ID: |
23672889 |
Appl. No.: |
10/392503 |
Filed: |
March 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10392503 |
Mar 20, 2003 |
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10092929 |
Mar 7, 2002 |
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10092929 |
Mar 7, 2002 |
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09434641 |
Nov 5, 1999 |
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09434641 |
Nov 5, 1999 |
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08947516 |
Sep 29, 1997 |
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6001985 |
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08947516 |
Sep 29, 1997 |
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08421982 |
Apr 14, 1995 |
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Current U.S.
Class: |
536/23.4 ;
514/3.8; 530/350; 536/23.72 |
Current CPC
Class: |
A61K 47/645 20170801;
A61K 38/00 20130101; C07K 2319/00 20130101; C07K 14/005 20130101;
C12N 2740/16322 20130101 |
Class at
Publication: |
514/12 ; 530/350;
536/23.72 |
International
Class: |
A61K 038/16; C07H
021/04; C07K 014/16 |
Claims
That which is claimed:
1. A fusion protein comprising: a first polypeptide sequence
corresponding to a portion of a protein functioning in replication
of a virus; and a second polypeptide sequence corresponding to a
virus inhibitory protein fragment, said fragment being sufficient
to inhibit replication of said virus.
2. A fusion protein comprising: a first polypeptide sequence
corresponding to a fragment of a viral protein; and a second
polypeptide sequence differing from said first sequence and
corresponding to a protein fragment, said fragment coupled to said
first sequence.
3. The fusion protein of claim 2, wherein said protein is delivered
to a virus.
4. The fusion protein of claim 2, wherein said protein is a
Vpr.
5. The fusion protein of claim 2, wherein said portion is a full
length amino acid sequence of said protein.
6. The fusion protein of claim 2, wherein said protein is Vpx.
7. The fusion protein of claim 3, wherein said virus is selected
from the group consisting of virions and virus-like particles,
particles containing at least one viral protein, retrovirus,
lentivirus, HIV, and SIV.
8. The fusion protein of claim 2, wherein said second polypeptide
is selected from the group consisting of a staphylococcal nuclease,
chloramphenicol acetyl transferase, protease, integrase, reverse
transcriptase, Vif, Nef and Gag.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S.
application Ser. No. 10/092,929 filed Mar. 7, 2002, which is
continuation of U.S. application Ser. No. 09/434,641, filed Nov. 5,
1999, which is a divisional application of U.S. application Ser.
No. 08/947,516, filed Sep. 29, 1997, now U.S. Pat. No. 6,001,985,
which is a file-wrapper continuation of patent application Ser. No.
08/421,982, filed Apr. 14, 1995, now abandoned; each of which is
herein incorporated 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) Vpx and Vpr 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.
BACKGROUND OF THE INVENTION
[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. Using the yeast
retrotransposon Tyl as a retrovirus assembly model, Natsoulis and
Boeke tested this approach as a novel means to interfere with viral
replication. 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.
[0005] The prior art lacks 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.
BRIEF SUMMARY OF THE INVENTION
[0006] Vpr and Vpx packaging is mediated by the Gag precursor and
thus must play an important role in HIV assembly processes. The
present invention shows that Vpr and Vpx can be used as vehicles to
target foreign proteins to HIV/SIV virions. 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.
[0007] 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 the 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 fusions remain enzymatically active. Thus,
targeting heterologous Vpr and Vpx fusion proteins, including
deleterious enzymes, to virions represents a new avenue toward
anti-HIV drug discovery.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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
[0013] 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 form 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.
[0014] FIG. 1 shows the construction of vpr1, vpr1SN/SN*, vpx2 and
vpx2SN/SN* expression plasmids. FIG. 1A shows the illustration of
the pTMvpr1 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-2ST vpx coding region was
amplified by PCR and ligated into pTM1 at the NcoI and BglIISmal
sites. FIG. 1C shows the illustration of the fusion junctions of
the pTM-vpr1SN/SN* expression plasmids. 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.
BamHI/XhoI DNA fragments containing SN and SN* were ligated into
BamHI/XhoI cut pTMvpx2. 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 pTMI polylinker (double underlined)
between the two coding regions.
[0015] FIG. 2 shows the expression of Vpr1- and VPX2-SN and SN*
fusion proteins in mammalian cells. FIG. 2A shows the pTM1,
pTM-vpr1, pTMvpr1SN and pTM-vpr1SN* 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, pTMvpx2SN 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.
[0016] 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-vpr1SN* 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
ultracentrifaged (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, pTMvpx2SN 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 blotted 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.
[0017] 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 Vprl SN. rVT7 infected (T7
expressing) HeLa cells were transfected with pTM-vprl SN alone and
in combination with pTM-gag2 and pTM-gagl. Pellets (VLPs, middle
and bottom panels) and cells (top panel) were prepared 24 hours
after transfection and examined by immunoblot analysis using
anti-Vprl (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 pTMvpx2SN alone and in combination
with pTM-gagl 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.
[0018] 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-vprl (2.5, 5
and 10 .mu.g) and pTM-vprl SN (2.5, 5 and 10 .mu.g), either
individually or together in combination with pTM-gag1 (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.
[0019] 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-gagl/pTM-vpr1SN*, 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 .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. Standards (A) were
prepared by dilution of purified staphylococcal nuclease (provided
by A. Mildvan) into cocktail buffer and assayed.
[0020] 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 pSXB1I/pTMvpx2SN*. 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 antiVpx (left) and anti-Gag
(right) antibodies.
[0021] FIG. 8 shows the incorporation of Vpr1SN into HIV-1 virions
by trans complementation. Culture supernatant virus from HLtat
cells transfected with pNL4-3 (HIV-1) and pNL4-3 R.sup.-(HIV-1 vpr
mutant) or cotransfected with pNL4-3/pLR2P-vpr1SN and
pNL4-3R.sup.-/pLR2P-vpr1SN was prepared for immunoblot analysis as
described above. Blots were probed with anti-SN (FIG. 8A), antiVprl
(FIG. 8B) and anti-Gag (FIG. 8C) antibodies.
[0022] FIG. 9 shows the inhibition of Vpr1/Vpx2-SN processing by an
HIV protease inhibitor. HIV-1 (pSG3) and HIV-2 (pSXBI) 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.
[0023] FIG. 10 shows the incorporation of enzymatically active
Vprl- 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. PCR amplified BamHI/XhoI DNA
fragments containing CAT were ligated into BglII/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 vprl/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 (HIV1-R), or cotransfected with pNL4-3/pLR2P-vpr1CAT and
pNL4-3R.sup.-/pLR2PVpr1 CAT was prepared as described above and
examined by immunoblot analysis. Replica blots were probed with
anti-Vprl (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 pSXBI/pLR2P-vpx2CAT was prepared as described above and
examined by immunoblot analysis. Replica blots were probed with
anti-Vpx2 (left) and antiGag (right) antibodies. FIG. IOD shows
that virion incorporated Vprl- and Vpx2-CAT fusion proteins possess
enzymatic activity. Viruses pelleted from HLtat cells transfected
with pSXB1 (HIV-2) or cotransfected with pSXB1/pLR2Pvpx2CAT and
pNL4-3/pLR2P-vpr1CAT were lysed and analyzed for CAT activity.
HIV-2 was included as a negative control.
[0024] 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-vprl SN 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.
DETAILED DESCRIPTION OF THE INVENTION
[0025] As used herein, the term "fusion protein" refers to either
the entire native protein amino acid sequence of Vpx (of any HIV-2
and SIV) and Vpr (of any HIV1 and SIV) 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 virus like particles.
[0026] As used herein, the term "virion" refers to HIV-1, HIV-2 and
SIV virus particles. As used herein, the term "virus-like particle"
refers to any composition of HIV-1, HIV-2 and SIV proteins other
than which exists naturally in naturally infected individuals or
monkey species that are capable of assembly and release from either
natural or immortalized cells that express these proteins.
[0027] As used herein, the term "transfect" refers to the
introduction of nucleic acids (either DNA or RNA) into eukaryotic
or prokaryotic cells or organisms. As used herein, the term
"virus-inhibitory protein" refers to any sequence of amino acids
that have been fused with Vpx or Vpr sequences that may alter in
any way the ability of HIV-1, HIV-2 or SIV viruses 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, Nef and Gag 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/SUV protease, integrase, reverse
transcriptase, Vif, Nef and Gag proteins), or any other non viral
protein that, when expressed as a fusion protein with Vpx or Vpr,
alter virus multiplication and spread in vitro or in vivo.
[0028] 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 (Vprl) 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 Vpr1 SN/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 VprlSN 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.
[0029] Western blot analysis of sucrose gradient purified virions
revealed that both Vprl 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.
[0030] 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
[0031] HeLa, HeLa-tat (HLtat) and CV-1 cells were maintained in
Dulbecco's Modified Eagle's Medium supplemented with 10% fetal
bovine serum (FBS). 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., J. Virol. 66:7104-7112
(1992). HIV-1.sub.YU2, HIV-1 pNL 4-3-R and pNL 4-3,
HIV-1.sub.HXB2D, HIV-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
[0032] To generate HIV-1 Vpr specific antibodies, the
HIV-1.sub.YU.sub..sup.-.sub.2 vpr open reading frame was amplified
by polymerase chain reaction (PCR) using primers (sense:
5'GCCACCTTTGTCGACTGTTAAAAAACT-3' SEQ ID No:1 and anti-sense:
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 (DH5.alpha.) 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., Gene 67:31-40
(1988). New Zealand White rabbits were immunized with 0.4 mg of
purified Vprl 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
[0033] A DNA fragment encompassing .sup.HIV-1HXB2D.sup.gag
(nucleotides 335-1837) was amplified by PCR using primers (sense:
5'AAGGAGAGCCATGGGTCGAGAGCG-3' SEQ ID NO: 3 and anti-sense:
5'GGGGATCCCTTTATTGTGACGAGGGG-3' SEQ ID NO: 4) containing NcoI and
BamHI restriction sites, respectively (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 'GGGGGGCCCCTACTGGTCTTTT- CC-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.
[0034] 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 'GAAGATCTACCATGGAAGCCCCAGAAGA-3' SEQ ID NO: 7 and anti-sense:
5'CGCGGATCCGTTAACATCTACTGGCTCCATTTCTTGCTC-3' SEQ ID NO: 8)
containing NcoI and HpaI/BamHI sites, respectively (underlined).
The reaction product was cut with NcoI and BamHII and ligated into
pTM1, generating a pTM-vpr1 (FIG. 12A). In order to fuse SN and SN*
in--frame with vprl, 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. 1C.
[0035] 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'TGCACTGCAGGAAGATCTTAGACCTGGAGGGGGAGGAGG-3' SEQ ID NO: 10)
containing NcoI and BglII sites, respectively (underlined). After
cleave 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. 1B). 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 Expression Plasmids
[0036] 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-2ROD, 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 vprl, 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 pLR2Pvpr1, pLR2P-vpr1 SN, 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 pLR2Pvpx2SN were removed and
substituted with a PCR amplified BglII/XhoI DNA fragment containing
CAT, generating pLR2P-vpr1CAT and pLR2P-vpx2CAT, respectively (FIG.
9A).
Example 5
Transfections
[0037] Transfections of proviral clones were performed in HLtat
cells using calcium phosphate DNA precipitation methods as
described by the manufacturer (Strategene). T7-based (PTMI)
expression constructs were transfected using Lipofectin (BioRad)
into rVT7 infected HeLa cells as described previously by Wu, et
al., J. Virol. 68:6161-6169 (1994). These methods were those
recommended by the manufacturer of the Lipofectin reagent.
Example 6
Western Immunoblot Analysis
[0038] Virions and virus-like particles (VLPs) were concentrated
from the supernatants of transfected or infected cells by
ultracentrigufation 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-HC1 (pH 6.8) 0.2% sodium dodecyl sulfate (SDS), 5%
2-mercaptoethanol, 10% glycerol], boiled and separated on 12.5%
polyacrylamide gels containing SDS. Following electrophoresis,
proteins were transferred to nitrocellulose (0.2 .mu.m; Schleicher
& Schuell) 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 7
SN Nuclease Activity Assay
[0039] Cells and viral pellets were resuspended in nuclease lysis
buffer (40 mM Tris-HCI, 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-HCI, pH 8.8, 10 mM CaC12, O.I 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 8
Expression of Vprl- and Vpx2-SN and SN* Fusion Proteins in
Mammalian Cells
[0040] 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-vprl SN/SN*, and pTM-vpx2SN/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
pTMI transfected cells included as controls. Thus, both SN and SN*
fusion proteins can be expressed in mammalian cells.
Example 9
Incorporation of Vprl- and Vpr2-SN/SN* Fusion Proteins into
Virus-Like Particles
[0041] 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 pTMvpr1SN*
were transfected into HeLa cells alone and in combination with the
HIV1 Gag expression plasmid, pTM-gag I. Twenty-four hours after
transfection, cell and VLP extracts were prepared and analyzed by
immunoblot analysis (FIG. 3A). Anti-Vpr antibody detected Vprl,
Vprl SN and Vprl SN* in cell lysates (top panel) and in pelleted
VLPs derived by coexpression with pTM-gag1 (middle panel). In the
absence of HIV-1 Gag expression, Vprl and VprlSN 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.
[0042] To demonstrate that Vpx2SN was similarly capable of
packaging into HIV2 VLPs, pTM-vpx2, pTM-vpx2SN and pTM-vpx2SN* were
transfected into HeLa cells alone and in combination with the HIV-2
Gag expression plasmid, pTMgag2. 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 Vpr1SN and Vpx2SN demonstrated
co-sedimentation of these fusion proteins with VLPs (data not
shown).
[0043] 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 HIV1 Gag
precursor for incorporation into HIV-1 VLPs. To show that the
association of Vpr1 SN and Vpx2SN with VLPs was not mediated by the
SN moiety, but was due to the Vpr and Vpx specific packaging
signals, pTM-vpr1SN and pTMvpx2SN 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-gagl. Similarly, Vpx2SN was detected in all
pTM-vpx2 transfected cells (FIG. 413, 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 Vpr1 SN and Vpx2SN
into VLPs requires interaction of the cognate Gag precursor
protein, just like native Vpr1 and Vpx2. While Vpr1 SN 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 Vpr1 SN and Vpx2SN
packaging was compared by competition analysis (FIG. 5). pTM-vpr1SN
and pTM-vpx2SN were cotransfected with pTM-gag1/pTM-vprl and
pTMgag2/pTMvpx2, respectively, using ratios that ranged from I:4 to
4:1 (FIG. 5A and FIG. 5B, left panels). For comparison, pTM-vpr1SN
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. 513, 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 10
Vpr1SN and Vpx2SN Fusion Proteins Possess Nuclease Activity
[0044] 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 Vpr1SN
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 VprlSN* and Vpx2SN* were included as negative
controls and dilutions of purified SN served as reference standards
(FIG. 6A). Both virion associated Vpr1 SN (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 11
Incorporation of Vpx2SN Fusion Protein into HIV-2 Virions
[0045] 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 HIV2 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-2ST
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. 713, 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.
[0046] To show packaging of Vpx2SN into HIV-2 virions, sucrose
gradient analysis was performed. Extracellular virus collected from
culture supematants of HLtat cells forty-eight hours after
cotransfection with pLR2P-vpx2SN and HIV2.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 fractions (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.
[0047] 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-2ST 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 antiGag (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 12
Incorporation of Vpr1SN into HIV-1 Virions
[0048] 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- (HIV-1-R) alone and in combination with PLR2P-vpr1SN were
concentrated by ultracentri fugation 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
alone. From expression in the rVT7 system, the full-length Vpr1 SN
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, Vpr1 SN 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).
[0049] To demonstrate more directly that cleavage of the Vprl- and
Vpx2-SN fusion proteins was mediated by the HIV protease, virus was
concentrated from pNL4-3-R-/pLR2P-vpr1 SN 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 p55gag (FIG. 9A).
Similarly, virions produced in the presence of L-689,502 also
contained greater amounts of the uncleaved species of Vpr1 SN and
Vpx2SN fusion proteins (FIG. 9B). Taken together, these results
demonstrate that Vprl- and Vpx2-SN fusion proteins are subject to
protease cleavage during or subsequent to virus assembly.
Example 13
Vprl-CAT and Vpr 2-CAT Fusion Protein Incorporation into HIV
Virions
[0050] 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-vpr1CAT and pLR2P-vpxCAT (FIG. 10A). pNL4-3/pLR2P-vpr1CAT,
pn14-3-R.sup.-/pLR2P-vpr1C- AT 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-vpr1CAT with both pNL4-3 and
pNL4-3-R.sup.- (FIG. 10B). This size is consistent with the
predicted molecular weight of the full-length Vpr1CAT 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-2ST and
pRL2P-vpx2CAT, in which anti-Vpx antibody detected 41 and 15 kDa
proteins (FIG. 10C). These results demonstrate that Vpr1CAT 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.
Example 14
Virion Incorporated SN and CAT Fusion Proteins are Enzymatically
Active
[0051] The ability of Vpr1 and Vpx2 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. 11A) 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.
[0052] 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
VprNpx fusion proteins, although cleavage by the viral protease may
inactivate the fusion partners.
[0053] The present invention demonstrated the capability of HIV1
Vpr and HIV2 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. 5, 7 and 8) indicated that the viral signals mediating their
packaging were not obstructed by the foreign components of the
fusion molecules. Likewise, virionassociated SN and CAT fusion
proteins remained enzymatically active.
[0054] 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 once 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 Vpr1 SN
(FIGS. 7 an 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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
10 1 27 DNA HIV-1 YU-2 vpr Primers 1 gccacctttg tcgactgtta aaaaact
27 2 24 DNA HIV-1 YU-2 vpr Primers 2 gtcctaggca agcttcctgg atgc 24
3 25 DNA HIV-1 HXB2Dgag Primers 3 aaggagagcc atgggtgcga gagcg 25 4
26 DNA HIV-1 HXB2Dgag Primers 4 ggggatccct ttattgtgac gagggg 26 5
25 DNA HIV-2ST gag Primers 5 attgtgggcc atgggcgcga gaaac 25 6 24
DNA HIV-2ST gag Primers 6 ggggggcccc tactggtctt ttcc 24 7 28 DNA
HIV-1 YU2 vpr Primers 7 gaagatctac catggaagcc ccagaaga 28 8 39 DNA
HIV-1 YU2 vpr Primers 8 cgcggatccg ttaacatcta ctggctccat ttcttgctc
39 9 25 DNA HIV-2ST vpx Primers 9 gtgcaacacc atggcaggcc ccaga 25 10
39 DNA HIV-2ST vpx Primers 10 tgcactgcag gaagatctta gacctggagg
gggaggagg 39
* * * * *