U.S. patent application number 09/817413 was filed with the patent office on 2002-05-02 for chimeric viral proteins.
Invention is credited to Koprowski, Hilary, Srinivisan, Alagarsamy.
Application Number | 20020052478 09/817413 |
Document ID | / |
Family ID | 21926890 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020052478 |
Kind Code |
A1 |
Srinivisan, Alagarsamy ; et
al. |
May 2, 2002 |
Chimeric viral proteins
Abstract
Chimeric viral proteins and nucleic acid constructs that code
for them and are useful as therapeutic agents are disclosed.
Inventors: |
Srinivisan, Alagarsamy;
(Glen Mills, PA) ; Koprowski, Hilary; (Wynnewood,
PA) |
Correspondence
Address: |
REED SMITH SHAW & MC CLAY LLP
2500 ONE LIBERTY PLACE
PHILADELPHIA
PA
19103
US
|
Family ID: |
21926890 |
Appl. No.: |
09/817413 |
Filed: |
March 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09817413 |
Mar 26, 2001 |
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09053941 |
Apr 2, 1998 |
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6271354 |
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60043380 |
Apr 3, 1997 |
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Current U.S.
Class: |
530/387.3 ;
530/350; 536/23.4; 536/23.72 |
Current CPC
Class: |
C07K 14/005 20130101;
A61K 48/00 20130101; A61K 39/00 20130101; C07K 2319/00 20130101;
C12N 2740/16322 20130101 |
Class at
Publication: |
530/387.3 ;
530/350; 536/23.72; 514/44; 536/23.4 |
International
Class: |
A61K 048/00; C07H
021/04; C07K 014/005; A01N 043/04; C12P 021/08 |
Claims
1. A nucleic acid construct comprising a nucleotide sequence coding
for a chimeric viral protein comprising: (a) a protein of a virus;
and (b) a polypeptide of said virus, said polypeptide joined by a
peptide linkage to said viral protein in said chimeric protein,
said polypeptide not normally joined by said peptide linkage to
said protein in said virus or in cells infected by said virus.
2. The nucleic acid construct of claim 1 that is a DNA
construct.
3. The nucleic acid construct of claim 1 comprising a nucleotide
sequence coding for: (a) a protein of a virus, said protein not
comprising a site for cleavage by a proteolytic enzyme of said
virus; (b) a polypeptide proteolytic cleavage site of said virus,
said cleavage site being a site for cleavage by a proteolytic
enzyme of said virus; such that said protein is covalently linked
by a peptide linkage to said polypeptide proteolytic cleavage
site.
4. The nucleic acid construct of claim 3, wherein the virus is an
animal virus or a human virus.
5. The nucleic acid construct of claim 4, wherein the virus is a
human virus.
6. The nucleic acid construct of claim 5 wherein the virus is
selected from the group consisting of: herpes simplex virus type I,
herpes simplex virus type II, human cytomegalovirus, human herpes
virus type, and human immunodeficiency virus (HIV).
7. The nucleic acid construct of claim 6 wherein the virus is human
immunodeficiency virus (HIV).
8. The nucleic acid construct of claim 7, wherein the protein of
the virus is not a capsid protein of the virus.
9. The nucleic acid construct of claim 8, wherein the viral protein
is the vpr protein.
10. The nucleic acid construct of claim 8, wherein the polypeptide
proteolytic cleavage site corresponds to an amino acid sequence
found in the Gag or Gag-Pol precursor proteins of HIV.
11. The nucleic acid construct of claim 1 comprising a nucleotide
sequence coding for: (a) a protein of a virus, said first protein
not being a protein that forms a dimeric proteolytic enzyme of said
virus; (b) a dimer interface polypeptide sequence of an enzyme of
said virus, said sequence being one by which monomers of said
enzyme combine to form the active dimeric enzyme, such that said
first protein is covalently linked by a peptide linkage to said
dimer interface polypeptide sequence.
12. The nucleic acid construct of claim 11, wherein the enzyme is
selected from the group consisting of: protease, DNA polymerase and
ribonucleotide reductase.
13. The nucleic acid construct of claim 12, wherein the dimer
interface polypeptide sequence is that of the HIV protease.
14. A nucleic acid construct comprising a nucleotide sequence
coding for a chimeric viral protein comprising: (a) a non-capsid
protein of a virus, the non-capsid protein not comprising a
polypeptide proteolytic cleavage site for cleavage by a proteolytic
enzyme; (b) a polypeptide of the virus, the polypeptide joined by a
peptide linkage to the viral protein in said chimeric protein,
wherein the polypeptide is not normally joined by the peptide
linkage to the non-capsid protein in said virus or in cells
infected by the virus.
15. The nucleic acid construct of claim 13 wherein the polypeptide
has the cleavage site for cleavage by the proteolytic enzyme.
16. The nucleic acid construct of claim 14 wherein the cleavage
site being a site for cleavage by the proteolytic enzyme of the
virus.
17. The nucleic acid construct of claim 13 wherein the virus is an
animal virus or a human virus.
18. The nucleic acid construct of claim 16 wherein the virus is a
human virus.
19. The nucleic acid construct of claim 17 wherein the virus is
selected from the group consisting of: herpes simplex virus type I,
herpes simplex virus type II, human cytomegalovirus, human herpes
virus type, and human immunodeficiency virus (HIV).
20. The nucleic acid construct of claim 18 wherein the virus is
human immunodeficiency virus (HIV).
21. The nucleic acid construct of claim 19 wherein the polypeptide
proteolytic cleavage site corresponds to an amino acid sequence
found in the Gag or Gag-Pol precursor proteins of HIV.
22. A chimeric viral protein of claim 20, wherein the non-capsid
protein is the vpr protein.
23. A process of interfering with the growth of a virus in an
animal or human, said process comprising administering to the
animal or human a nucleic acid construct of claim 1.
24. The process of claim 23 wherein the animal or human has not
been infected with the virus.
25. The process of claim 23 wherein the animal or human has been
infected with the virus.
26. The process of claim 23 wherein the construct is administered
so as to become integrated into a cell of said animal or human.
27. The process of claim 26 wherein the cell is selected from the
group consisting of a bone marrow cell and a blood cell.
28. The process of claim 27 wherein the cell is a lymphocyte.
29. The process of interfering with the growth of a virus in an
animal or human, said process comprising administering to the
animal or human a nucleic acid construct of claim 14.
Description
[0001] This application is a division of U.S. Application No.
09/053,941 filed Apr. 2, 1998 which claims the benefit of U.S.
Provisional Application No. 60/043,380, filed Apr. 3, 1997, which
application is incorporated herein by reference.
[0002] This work was supported by funds from the National
Institutes of Health (AI29306). It was also supported by a grant
from the Commonwealth of Pennsylvania to the Biotechnology
Foundation, Inc., and a grant from the Biomedical Research Support
Committee and institutional funds.
BACKGROUND
[0003] The field of the invention is anti-viral agents.
[0004] Effective antiviral agents will be of great value in
controlling virus replication and delaying the onset of
HIV-1-related disease symptoms. Current therapy involves the use of
antiviral agents that target the enzymatic functions of the virus,
resulting in the emergence of resistant viruses to these agents,
thus lowering effectiveness.
[0005] HIV-1 is a member of the lentivirus family of retroviruses.
Upon infection with the virus, individuals exhibit a variable onset
of AIDS-related diseases (Levy, 1993.). Recent studies have shown
that even in the midst of the clinically latent period, there is no
virological latency in the infected individuals (Pantaleo et al.,
1993; Embretson et al., 1993). Given this scenario, it has been
suggested that inhibitors of virus replication will be of immense
value in the onset and control of AIDS-related diseases in persons
infected with HIV-1 (Ridky et al., 1995; Miller et al., 1996;
Mellors et al., 1996; Crowe et al., 1996).
[0006] The HIV-1 life cycle shares several features common to all
retroviruses. These features include virus attachment to a specific
receptor, penetration, uncoating, reverse transcription,
translocation of viral DNA from the cytoplasm to the nucleus,
integration, expression of viral proteins, assembly, and maturation
of virus particles (1). Virally encoded enzymatic activities, which
are essential for the processes associated with virus infection,
have all been used as targets for developing agents that interfere
with the virus replication (2-4). Unfortunately, the use of such
antiviral agents has also resulted in the emergence of
drug-resistant viruses (5-7). In comparison to the monotherapy,
combination therapy involving multiple inhibitors has been shown to
be effective (8-11). The emergence of drug-resistant viruses,
however, will remain a problem with the continued use of antiviral
agents to target viral enzymes. Hence, alternative strategies to
contain HIV-1 replication are warranted. Toward this goal, an
approach to generate a novel anti-HIV-1 agent from within the virus
has been considered.
[0007] Unlike the simple retroviruses, the HIV-1 genome contains
six auxiliary genes in addition to the gag, pol and env common to
all retroviruses. The processes associated with virus infection are
carried out by different viral gene products, which makes these
proteins potential targets for antiretroviral therapy. These
processes include: a) binding to a receptor and virus
internalization, b) reverse transcription and transport of viral
DNA to the nucleus for integration, c) expression of viral proteins
and d) assembly and releases of viral particles from infected cells
(Levy, 1993).
[0008] Among the auxiliary gene products of HIV-1, vpr, vif, and
nef have been shown to be associated with virus particles to a
varying extent (12-16). The virion-associated protein Vpr has been
an intensive area of interest with respect to understanding the
role of Vpr in virus infection. Vpr coding sequences (96 aa) are
found to overlap Vif at the 5' end and Tat at the 3' end (17).
Characteristic features of Vpr include virion incorporation, cell
cycle arrest at the G.sub.2 stage, nuclear localization,
participation in transport of the prointegration complex,
demonstration of cation channel activity, and interaction with
several candidate cellular proteins (18-28). Additionally, work
from our laboratory and others has shown that Vpr is essential for
optimum infection of macrophages (29, 30). Mutational analysis of
Vpr has revealed the presence of critical domains needed for its
virion incorporation and the importance of the predicted helical
domain (amino acids 17-34) in such an event (22, 31-38). The virion
specificity and abundance of Vpr in viral particles provide avenues
for localizing antiviral agents to progeny virus, giving the
ability to interfere with the assembly, maturation, and infectivity
of HIV-1.
[0009] Upon initial synthesis as a polyprotein precursor, the HIV-1
aspartyl protease has the unique ability to autocatalyze its own
cleavage from the Pr160 polyprotein precursor. After its release,
the protease is then able to catalyze the cleavage at other sites
generating the mature Gag protein, p17, p24, p7, and p6 and the
reverse transcriptase (RT) and integrase enzymes. The specific
cleavage sites between the proteins in the polyprotein precursor
recognized by HIV-1 protease are highly conserved among viral
isolates (39).
[0010] In the present invention, in order to generate an effective
anti-HIV-1 agent from within the virus, we have combined the
protease cleavage site residues found in the Gag and Gag-Pol
precursor proteins and the virion-specific feature of Vpr. The
rationale for this approach is that an inappropriate presentation
of chimeric Vpr (Vpr-C) proteins containing protease cleavage
signal sequences might interfere with the processing of bona fide
viral precursor polyproteins leading to the generation of
incompletely processed noninfectious virus particles (2). The
differential amount of Gag, Gag-Pol, and Vpr proteins present in
the virus particles supports the feasibility of such an approach.
The results demonstrate that the above strategy is effective in
interfering with HIV-1 virus replication.
[0011] As regards a second aspect of the invention, it is relevant
that HIV-1 PR is an obligatory homodimer, with the active site made
from two monomers containing DTG amino acid residues brought into
proximity through regions in the NH.sub.2 and COOH termini of the
PR monomers, known as the dimer interface (Babe et al., 1991). The
dimer interface region of ribonucleotide reductase and DNA
polymerase from herpes simplex virus and HIV-1 RT have been
investigated as potential targets for developing antiviral agents
(Dutia et al., 1986; Cohen et al., 1986; Divita et al., 1994;
Digard et al., 1995). It has been suggested that peptides
corresponding to the dimer interface structure between the HIV-1 PR
subunits may interfere with the generation of an enzymatically
active molecule. It is estimated that the C-terminal 4 residues of
PR contribute up to 50% of the intersubunit interaction in the
dimer (Weber, 1990). A synthetic peptide corresponding to this
domain showed dissociative inhibition of HIVI PR in vitro and a
weak inhibition of viral replication in cell cultures (Zhang et
al., 1991; Schramm et al., 1991; 1993; Babe et al., 1992).
[0012] The approach utilized in the second aspect of the invention
takes advantage of the dimerization feature of PR and localizes a
peptide corresponding to the dimer interface domain (The 4-amino
acid sequence, TLNF as represented by the standard single-letter
codes for amino acids) in the virus particle through chimeric Vpr.
Our concept involves the incorporation of chimeric Vpr into the
virus particle along with the Gag and Gag-Pol precursors where it
is likely to interfere with the formation of active PR. The lack of
availability of active PR will ultimately lead to the generation of
immature, non-infectious virus particles due to incomplete
processing of viral proteins. The data presented here shows that
the presence of chimeric Vpr in viral particles indeed resulted in
reduced levels of virus replication.
SUMMARY OF THE INVENTION
[0013] In a most general aspect, the invention is a chimeric viral
protein comprising: 1) a first protein of a virus; and 2) a
polypeptide of said virus, said polypeptide joined by a peptide
linkage to said first viral protein in said chimeric protein, said
polypeptide not normally joined by said peptide linkage to said
polypeptide in said virus or in cells infected by said virus.
[0014] In one aspect, the invention is a chimeric viral protein
comprising:l) a first protein of a virus, said first protein not
compromising a site for cleavage by a proteolytic enzyme of said
virus; 2) a polypeptide proteolytic cleavage site, of said virus,
said cleavage site being a site for cleavage by a proteolytic
enzyme of said virus such that said first protein is covalently
linked by a peptide linkage to said polypeptide proteolytic
cleavage site.
[0015] In a second aspect, the invention is a chimeric viral
protein comprising: 1) a first protein of a virus, said first
protein not being a protein that forms a dimeric proteolytic enzyme
of said virus; 2) a dimer interface polypeptide sequence of an
enzyme of said virus, said sequence being one by which monomers of
said enzyme combine to form the active dimeric enzyme, such that
said first protein is covalently linked by a peptide linkage to
said dimer interface polypeptide sequence.
[0016] Related aspects of the inventions are nucleic acid
constructs that code for the chimeric protein, and the process of
administering them as therapeutic agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1. Minus strand primers (5'-3') used to generate Vpr-C.
Primers include restriction site, stop codon (*), sequences
specific for the cleavage site (solid rectangle), and sequences
specific for the 3' end of Vpr (open rectangle).
[0018] FIG. 2. (A) Schematic representation of the Gag, and Gag-Pol
precursors of HIV-1 indicating protease cleavage sites 1-9 (bold).
The Vpr-C proteins contain the corresponding cleavage signal found
in the Gag and Gag-Pol precursors indicated by number and
abbreviation for the site, fused in-frame to the C terminus of Vpr
resulting in a protein of 106 aa.
[0019] Likewise, all Vpr-C constructs received a Flag epitope
(DYKDDDDK, Asp-Tyr-Asp-Asp-Asp-Asp-Tyr) immediately following the
cleavage site. The asterisk indicated the construct containing the
Flag epitope alone. The dashed line indicates site of ribosomal
frame shift. (B) Proviral clone pNL4-3 cleaved with EcoRI/Sal/I in
Vpr coding region to allow insertion of EcoRI/XhoI generated
fragment from Vpr-C (NLVRP-C). Vpr-C- derived fragments contain a
stop codon (*) so sequences downstream of SalI are out of frame.
NLVPR-C HYGRO contains an SV-Hygr cassette in the env gene for
selection of positive clones in single-round replication assay.
[0020] FIG. 3. RIPA analysis of in vitro transcribed and translated
Vpr-C proteins. Antiserum to Vpr was used as described. Vpr-C
proteins exhibit a shift in migration and appear around 18 kDa.
Designation of the Vpr constructs are described in legend to FIG.
2.
[0021] FIG. 4. Immunoblot analysis of HeLa cells transfected with
Vpr-C constructs. (A) Analysis of cell lysates showed that Vpr-C
and Gag proteins are produced and migrate to the expected 18-kDa
and 55-kDa positions, respectively. Observed on the original photos
of the gels, but not necessarily on reproductions of those photos,
were weak bands as follows: For each of 7/1-F, 1/6-F, TF/PR-F, a
single weak band between the band migrating between the 51.6 and 66
kD markers and the major band migrating roughly mid way between the
34.1 kD marker and the 51.6 marker.(B) Analysis of cell
supernatants revealed that the Vpr-C retains its ability to be
incorporated into virus-like particles. Observed on the original
photos of the gels, but not necessarily on reproductions of those
photos, were weak bands as follows: In the lane for 24/2-F, three
weak bands for proteins with molecular weights slightly less than
66 kD, migrating close to the 51.5 kD marker; In the lane for
7/1-F, a very weak band close to the 51.6 kD marker and a weak band
mid way between the 34.1 and 51.6 markers, and for TF/PR-F, 5 weak
bands ranging from about the 51.6 marker to close to the 34.1
marker.
[0022] FIG. 5. Schematic representation of protease monomer and
chimeric Vpr. (A) Structure of protease monomer (99 aa residues).
Amino acid residues corresponding to the dimer interface region of
active protease are indicated. (B) Structure of wild type (96 aa
residues) and chimeric Vpr. Vpr-PC1 (100 aa residues) contains 4
amino acids from the C-terminus of protease added to the coding
sequences of Vpr. Vpr-H-PC1 (105 aa residues) contains a flexible
hinge region in between the Vpr and protease coding sequences.
Theses constructs were generated in a plasmid vector designated
pCDNA3 with the coding sequences of the Vpr chimera flanked by Hind
III and Xho I restriction sites at the 5' and 3' end,
respectively.
[0023] FIG. 6. RIPA analysis of in vitro-translated Vpr, Vpr-PC1,
and Vpr-H-PC1 proteins. Antiserum against full-length Vpr was used.
Details of Vpr-PC1 and Vpr-H-PC1 are as described in FIG. 5. As
expected, the pCDNA3 vector alone did not show a band in the 14 kDa
range as was observed with the wild type Vpr plasmid.
[0024] FIG. 7. Incorporation of chimeric Vpr into virus-like
particles directed by HIV-1 Gag as described in materials and
methods. Analysis of cell supernatants of transfected HeLa cells
revealed that Vpr-PKA,Vpr-PC1 and Vpr-H-PC1 retained the
incorporation phenotype of wild-type Vpr. (lane 1), wt Vpr; (lane
2), Vpr-PKA (control); (lane 3), Vpr-PC1; and (lane 4),
Vpr-HPC1.
[0025] FIG. 8. Generation of HIV-1 proviral DNA containing Vpr-PCl
and VprH-PC1 sequences. Proviral clone-designated NL4-3 was cleaved
with EcoRI/Sal I in Vpr coding region to allow insertion of
EcoRI/Xho I from VprPCl or Vpr-H-PC1. Since the fragment generated
with EcoR I and Xho I from Vpr-PC1 and Vpr-H-PC1 carries a
termination codon following the coding sequences, the duplication
of sequences created in the proviral DNA is not translated. It is
also important to note that the strategy used here does not
interfere with the overlapping region of vpr and tat coding
sequences. NLVpr-PC1-HYGRO and NLVpr-H-PC1 HYGRO contain an
SV40-Hygr cassette in the env gene for selection of positive clones
in a single-round replication assay. The wild-type, Vpr-PC1 and
Vpr-H-PC1 containing proviral DNAs were cleaved with the Nhe I
restriction enzyme, and a Hygr cassette under the control of the
SV40 early promoter was introduced thus eliminating env gene
expression. FIG. 8 is FIG. 8A plus FIG. 8B.
[0026] FIG. 9. Electron micrographs of cells transfected with
NLVpr-H-PC1 constructs. A) Low power magnification showing an
abundance of viral buds (arrows) at the cell membrane. Bar, lum. B)
Enlarged area of (A) showing whole viral particles (Arrowheads)
within a membrane-bound vacuole. Note the viral buds (arrows). Bar,
100 nm. C,D) Mature viral particles attached to the cell membranes
(arrowheads). Bars, 100 nm.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations
[0027] Vpr-C, chimeric Vpr; RT, reverse transcriptase; RIPA,
radioimmunoprecipitation assay; SV40, simian virus 40;
SV-Hyg.sup.r, SV40 hygromycin gene cassette; MLV, murine leukemia
virus. Where amino acid sequences are denoted by both the single
letter codes for amino acids and the three letter codes for amino
acids, and there is a discrepancy between the two, the single
letter code is most likely the correct one.
Aspects of the Invention
[0028] In a most general aspect, the invention is a chimeric viral
protein having: 1) a first protein of a virus; 2) a polypeptide of
said virus, and 3) said polypeptide joined by a peptide linkage to
said first viral protein in said chimeric protein, said polypeptide
not normally joined by said peptide linkage to said polypeptide in
said virus or in cells infected by said virus.
[0029] The polypeptide is preferably not more than 40 amino acids
in length, most preferably not more than 10 amino acids in
length.
[0030] One aspect of the invention is a chimeric viral protein
having:1) a first protein of a virus, said first protein not
compromising a site for cleavage by a proteolytic enzyme of said
virus; 2) a polypeptide proteolytic cleavage site, of said virus,
said cleavage site being a site for cleavage by a proteolytic
enzyme of said virus; such that said first protein is covalently
linked by a peptide linkage to said polypeptide proteolytic
cleavage site.
[0031] In a particular aspect of the invention, the first protein
of the virus is not a capsid protein of the virus.
[0032] In another particular aspect, the polypeptide proteolytic
cleavage site corresponds to an amino acid sequence found in the
Gag or Gag-Pol proteins of HIV.
[0033] In another aspect of the invention is a chimeric viral
protein having: 1) a first protein of a virus, said first protein
not being a protein that forms a dimeric proteolytic enzyme of said
virus; 2) a dimer interface polypeptide sequence of an enzyme of
said virus, said sequence being one by which monomers of said
enzyme combine to form the active dimeric enzyme, such that said
first protein is covalently linked by a peptide linkage to said
dimer interface polypeptide sequence. For example, the enzyme can
be selected from the group, protease, DNA polymerase,
ribonucleotide reductase.
[0034] A peptide linkage may be a single peptide bond, a single
amino acid or a peptide (preferably less than 100 amino acids in
length). If the peptide linkage is an intervening amino acid, the
amino acid is joined by a single peptide bond to the first viral
protein and by a single peptide bond to the polypeptide proteolytic
cleavage site or the dimer interface polypeptide sequence. If the
peptide linkage is an intervening peptide, that intervening peptide
is joined by a single peptide bond to the first viral protein and
by a single peptide bond to the polypeptide proteolytic cleavage
site or the dimer interface polypeptide sequence.
[0035] The chimeric protein is constructed so that it is not
identical to a protein normally found in the virus or normally
created by the virus nucleic acid upon infection of cells.
"Normally" refers to the situation where the chimeric protein is
not the result of human intervention, such as the creation by a
human of a chimeric nucleic acid construct coding for the chimeric
protein.
[0036] The chimeric viral protein is preferably one wherein the
virus referred to is an animal virus or a human virus, more
preferably a human virus, most preferably selected from the group:
herpes simplex virus type I, herpes simplex virus type II, human
cytomegalovirus, human virus type, human immunodeficiency virus
(HIV). Another preferred group, one that partially overlaps the
preceding group, are retroviruses, most preferably
lentiviruuses.
[0037] In particular embodiments, a chimeric viral protein is one
wherein the first protein of the virus is not a capsid protein of
the virus.
[0038] A related invention is a nucleic acid construct comprising a
nucleotide sequence coding for an aforementioned chimeric viral
protein, especially a nucleic acid construct that is a DNA
construct.
[0039] Another related invention is the process of interfering with
the growth of a virus in an animal or human, said process
comprising administering to the animal or human a nucleic acid
construct comprising a necleic acid sequence coding for the
aforementioned chemeric protein. In one embodiment of the process,
the animal or human has not been infected with the virus. In
another embodiment, of the process, the animal or human has been
infected with the virus. For example, the construct is administered
so as to become integrated into a cell of an animal or human,
examples of cells being a bone marrow cell or blood cell, such as a
lymphocyte.
EXAMPLES
Example 1
Materials and Methods for Example 2 Plasmids
[0040] Cloning of wild-type and Vpr-C was carred out using the
pCDNA3 expression vector as described (31-35). DNA fragments were
amplified through PCR using the proviral clone pNL4-3 with primers
containing HindIII and XhoI at the 5' and 3' end of the Vpr coding
region, respectively. Sequences corresponding to the protease
cleavage site residues were added to frame (10 aa) to the C
terminus of Vpr coding sequences as part of the minus strand primer
(see FIGS. 1A and 2A). Sequences representing the Flag epitope (F)
were also added to the C terminus of Vpr. All recombinant plasmids
were verified by restriction enzyme cleavage DNA sequence
analysis.
[0041] Vpr-C fragments were prepared by excision from the
respective expression vector at the EcoRI and XhoI sites for
insertion into the proviral DNA, pNL4-3, cleaved at the unique
sites EcoRI and SalI (see FIG. 2B). This strategy does not
interfere with the overlap region of vif and tat.
In Vitro Transcription/Translation and RIA Analysis of Vpr-C
Proteins
[0042] The coupled T7 transcription/translation system (Promega)
was used for characterizing the expression of the Vpr-C clones.
Incubation conditions were followed according to manufacturer's
instructions.
[0043] Radioimmunoprecipitation assay (RIPA) analysis if in vitro
translated proteins was carried out using polyclonal antiserum to
Vpr as described (35).
Infection/Transfection, Metabolic Labeling, Immunoprecipitation,
and Western Blot Analysis
[0044] For expression studies, the recombinant vaccinia virus
vTF7-3 that expresses T7 RNA polymerase in infected cells was used.
HeLa cells at 10.sup.6 cells per 35-mm tissue culture dish were
first infected with the virus at a multiplicity of infection of 10
for 1 hr and subsequently transfected via the Lipofectin method
(Life Technologies, Gaithersburg, Md.) with the Vpr-C expression
vectors in conjunction with an HIV-1 Gag expression vectors for
virion incorporation analysis. Transfected cells were washed in PBS
and starved in DMEM (without sera, Met and Cys) for a total of 1
hr, followed by labeling with .sup.35S protein labeling mix (Du
Pont/NEN) at 200 .mu.Ci/ml (1.2 Ci/mmol; 1 Ci=37 GBq) for a total
of 5 hr. The culture medium, cleared of cellular debris by low
speed centrifugation, was subsequently centrifuged at 25,000 rpm
for 90 min, and virus-like-particles were suspended in RIPA buffer.
Cells were washed twice in PBS and lysed in RIPA buffer as above.
Immunoprecipitation analysis subsequently followed using polyclonal
HIV-1 Vpr, Gag, and Flag epitope antibodies, respectively.
Immunoprecipitated proteins were seperated on 15% SDS/PAGE and
immunoblot analysis was carried out as described (Santa Cruz
Biotechnology).
Generation of Virus upon Transfection of HIV-1 Proviral DNA and
Virus Infectivity Studies
[0045] Both wild-type-and Vpr-Ccontaining proviral DNA were
transfected into rhabdomayosarcoma cells as described (40). Virus
particles released into the culture medium were harvested 72-120 hr
posttransfection and quantitated by RT and p24 antigen assay (40).
The virus infectivity studies were carried out using established
CD4.sup.+ CEM cells as targets. Three million cells were incubated
with virus innoculum, normalized on the basis of RT activity or p24
antigen levels, for 2 hr at 37.degree. C. Infected cells were then
washed and resuspended in RPMI 1640 medium. Aliquots from infected
cultures were take once a week and split to keep the cell
concentration at one million cells/ml.
Single Cycle Replication Assay
[0046] Using a previously published strategy, proviral DNAs
containing Vpr-C were cleaved with the Nhel restriction
endonuclease, and a simian virus 40 (SV40) early promotor/enhancer
hygromycin gene cassette (SV-Hyg.sup.r) was inserted, leading to
the disruption of the env gene (41-43). pED84, which contains the
insertion of the SV-Hgy.sup.r cassette in the env gene of pNL4-3
was used as the control plasmid for transfection experiments and
contains a wild-type vpr gene (44). To generate virus particles
capable of only a single round of replication, cotransfection of
Cos cells was performed with the Vpr-C modified proviral clones
(NLVpr-C-HYGRO) and an amphotropic murine leukemia virus (A-MLV)
envelope glycoprotein (Env-gp) expression plasmid, pSV-A-MLV-env
(see FIG. 2B) (45).
[0047] Virus particles released into the medium were harvested 72
hr posttransfection and cleared of cellular debris by low speed
centrifugation. An aliquot was used to infect HeLa T4 cells. At 48
hr postinfection, selection of hygromycin resistance was initiated
with media containing 200 mg/ml hygromycin B. After 9-11 days,
hygromycin-resistant colonies were stained with 0.5% crystal violet
in 50% methanol and counted.
Example 2
Results
Construction of Vpr-C Containing HIV-1 Protease Cleavage Signal
Sequences
[0048] The structural proteins Gag and Gag-Pol of HIV-1 are
synthesized as precursor polyproteins and contain a total of 12
cleavage sites recognized by the virus-encoded protease allowing
for precursor processing and virus maturation (46). The specificity
of HIV-1 protease is found to lie in the detection and cleavage of
a scissile bond (Met-Met, Leu-Ala/Phe, Tyr-Pro, Phe-Pro/Tyr/Leu)
within the minimim context of four residues 5' (P1-P4) and three
residues 3' (P1'-P3') to the site of cleavage (2, 47-49). In this
study, nine major sites for generating Vpr-C were selected. Primers
that comprise sequences corresponding to the nine cleavage sites,
followed by a termination codon, were synthesized to allow for the
addition of sequences at the C terminus of Vpr by PCR (FIG. 1). In
addition, following each of the cleavage sites, and 8 aa Flag
epitope was added to aid in antibody detection of the chimeric
proteins (23). As a control, we have generated a Vpr-C containing
only a Flag epitope at the C terminus. The proviral clone pNL4-3
was used as the template for generating replication-competent
proviral clones containing the Vpr-C. The details of cleavage site
residues used for generating Vpr-C are presented in FIG. 2A. The
designation of Vpr-C is indicated by the abbreviation for the
respective protease cleavage site. The cleavage signals added onto
our Vpr-C proteins include the P5 and P5' residues specific to each
of the respective sites, Vpr-C clones were verified for sequence
integrity.
Expression and Virion Incorporation of Vpr-C
[0049] We used an in vitro T7 expression system to verify the
expression of each of the Vpr-C proteins. In vitro translated Vpr-C
proteins were immunoprecipitated with polyclonal Vpr antiserum. As
expected, each Vpr-C protein was expressed at levels equal to the
wild-type Vpr protein (FIG. 3). As reported earlier, the wild-type
Vpr migrates to 14 kDa (32). The different Vpr-C proteins displayed
altered mobility in comparison to wild-type Vpr. This may be due to
the added signal sequences containing highly acidic and hydrophobic
residues. The Vpr-C Flag protein (Vpr-F) with the Flag addition,
made mostly of acidic residues, also migrated differently than
wild-type Vpr (data not shown).
[0050] To verify that the Vpr-C proteins retain the ability to
incorporate into virus-like particles directed by HIV-1 Gag and to
monitor the expression of Vpr-C in cells, we employed a vaccinia
virus T7 RNA polymerase expression system (vTF7-3). vTF7-3-infected
HeLa cells were transfected with wild-type Vpr or Vpr-C expression
plasmids containing the Flag epitope (F) in combination with the
Gag expression vector pCDGag by the Lipofectin method. Immunoblot
analysis of Vpr-F, Vpr-C-F, and Gag was performed in both cells
lysates and culture media with anti-Flag and anti-Gag antiserum 24
hr posttransfection (FIG. 4). Results of the cell lysate showed the
presence of both Gag and Vpr (FIG. 4A). The culture medium
immunoblot indicated that the Vpr-C proteins are incorporated into
virus-like particles (FIG. 4B). As expected, the mock, control
plasmid pCDNA-3 and pCDVprA cells failed to show a corresponding
protein.
Effect of Vpr-C in HIV-1 Infection of CEM Cells
[0051] Upon characterization of Vpr-C constructs, the Vpr-C protein
coding sequences were introduced into HIV-1-NL43 proviral DNA
(NLVpr-C) (FIG. 2B). To avoid disruption of the overlap of vpr with
tat at 3' end in the pNLVpr-C construct, the unique EcoRI and Sal/I
restriction endonuclease cleavage sites were used to introduce the
3' end of chimeric Vpr from the recombinant plasmids.
[0052] The proviral DNA NL4-3 and NL-Vpr-C were transfected into
rhabdomayosarcoma cells to generate viruses for evaluating the
effect of Vpr-C at the level of viral replication. Virus released
into the medium was collected 5 days posttransfection and
quantitated by an RT assay (40). Equivalent RT activity infected
cells were monitored for virus replication for nearly 30 days.
Cultures infected with wild-type pNL4-3 showed peak virus
production at 15-21 days postinfection, as is generally observed
with a spreading infection. Similar results were noted using virus
derived from pNL4-3 Vpr-F (data not shown). The replication of the
virus particles derived from proviral DNA containing NL-Vpr-C was
altered in comparison to the control (Table 1). The chimeric
viruses exhibited a delayed kinetics of virus spread and very
little replication was evident for up to 14 days, suggesting the
presence of a mixture of infectious and noninfectious virus
particles. Strikingly, the NLVpr-24/2 virus showed no viral
replication for up to 28 days. In general, the infectivity assays
carried out in CEM cells represent a spreading infection and the
results generated may reflect a cumulative effect over multiple
rounds of infection.
Utilization of Single-Round Replication Assay to Evaluate the
Effect of Vpr-C
[0053] To precisley evaluate the effect of Vpr-C in a single cycle
of virus replication a single-round replication assay was
established as described (41-44). For this purpose, NL4-3 and
NL4-Vpr-C proviral DNA were cleaved at the NheI restriction enzyme
cleavage site where a hygromycin gene cassette (SV-Hyg.sup.r) under
the control of the SV40 early promoter was inserted (NLVpr-CHYGRO)
(FIG. 2B) because the SV-H cassette was inserted in the env gene
leading to the disruption of Env expression, virus particles
containing amphotropic envelope were generated by
trans-complementation using the MLV envelope expression plasmid by
cotransfection.
[0054] The virus particles present in the supernatant were used to
infect HeLa T4 cells and the infected cells were selected in media
containing hygromycin B as described (41-44). The number of
Hyg.sup.r-resistant colonies in the presence of the antibiotic
reflects the ability of a pseudotyped particle to infect and
integrated into the cellular genome. The results are presented in
Table 2 and correlated well with the multiple rounds of replication
assay.
[0055] In an attempt to increase the efficiency of Vpr-C as
pseudosubstrates for protease, we have introduced amino acids
constituting a flexible hinge region (Gly-Gly-Ser-Ser-Gly)
immediately 5' to the cleavage signal of the 17/24 construct (FIG.
2A). This construct was chosen to receive a hinge based
1TABLE 1 Effect of Vpr-C on virus Replication T activity in culture
supernatant, cpm/.mu.l Virus derived from designated 14 days after
21 days after 28 days after proviral DNA infection infection
infection PNL43* 1082 1678 1380 NLVpr 1/6 49 219 828 NLVpr 24/2 0
67 0 NLVpr 2/7 287 1883 1755 NLVprPR/RT 29 2845 2450 NLVpr 17/24 28
2133 1366 NLVpr RT/Rnase 193 2116 1248 NLVpr 7/1 299 1965 2132
NLVpr TF/PR 681 652 1016 *The replication pattern of virus derived
from NLVPR-F was similar to pNL4-3.
[0056]
2TABLE 2 Effect of Vpr-C in a single round replication Proviral
Titers % (+) % (+) Vpr clones CFU/ml* inhibition up-regulation ED84
1536 NL 1/6 1088 30 NL 2/7 928 40 NL H 17/24 1088 30 NL PR/RT 2608
169 NL TF/PR 1072 31 NL 17/24 1248 29 NL 7/1 1936 126 NL 24/2 0 100
*No Hygr colonies were observed for any of the proviral clones when
the trans-complementation was preformed without pSV-A-MLV-env,
pSV-clones when the trans-complementation was preformed without
pSV-A-MLV-env, pSV-A-MLV-env by itself, or mock transfected.
*Extent of inhibition and upregulation was calculated in comparison
to pED84 control proviral DNA. *The virus derived from NLVPR-F
proviral DNA showed replication results similar to virus from
pED84.
[0057] on its weak performance and from previous reports that the
Tyr-Pro scissile bond it contains acts as a late site for cleavage
making it one of the less efficient sites for protease cleavage
(50). Results generated from the H17/24 chimera indicated an
enhancement of the inhibitory affect in the single round infection
assay; however, it still lacks the total inhibition seen with
Vpr-24/2.
Discussion
[0058] Currently, there are several drugs that have been approved
by the Federal Drug Administration to treat HIV-1 infected
individuals, all of which either target the viral RT or protease
enzymatic activities of the virus. The continued treatment of
virus-infected individuals with these drugs has led to the
identification of viruses that exhibit partial to full resistance
to treatment as a result of specific changes in the target enzymes
(51). In the absence of a successful vaccine to prevent HIV-1
infection, various alternative approaches have been proposed and
are being actively investigated (8-11). These include the capsid
fusion approach where a toxic gene product can be fused to the
capsid protein for inactivating the virion components, chimeric
receptor molecules targeted to Env, and the use of trans-dominant
mutants targeting Gag, Rev, Tat, and Env for inactivating the virus
at different stages of the life cycle (52-57).
[0059] The virion association of nonstructural proteins encoded by
HIV-1 provides a unique opportunity to attack the virus particle in
trans, and are advantageous over structural protein based antiviral
approaches (52-54, 57). Along these lines, Kappes and coworkers
(35, 58, 59) have generated chimeric proteins based on HIV-1 Vpr
and HIV-2 Vpx utilizing stapylococcal nuclease and wild-type and
mutated HIV-1 protease fused in-frame to these proteins. In our
studies, we have generated a chimeric protein based on Vpr
utilizing the conserved protease cleavage site sequences from the
Gag and Gag-Pol precursor polyproteins as a fusion partner. These
sequences are efficiently cleaved by HIV-1 protease when presented
as peptide substrates (46-49, 60). The interesting features of the
chimeric proteins generated here are the minimal addition of
residues (10 residues), no toxicity due to added sequences, and the
likelihood of Vpr-C to behave like the wild-type Vpr protein due to
the minimal increase in size. The strategy outlined here brings the
chimeric protein closer to the target protein in the virus
particle. It is likely that the ability of Vpr-C to serve as a
pseudosubstrate for HIV-1 protease can lead to the exhaustion of
protease activity. This would, in essence, reduce the effectiveness
of protease to participate in the maturation of the virus particle
by not acting on the bona fide viral precursor proteins.
[0060] The results generated with Vpr-C showed that the chimeric
protein retains the ability to get incorporated into the virus
particle. In the context of the proviral DNA, the Vpr-C suppressed
HIV-1 replication both in multiple- and single-round replication
assays. As the single-round replication assay involves the
establishment of cells resistant to hygromycin, one concern was
whether the wild-type Vpr or Vpr-C would complicate the results due
to cell cycle arrest. This does not seem to be the case due to the
positioning of the selectable marker (hygromycin gene) which is
under the control of the SV40 promoter. The concordance between the
results from the single- and multiple-round replication assays also
argues against such a possibility. In addition, further support for
such an interpretation is seen with the variable replication
results observed with the different Vpr-C containing viruses.
[0061] Considering the stability of chimeric vpr sequences in the
viral genome over several rounds of replication (data not shown),
the kinetics of the replication pattern suggests that the viral
population may contain a mixture of replication competent and
defective viruses. Suprisingly, we noted that some Vpr-C proteins
did not have influence on viral replication ans some even showed an
up-regulation of viral replication (Tables 1 and 2). Complete
inhibition was observed only with the Vpr chimera 24/2 and moderate
inhibition was observed with Vpr chimeras 1/6, 2/7, 17/24, and
trans-frame (TF)/PR. The cleavage of the 24/2 site is important in
that it serves as a regulator for the sequential processing of the
Gag precursor, and is unique in that it is the only site of the
nine recognized by the protease to have glutamic acid at the P2'
position (61).
[0062] The inhibition of viral replication observed with viruses
containing Vpr-C may be due to the ability of Vpr-C to overwhelm
the protease activity. Biochemical analysis of virus particles with
respect to the status of the precursor proteins is likely to
provide information regarding the mechanism of inhibition. Earlier
biochemical studies showed that 2,500 Gag and .about.5-10% of
Gag-Pol molecules in relation to Gag are present in each virion
(1,31). Though the exact number of Vpr molecules present in the
virus particle has not been determined for HIV-1, studies on HIV-2
indicate that Vpx, a protein related to Vpr, is present in
equimolar concentration to that of p28 in virus particles (62).
Such a scenario in HIV-1 is likely to lead to the presence of an
enormous number of Vpr-C pseudosubstrates for protease to act on
within the virus particle and may interfere with the processing of
the authentic viral precursor proteins. It has been shown that
partial inhibition of Gag and Gag-Pol processing results in
aberrantly assembled viruses (2). The variable inhibition we
observed regarding viral replication suggests that the ability of
Vpr-C to serve as a pseudosubstrate for protease may vary as
observed with oligopeptide substrates corresponding to cleavage
sites (46,47,50,60,63). Furthermore, previous work on protease
cleavage in the context of corresponding peptides and protein
precursors has revealed the importance of either residues or
conformational determinants within the Gag and Gag-Pol precursors
that affect the order of cleavage and the actual cleavage event of
the target substrates (50,61,64,65). Because the cleavage signals
in Vpr-C are presented out of the context of the Gag precursors,
the absence of both up and downstream determinants may have
prevented Vpr-C from being a substrate and competitor for the
protease cleavage site. This would ultimately lead to the lack of
influence on viral maturation and infectivity.
[0063] The threshold level of protease required for maturation of
the virus particle is not known. Studies involving protease
inhibitors have shown that the enzyme needs only a 50-fold
reduction in activity whereas a 25-fold reduction still allows for
processing and subsequently the production of infectious virus
(66). It is plausible that the constructs showing no effect did not
offer enough competition to prevent processing from occurring.
Likewise the partial inhibitory constructs may not have reached the
50-fold reduction threshold, but went farther than those chimeras
with no apparent effect. The high level of replication observed
with virus derived from certain Vpr-C containing proviral DNA is
intriguing. Because equal amounts of virus were used as innoculum
to infect CEM cells, it is likely that some Vpr-C pseudosubstrates
enhanced the level of virus production by activating protease.
Alternatively, the increased protease activity may act on an as yet
unidentified step in viral replication.
[0064] We have shown that a novel class HIV-1 agents can be
generated with the desirable end result of eliminating virus
infection. We have termed this class of agents,"anti- HIV agents
from within". The studies carried out with the virus derived from
proviral DNA containing Vpr-C provided evidence in support of this
strategy. The disadvantage in the use of Vpr as fusion partner for
generating a chimeric protein is its ability to arrest cells at the
G.sub.2 stage of the cell cycle. In this regard, work from our
laboratory and others (ref. 22) has demonstrated the the cell cycle
arrest can be abolished by introducing changes at the C terminus of
Vpr, and yet still retain virion incorporation.In addition, the
Vpr-C proteins will also prove to be useful for dissecting the
steps involved in virus maturation.
[0065] These results show that chimeric proteins generated from
within HIV-1 have the ability to suppress HIV-1 replication and
make ideal agents for gene therapy or intracellular immunization to
treat HIV-1 infection.
[0066] We would like to express our thanks to Dr.
[0067] Antiono Panganiban (University of Wisconsin, Madison) for
the gift of pED84. pSV-A-MLV-env was obtained from the AIDS
Research And Reference Reagent Program of the National Institute of
Health.
Example 3
Materials and Methods for Example 4
Recombinant Plasmids
[0068] Construction of the Vpr expression plasmid was carried out
as described (Mahalingam et al., 1995a-e). Vpr coding sequences
were amplified using primers containing Hind III and Xho I
restriction enzyme recognition sequences at the 5' and 3' end,
respectively. The DNA fragment generated through PCR was cloned
into a pCDNA3 plasmid vector. For the generation of the chimeric
construct containing Vpr and the C-terminus of PR, four aa residues
from the C-terminus corresponding to the dimer interface structure
of PR were added in frame to the C-terminus of Vpr (Vpr-P1). A
similar strategy was also used for the construction of the
Vpr-H-PC1 plasmid, in which a flexible hinge region was added (The
5-amino acid sequence G-G-S-S-G, as represented by the standard
single-letter codes for amino acid; Gly-Gly-Ser-Ser-Gly) between
the Vpr coding sequences and the dimer interface domain. All of the
recombinant plasmids were verified by DNA sequence analysis.
[0069] The chimeric Vpr sequences were introduced into the HIV-1
proviral DNA designated NL4-3 (Adachi et al., 1986). The DNA
fragment encompassing the 3' end of the chimeric Vpr generated by
cleavage with EcoR I and Xho I from the expression plasmid was
cloned into the proviral DNA, cleaved at the unique EcoR I and Sal
I site. This strategy does not interfere with the overlap of vpr
and tat, as the Vpr fragment contains a termination codon.
Expression and Incorporation into Virus-like Particles
[0070] The expression of the Vpr-PC1 and Vpr-H-PC1 proteins was
analyzed using an in vitro transcription-coupled translation system
in accordance with the manufacturer (Promega). The translated
protein was subjected to radioimmunoprecipitation (RIPA) as
described (Mahalingam et al., 1995d). For the expression of Vpr in
cells, and Vpr incorporation into virus particles, we have used the
vaccinia virus T7 polymerase system (Mahalingam et al., 1995a-e).
Vpr and Gag expression plasmids were transfected alone and in
combination into HeLa cells. Cell lysates and culture supernatants
were subjected to RIPA using antibodies to Gag and Vpr as
previously described (Mahalingam et al., 1995d.)
Virus Infection Studies
[0071] Proviral DNA containing Vpr, Vpr-PC1, Vpr-H-PC1, or other
chimeric Vpr was transfected into RD or HeLa cells. Virus released
in the culture medium was collected at the end of 72-120 hours and
quantitated by an RT assay (Nagashunmugam et al., 1992). An
equivalent amount of virus based on RT activity values was used to
infect the CEM cells for viral replication studies. The culture
supernatant from infected cells was monitored periodically for
virus production.
Single Cycle Replication Assay
[0072] The chimeric Vpr containing proviral DNA (NLVpr-PC1 and
NLVpr-H-PC1), or wild type NL4-3 DNA, was cleaved at the Nhe I site
and a Hygr gene under the control of SV40 early promoter was
inserted into env which disrupts its expression. Co-transfection of
the modified proviral DNA and the murine amphotropic env expression
plasmid results in the generation of virus particles capable of
only a single round of replication (Rizvi et al., 1996). The virus
particles released into the culture supernatant were centrifuged,
resuspended in medium from which an aliquot was used to infect HeLa
cells in the presence of DEAE-dextran. The infected cells were
washed 48 hours after infection and placed in medium containing
hygromycin. At the end of 14 days, colonies of cells resistant to
hygromycin were stained and counted. In addition, the infectivity
of the virus particles was also tested by using a MAGI assay as
described (Kimpton and Emerman, 1992). This assay provides a
measure of infection of a cell by the induction on an endogenous
Pgalactosidase gene under transcriptional control of the HIV-1
LTR.
Electron Microscopy (EM)
[0073] After transfection, cells were fixed for EM with 2.5%
glutaraldehyde, postfixed with osmium tetroxide and embedded in
Epon/Araldite. Sections were then stained with lead citrate and
uranyl acetate (Nagashunmugam et al., 1992).
Example 4
Results
Chimeric Vpr Maintains the Phenotype of the Wild-type Vpr
Protein
[0074] Based on structural and enzyme inhibition studies involving
peptides derived from HIV-1 PR, we fused the C-terminal four
residues (TLNF, Thr-Leu-Asn-Phe) of protease in frame to the
C-terminus of Vpr (FIG. 5) designated Vpr-PC1. There is a
possibility that the PR coding sequences present in Vpr-PC1 may not
be accessible to its target Gag-Pol and/or partially cleaved
precursor proteins due to the direct fusion to the C-terminus of
Vpr. In order to give flexibility to the residues added to the Vpr,
an additional construct was generated where a flexible hinge region
was,introduced between Vpr and TLNF (Vpr-H-PC1). For the generation
of a negative control, residues unrelated to PR from the protein
kinase domain (PKA) (R-R-A-S-V, Arg-Arg-Ala-Ser-Val) and the FLAG
epitope (D-Y-K-D-D-D-D-K, Asp-Tyr-Asp-Asp-Asp-Asp-Tyr) were fused
in-frame with the C-terminus of Vpr.
[0075] The characteristics of Vpr-PC1 and Vpr-H-PC1 (chimeric Vpr)
were then tested using an in vitro transcription-coupled
translation system. The translated protein was immunoprecipitated
utilizing antibodies to full-length Vpr as described (Mahalingam et
al., 1995a). The Vpr-PC1 and Vpr-H-PC1 vectors showed proteins with
altered migration in comparison to the wild type Vpr in the gel,
which may be due to the addition of amino acid residues at the
C-terminus (FIG. 6).
[0076] Since incorporation of chimeric Vpr proteins into virus
particles is vital to our strategy, we analyzed the virion
incorporation properties of Vpr-PC1 and Vpr-H-PC1, using the
recombinant vaccinia virus containing T7 polymerase to drive
protein expression as described (Mahalingam et al., 1995a-e).
Co-transfection of Gag and Vpr, Vpr-PKA, Vpr-PC1, or Vpr-H-PC1
plasmids into vaccinia virus infected Hela cells showed expression
of appropriate proteins within cells.
[0077] Analysis of the virus-like particles released from cells by
RIPA indicated two bands corresponding to Gag and Vpr (FIG. 7).
Thus, It is evident that the Vpr-PC1 and Vpr-H-PC1 proteins retain
the ability to be incorporated into virus particles.
Effect of Chimeric Vpr on HIV-1 Replication
[0078] To evaluate the effect of Vpr-PC1 and Vpr-H-PC1 on viral
replication, we have generated HIV-1 proviral DNA containing the
sequences encoding chimeric Vpr. We have used HIV-1 proviral DNA
(designated NL4-3) for this purpose, as it contains all the
functional auxiliary genes encoded by the virus (Adachi et al.,
1986). Vpr-PC1 and Vpr-H-PC1 coding sequences were cleaved using
EcoR I and Xho I restriction enzymes, and the released fragment was
ligated to the proviral DNA cleaved with EcoR I and Sal I (FIG. 8).
The chimeric Vpr in the proviral DNA was verified by DNA sequence
analysis.
[0079] The proviral DNA containing Vpr-PC1 and Vpr-H-PC1 was
transfected into either human rhabdomyosarcoma (RD) or HeLa cells
to generate viruses for biological studies. The virus particles
were quantitated by RT and/or HIV1 p24 antigen assays with
subsequent infection (viral replication assay) being carried out
using established CD4+ CEM cells as targets. The virus infection
was initiated with an equal amount of virus based on RT activity,
with infected cells being monitored for up to a month. The virus
derived from wild type NL4-3 replicated as expected, with a peak
virus production at 15-20 days after infection. In contrast, the
replication studies carried out with the viruses derived from
proviral DNA containing Vpr-PC1 or Vpr-H-PC1 showed a delayed
kinetic pattern and registered a low level of virus production in
comparison to the control (Table 3). These results suggest that
viruses generated from Vpr-PC1 and Vpr-H-PC1 containing proviral
DNA may harbor a mixture of non-infectious and infectious
particles. The viral replication assay using CEM cells involves a
spreading infection, which needs to be considered when interpreting
the observed results. Subsequently, this assay does not allow the
effect of Vpr-PC1 and Vpr-H-PC1 to be evaluated quantitatively when
a mixture of viral populations exists.
[0080] Therefore, to precisely quantitate the effect of Vpr-PC1 and
Vpr-H-PC1 on viral replication, we used a single-round replication
assay. Limiting the virus to a single replication event would allow
one to observe direct and immediate effects of the chimeric
proteins on the infectivity of the virus produced. Co-transfection
of NLVpr-PC1-HYGRO or NLVpr-H-PC1-HYGRO, in which the endogenous
env is disrupted with a hygromycin (Hygr) resistance gene/marker
under SV40 promoter, with an amphotropic Env expression plasmid
into cells, resulted in the release of virus particles capable of
initiating infection (FIG. 8). However, the viruses generated are
replicationdefective due to the absence of a trans-complemented env
gene in the target cells. The cells will confer resistance to
hygromycin when grown in the selective medium if they contain the
proviral DNA, possible only if successful infection occurred. In
this assay, each Hygr colony of HeLa cells represented an infection
event. When the numbers of colonies were compared to the control,
the virus derived from NLVpr-PC1 and NLVpr-HPC1 registered an
inhibition of 66 and 80%, respectively (Table 4). Likewise, when
the virus particles were tested using another single cycle assay
based on .beta.-galactosidase expression, the MAGI assay, the
results confirmed the earlier findings by showing 82 and 92%
inhibition for NLVpr-PC1 and NLVpr-HPC1, respectively (data not
shown).
[0081] As viral particles go through an ordered processing of
precursor proteins to attain their characteristic morphology, it
was of interest to examine the structure and maturation of viral
particles produced by the chimeric Vpr-containing proviral DNA. One
would expect a correlation between lack of infectivity and aberrant
virus morphology. While virus particles with typical lentivirus
morphology were observed in cell cultures transfected with both
wild-type and chimeric proviral DNA, the number of typical
particles was considerably less in the case of the latter.
Moreover, cells transfected with chimeric proviral DNA showed an
abnormal accumulation of viral buds and ring-shaped particles (FIG.
9A). Some of these cells had mature particles within membrane-bound
vacuoles (FIG. 9B). Also of interest was the unusual presence of
viral particles containing a mature core still attached to the cell
membrane in the cells transfected with NLVpr-PC1 and NLVpr-H-PC1
(FIGS. 9C, D).
Discussion
[0082] The studies presented here outline an unique strategy to
contain HIV-1 replication based on the colocalization of the
antiviral agent with the target protein in the virus particles. The
processing of the viral precursor proteins, mediated by HIV-1 PR,
has been shown to take place in two different compartments
(Hunter,1994; Kohl et al., 1988). The intracellular processing of
viral proteins by PR is likely to exclude the processed proteins
from associating with the budding virus particles. The processing
of proteins that occurs in the virus particles converts the
immature into mature infectious particles (Katz and Skalka,1994;
Kohl et al., 1988). Since the latter process is carried out in an
isolated compartment away from host cells, the concept of
virion-specific therapeutic molecules is appealing as an approach
to disrupt virus replication. The generation of an infectious virus
particle is strictly dependent upon the completion of processing by
the viral PR (Katz and Skalka,1994; Debouck, 1992; Katoh et al.,
1989; Kaplan et al., 1993). Therefore, the crucial role and the
specificity of PR for viral proteins have generated intense efforts
to identify effective inhibitors targeting the enzyme to prevent
virus maturation.
[0083] The virion-specific therapeutic molecule generated from
within provides high specificity for its incorporation into the
virus particles. Among the three auxiliary gene products that are
incorporated into HIV-1 viral particles, Vif, Vpr and Nef (Trono,
1995; Subbramanian et al., 1994; Welker et al., 1996; Pandori et
al., 1996; Natsoulis et al., 1991), the Vpr molecule, due to its
small size, abundance, and stability, best meets the criteria for a
vehicle to deliver a peptide of interest into the virus particle.
Since Vpr does not influence virus morphogenesis, this approach is
more advantageous than the capsid fusion approach, where the
exogenous sequences targeting the virus are fused to the capsid
(Natsoulis et al., 1995; Wills and Craven, 1991; Kaplan et al.,
1994).
[0084] The extent of incorporation of viral proteins such as Gag,
Gag-Pol and chimeric Vpr further lends credence to the strategy
outlined here. It is reported that each virus particle contains
2500 copies of Gag and approximately 1/10 or 1/20 of Gag-Pol
polyproteins (Hunter et al., 1994; Wills, 1991). Though the exact
number is not available for HIV-1 Vpr, it is present in high
amounts, as the Vpr-related Vpx has been shown to be present in
equimolar concentration as that of p24 of HIV-2 Gag (Henderson et
al., 1988). Such a scenario presents a situation in which the
concentration of Vpr-PC1 and Vpr-H-PC1 proteins is in excess of
that of Gag-Pol, and may lead to a reduction in the level of active
PR available.
[0085] Viral populations derived from the HIV-1 proviral DNA
containing chimeric Vpr showed lower replication in comparison to
wild type HIV-1 proviral DNA. Further, the extent of replication
was lower with chimeric Vpr containing a flexible hinge region
between Vpr and PR coding sequences (Vpr-H-PC1). The addition of
hinge region residues (G-G-S-S-G, Gly-Gly-Ser-Ser-Gly) may provide
flexibility to the dimer interface domain so that the chimeric Vpr
molecules are able to form stable structures with PR monomers. This
results in inactive PR, and leads to the generation of
non-infectious virus particles. This is indeed supported by the
data from both multiple and single round replication assays.
[0086] The mechanisms by which Vpr-PC1 and Vpr-H-PC1 bring about
this effect on virus infectivity is not clear. The reduced
availability of active PR and interference with virion maturation
events through incomplete processing of precursor viral proteins
may account for the inhibitory effects observed. Analysis of viral
proteins in cells and in virus particles may provide clues as to
the mechanism involved in reduced virus infectivity.
[0087] Ultrastructural studies revealed abnormalities at the
budding stage and at the time of particle release. These data are
in agreement with the observation showing a link between the level
of PR activity and virion release (Kaplan et al., 1994). Zhang et
al. (1991), have shown that a peptide corresponding to the
C-terminus of PR inhibited PR activity in vitro . Similarly, Bab6
et al. (1992), noted that a 13 amino acid peptide representing N-
and C-termini of PR inhibited PR activity and prevented the
assembly of PR dimers in refolding experiments in vitro. In
contrast to the data reported here, only a weak effect was noted
with respect to virus replication in MT4 cells using a peptide from
the dimer interface structure (Schramm et al., 1991). This may be
due to the inefficient localization of the peptide in the virus
particle. It is likely that the presentation of a dimer interface
domain in the context of a protein with specificity for
incorporation into virus particles may bring the domain close to
the target precursor Gag-Pol protein containing PR. The novel
agents described here may provide useful information for the
development of peptide mimetics to contain HIV-1 replication.
[0088] Interestingly, Vpr-PC1 and Vpr-H-PC1 lack the cell cycle
arrest function which enables the use of modified Vpr as an
effective and specific delivery vehicle for genetic approaches to
treat HIV-1 infection.
3TABLE 3 Effect of Vpr-C on Virus Replication RT activity in
culture supernatant, cpm/.mu.l 5 days 9 days 16 days 20 days 26
days Virus Derived from after after after after after Designated
proviral infec- infec- infec- infec- infec- DNA tion tion tion tion
tion *NL4-3 42 3999 7513 10764 2184 NLVpr-PC1 0 0 4127 5504 3888
NLVpr-H-PC1 0 0 758 5162 4611 *The replication pattern of virus
derived from NLVpr-F and NLVpr- *The replication pattern of virus
derived from NLVpr-F and NLVpr-PKA was similar to NL4-3
[0089]
4TABLE 4 Effect of Vpr-C on Virus infectivity in a single round of
replication Proviral VPR clones Titers CFU/ml* % (+)
Inhibition.dagger. ED84.dagger..dagger. 1760 0 NLVpr-PC1 HYG 590 66
NLVpr-H-PC1 HYG 352 80 *No Hygr colonies were observed for any of
the proviral clones when the trans-complementation was performed
without pSDV-A-MLV-env, pSV-A-ML-env by itself, or mock
transfected. .dagger.Extent of inhibition was calculated in
comparison to ED84 control proviral DNA. .dagger..dagger.The virus
derived from NLVpr-F and NLVpr-PKA proviral DNA showed replication
results similar to virus from ED84 control.
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Sequence CWU 0
0
* * * * *