Transcription of the human immunodeficiency virus
type-1 (HIV-1) genome is controlled by cooperative interaction of viral encoded proteins and host regulatory proteins. In this study, we have
examined the capacity of the viral auxiliary protein, Vpr, to modulate
transcriptional activity of the HIV-1 promoter sequence located within
the long terminal repeat (LTR). We demonstrate that ectopic expression
of Vpr in human astrocytic cells, U-87MG, enhances the basal activity
of the viral promoter in transfected cells and that the GC-rich
sequences, spanning nucleotides
80 to
43, are important for this
activity. Since this region serves as the target for p53-induced
suppression of LTR activity and interacts with the ubiquitous
transcription factor, Sp1, we examined the cooperative activity of Vpr,
p53, and Sp1 upon LTR transcription. Results from co-transfection
studies indicated that overexpression of wild type p53, but not mutant
p53, decreases the level of activation of the LTR by Vpr.
Transcriptional activation of the LTR by Vpr required the presence of
Sp1 since overexpression of Vpr in cells with no endogenous Sp1 failed
to augment LTR activity. Results from protein-protein interaction
studies indicated that Vpr is associated with both p53 and Sp1 in cells
with ectopic expression of these proteins. Moreover, it was evident
that p53 and Sp1 interact with each other in these cells. These
functional and structural studies provided a working model on the
cooperative interaction of Vpr with cellular proteins Sp1 and p53 and
control of viral gene transcription at immediate early stage of
infection prior to the participation of other viral regulatory
proteins.
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INTRODUCTION |
The genome of the human immunodeficiency virus type 1 (HIV-1)1 contains, in
addition to common retroviral genes encoding structural proteins gag,
pol, and env, several auxiliary genes with a different degree of
importance for replication of the viral genome. While the auxiliary
genes responsible for production of Tat and Rev are essential for viral
gene expression and replication, the remaining group of this family,
so-called accessory genes which include vpr, vpu, nef, and
vif, are dispensable for virus replication in cell culture
(1). Mutagenesis studies, however, have indicated that any alterations
in these genes may have an impact upon the replication properties of
HIV-1.
Vpr, the 96-amino acid long viral protein, represents one of the most
studied accessory proteins. This small protein is of particular
interest among the other HIV-1 accessory proteins because of its
association with the virus particles and its unique structural feature
(2-4). Results from biochemical and genetic studies have revealed
several interesting properties of this protein. For example, in
infected cells, Vpr is localized, accumulates in nuclei (5, 6), and has
the ability to interact with several cellular regulatory proteins (6).
This protein can arrest cells at the G2 stage of the cell
cycle by affecting cdc 2/cyclin B expression and activity (7-13). In
addition, earlier studies have indicated that Vpr has the ability to
augment transcription of the HIV-1 LTR in cell-free in vitro
transcription assay (14). These observations led to the assumption that
the envelop-incorporated Vpr, by up-regulating viral gene expression in
newly infected cells at the immediate early phase, increases expression
of other viral genes including the potent transcription transactivator,
such as Tat, in the subsequent lytic cycle. Whereas the mechanism
whereby Tat exerts its regulatory action upon virus gene transcription
is greatly understood (for review, see Ref. 15), the pathway by which
Vpr induces promoter activity of HIV-1 remains elusive. In previous
studies, results from in vitro transcription assay suggested
that Vpr may induce transcription of the HIV-1 promoter through the
GC-rich motif of the LTR (14), which serves as the binding site for the
ubiquitous cellular transcription factor, Sp1 (16), and that
interaction of Vpr and Sp1 may be important for the observed activity
(14). Moreover, results from protein-protein studies indicated in
vitro association of Vpr and TFIIB (17). These observations
demonstrate that high level expression of the viral genome may require
cooperative interaction of several viral proteins, such as Tat, Vpr,
and the cellular proteins that may share a common target motif within the viral genome. The ability of Vpr to stimulate the HIV-1 promoter through the GC-rich motif, a region which has been demonstrated to
negatively respond to the p53 tumor suppressor protein, prompted us to
launch a series of functional and structural studies to assess the
ability of Vpr to regulate HIV-1 promoter activity in cell culture and
to determine the importance of the GC-rich binding protein and p53 in
this regulatory event.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
The plasmid constructs containing
various sequences of the HIV-1 LTR upstream of the reporter CAT gene
have been described previously (18). The reporter constructs with an
internal deletion in the GC-rich motif were provided by Dr. S. Zeichner
NCI, National Institutes of Health, (Bethesda, MD) (19). The pcDNA
Vpr expression plasmid containing the CMV promoter expressing Vpr was
provided by Dr. A. Srinivasan (Thomas Jefferson University,
Philadelphia, PA) and has been described previously (20). The Sp1
expression vector was generously provided by Dr. R. J. Tjian
(University of California Berkeley). The CMV-p53 and mutant p53
expression vectors have been described previously (21) and were kindly provided by Dr. R. Gartenhaus (Allegheny University of the Health Sciences, Philadelphia, PA). pPac-Sp1 and pPac-p53 represent plasmids in which cDNAs corresponding to Sp1 and p53, respectively, are expressed by the actin promoter in SL2 cells. The MCK-CAT plasmid containing muscle-specific creatine kinase promoter fused to the CAT
gene has been described previously (22).
Cell Culture, Transfection, and CAT Assay--
The human
astrocytic glial cell line, U-87MG (ATCC, Manassas, VA), was maintained
in Dulbecco's minimal essential medium supplemented with 10% fetal
calf serum (Life Technologies, Inc.). The stable clonal cell line,
GM47.23, that conditionally expresses wild-type p53 following treatment
of cells with dexamethasone was derived from human glioblastoma cells
(23). All cells were grown in the presence of penicillin-streptomycin
(100 units/ml). Drosophila melanogaster Schneider cell line,
SL2, was kindly provided by Dr. J. Jaynes (Thomas Jefferson
University). These cells were grown in Schneider medium (Life
Technologies, Inc.) supplemented with antibiotics and 12%
heat-inactivated fetal calf serum (24).
For transfection, approximately 5 × 105 cells were
seeded on 60-mm plates, and after 16 h, cells were transfected
with various plasmids as specified by the calcium phosphate
precipitation method (25). In general, the transfection mixture
contained 500 ng of the reporter plasmid either alone or with 5 µg of
the expression plasmids, and the final concentration of the promoter
DNA construct was normalized by the addition of empty expression
plasmid, pcDNA3. CAT activity was determined after 36-48 h
posttransfection by the previously described method (26). Each
transfection was done in duplicate and repeated 2-4 times with at
least three different plasmid preparations to ensure the
reproducibility of the results.
Antibodies--
Monoclonal antibodies for detection of p53 were
purchased from Oncogene Research Products (Cambridge, MA). pAb 421 (Ab-1) and pAb 240 (Ab-3) are mouse monoclonal antibodies which
recognize the amino acid residues 371-380 (27) and 213-217 (28) of
human p53, respectively. Polyclonal anti-flag antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). This antibody recognizes the flag tag sequence that is positioned at the C terminus of Vpr derived by pCMV-Vpr (29). Polyclonal antibody for detection of
Sp1 was purchased from Santa Cruz Biotechnology.
Immunoprecipitation and Western Blot Analyses--
U-87MG and
Drosophila SL2 cells were transfected with either the
pCMV-Vpr flag, pCMV-p53, or pCMV-Sp1 (for U-87MG cells) and pPac-Sp1 or
pPac-p53 (for Schneider cells) alone or in various combinations by the
calcium phosphate precipitation method (25). Transfectants were
trypsinized and subjected to immunoprecipitation and/or Western
analysis as described previously (30). Silver staining was performed
according to previously published procedures (31).
Treatment of GM47.23 cells with dexamethasone at a final concentration
of 1 µM was done according to the procedure described previously (23). Analysis of p53 production and HIV-1 LTR activity was
performed according to the procedures described above.
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RESULTS AND DISCUSSION |
Vpr is a 96-amino acid protein encoded by the HIV-1 genome that
has the ability to increase the rate of replication of the virus in
T-cell lines and peripheral blood mononuclear cells (PBMCs) (32, 33).
Earlier studies have indicated that Vpr has a transcriptional activity
and induces expression of the viral LTR and several cellular genes
(34). While activation of the viral promoter by Vpr may be mediated by
TFIIB, a component of the transcription initiation complex (17),
earlier studies pointed to the importance of the GC-rich motif located
upstream of the transcription start site, in the observed activation of
the HIV-1 gene by this protein. Here, we performed transfection studies
utilizing full-length and deletion promoter constructs derived from the
HIV-1 regulatory region to evaluate the ability of Vpr in enhancing the
activity of HIV-1 LTR in the human astrocytic cell line, U-87MG.
Results from this study indicated that ectopic expression of Vpr in
U-87MG cells transfected with the reporter plasmid containing the LTR promoter sequences from
458 to +80,
117 to +80, and
80 to +80 results in 6.4-7.0-fold activation of the viral promoter (Fig. 1). Removal of the sequences between
80
to
48, that deletes the GC-rich motif, completely abrogated Vpr
responsiveness and LTR activation in these cells. The use of 3'
deletion constructs that remove the sequences between +80 to +3
suggested that the sequences positioned downstream from the
transcription start site (+1) may not play an important role in
Vpr-induced activation of the LTR in these cells (Fig. 1). Results from
studies utilizing internal deletion constructs of the LTR that remove
the sequences between
80 to
48 from the full-length (3 and 7), and
the 5' deletion constructs (5 and 9) as depicted in Fig. 1, in the
transfection experiments verified that the sequences between
43 to
80 are essential for induction of LTR transcription by Vpr in
astrocytic cells.

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Fig. 1.
Functional analysis of the effect of Vpr on
HIV-1 LTR transcription. Left panel, schematic
representation of various reporter constructs with HIV-1-LTR sequences
fused to CAT gene. Numbers on the left describe
the end point nucleotide sequence of the LTR. The symbols depict two
copies of B (ovals) and three copies of the SP1 binding
site (diamonds). To determine transcription activity of each
construct, U-87MG cells were transfected with 500 ng of the reporter
construct in the absence or presence of 5 µg of Vpr expression
plasmid, and 48 h later, cell extracts were subjected to CAT
analysis. Right panel, -fold activation is expressed
relative to the value obtained for each construct in the absence of
Vpr. The numbers represent an average of several
transfection assays and indicate the -fold stimulation induced by Vpr.
Standard deviations are presented on the bar graphs.
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In earlier studies, we and others (35-37) demonstrated that the p53
tumor suppressor gene product has the ability to decrease transcriptional activity of the HIV-1 LTR in cell culture and in a
cell-free transcription system and that the sequences spanning the
GC-rich motif of the LTR are important for this activity (35). The
ability of Vpr to stimulate transcription of the LTR through the
GC-rich sequences prompted us to examine the potential cooperative action of Vpr and p53 on transcriptional activity of the HIV-1 promoter. In the first series of studies, U-87MG cells were
co-transfected with the LTR reporter plasmid (LTR-CAT) alone or
together with plasmids expressing either wild-type or mutant p53. As
shown in Fig. 2A, in accord
with the previous observations (35) where overexpression of wild-type
p53 decreased transcription of the viral promoter, production of mutant
p53 that contains a mutation in amino acid 143 of the protein led to an
increase in HIV-1 promoter activity. Of note, mutation in the amino
acid residue 143 completely abrogates transcriptional activity of p53
(38). Next, cells were transfected with LTR-CAT and an optimum amount
of Vpr expression plasmid in the absence and presence of plasmids
expressing either wild-type or mutant p53. As illustrated in Fig.
2B, overexpression of wild-type p53 decreased the level of
activation of the LTR by Vpr. Under similar experimental conditions,
mutant p53 showed no significant effect on Vpr-induced transcription of
the LTR.

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Fig. 2.
Regulation of HIV-1 gene expression by Vpr,
wild type (p53 wt), and mutant p53 (p53 mut.)
in U-87MG cells. Transient expression assays were performed by
co-transfection of various HIV-1-CAT reporter constructs (500 ng of
DNA) together with an increasing concentration of plasmids expressing
wt p53 and mutant 143-p53 (0.5, 1, and 2.5 µg) (A) or 5 µg of Vpr as indicated (B). Histograms represent CAT
activity expressed relative to the value obtained with the LTR-CAT
reporter construct. Numbers correspond to an average of
multiple independent experiments. The standard deviations did not
exceed 15%.
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In a different approach, to assess the effect of p53 on the activation
of the LTR by Vpr, we utilized the human glial cell line, GM47.23. This
cell line has the ability to conditionally overexpress wild-type p53
once the cells are maintained in media supplemented by dexamethasone
(23). Treatment of the cells with dexamethasone resulted in the highest
level of p53 production after 18 h, as examined by Western blot
analysis of the protein extract obtained from the cells treated with
dexamethasone for 12, 18, and 24 h (Fig.
3A). The effect of p53 on LTR
promoter activation by Vpr was then examined following transfection of these cells with the LTR-CAT construct alone or together with the Vpr
expression plasmid at different time intervals after dexamethasone treatment. Results from these studies revealed that treatment of the
cells with dexamethasone decreased basal and Vpr-mediated transcriptional activity of the viral promoter and that the maximum level of inhibition was achieved at 18 h when the level of p53 is
at the highest (Fig. 3, B and C). It should be
noted that treatment of the cells with dexamethasone had no significant
effect on the basal transcription of the HIV-1 LTR in these
cells.2 These observations,
along with the previous co-transfection data, strongly suggest that
functional interaction between Vpr and p53 may modulate the level of
Vpr transcriptional activation upon the LTR promoter.

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Fig. 3.
Inhibition of Vpr-induced activation of
HIV-1-LTR by wild type p53. A, Western blot analysis
representing expression of p53 protein in nuclear extracts obtained
from GM47.23 cells treated with 1 µM dexamethasone
(Dex.) for the indicated time prior to extraction of
proteins. In each lane, 30 µg of protein were loaded.
B, cells were co-transfected with the LTR-CAT construct and
the Vpr expression plasmid. The transfected pool of cells was
distributed into replicate plates, and 24 h later, cells were
treated with 1 µM dexamethasone for the indicated times
in a staggered manner such that the CAT assays were performed at the
same time. -Fold inhibition of LTR activity by wild type p53 is shown
following treatment. C, a representative of CAT assay
performed using extracts from cells untreated (-Dex) or
treated with dexamethasone (+Dex) at peak p53 expression (18 h) is shown.
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As described previously, p53 has been shown to act as a transcriptional
activator and repressor in a variety of cells. To examine the effect of
Vpr in the activation of the p53 responsive promoter, we utilized
MCK-CAT, which contains the p53 responsive element, and its
transcriptional activity is increased by the wild-type p53 (39). In
this study, cells were transfected with the reporter MCK-CAT constructs
and the wild-type p53 expression plasmid in the absence and presence of
the Vpr expression plasmid. As shown in Fig.
4, a substantial increase in the
transcriptional activity of MCK promoter was obtained in cells
expressing wild-type p53. Co-expression of wild-type p53 and Vpr
decreased the level of p53 activation of the MCK promoter in the
transfected cells. Of note, expression of Vpr alone in these cells
resulted in a modest (2-fold) induction of the MCK promoter. This
activation may be attributed to the presence of a GC-rich sequence
which resides within the MCK promoter (40). Co-expression of mutant p53
and Vpr in the cells showed a similar effect on the MCK promoter as
that observed in cells expressing Vpr alone. These data further indicate that functional interaction between p53 and Vpr may not be
restricted to the LTR promoter as evidenced by the cooperative action
of these two proteins upon the gene that is positively regulated by
p53. The finding that these two proteins in combination exert a
negative effect upon LTR activity is reminiscent of the interplay
between p53 and SP1 in activation of HIV-1-LTR (41), where the
influence on promoter activity was attributed to alteration in the
interaction of SP1 with DNA by p53.

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Fig. 4.
Vpr suppresses p53-induced transactivation of
MCK-CAT reporter construct. U-87MG cells were co-transfected with
the 3300-base pair MCK reporter plasmid that contains 3300 base pairs
of the MCK upstream control region and 5 µg of the expression
plasmids corresponding to p53 wild type (p53 wt), mutant
(mut.), and Vpr in various combinations as indicated. Cell
extracts were then subjected to CAT analysis. The bars
represent -fold activation of the reporter gene compared with the basal
level set at 1.0. The standard deviations are presented on each
bar graph.
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Since both p53 and Vpr exerted their regulatory action on the HIV-1 LTR
through the GC-rich motif, a region which is the target for binding of
the ubiquitous transcription factor, Sp1, in the next series of studies
we examined the involvement of Sp1 in the observed regulatory event. In
this study, SL2 cells, which have no endogenous Sp1, were transfected
with LTR-CAT alone or in various combinations with expression plasmids
that produce Vpr, wild-type p53, and Sp1. Results from these studies
indicated that in contrast to the data obtained from the U-87MG cells
(Figs. 3 and 5A), ectopic expression of Vpr and p53 in these cells exhibits no significant effect
on LTR promoter activity (Fig. 5B). However, once the
plasmid expressing Sp1 was included in the transfection mixture, the
level of LTR activity was drastically elevated. Co-production of Vpr and Sp1 resulted in transcriptional activation of the LTR to a level
that is comparable with that seen upon expression of Sp1 alone in these
cells. Of interest, expression of p53 significantly decreased the level
of transcriptional activation of the LTR by Sp1 and Sp1 plus Vpr. These
observations suggest that activation of the LTR by Sp1 may be modulated
by p53 and that Sp1 function can be counter prevented by p53. As
mentioned earlier, these findings corroborate with previous
observations indicating that p53 has the ability to alter the DNA
binding ability of Sp1 (41). Also, it is evident that Vpr is incapable
of augmenting LTR transcription in the absence of Sp1. Further, the
data presented in Fig. 5 suggest that yet another protein(s) present in
mammalian cells may be involved in Vpr activation of the HIV-1-LTR.

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Fig. 5.
Interplay between Vpr and p53 in the absence
of Sp1 protein. A, the activity of the Flag-tagged Vpr
was examined in U-87MG and compared with that of Vpr. Transfections
were performed as described in Fig. 1. B,
Drosophila Schneider SL2 cells that are deficient in
endogenous Sp1 were used in these studies. Five hundred nanograms of
the LTR-CAT promoter construct were co-transfected together with either
empty pPac-0 plasmid or 5 µg each of the pPac-Sp1, pPac-p53, or
CMV-Vpr expression plasmids. Cell extracts were prepared 48 h
posttransfection and were subjected to CAT analysis. Histograms show
the -fold promoter activation assessed by CAT activity expressed
relative to the value obtained with the reporter construct alone.
Standard deviations did not exceed 15%.
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In the next series of experiments, we investigated the interaction of
Vpr with Sp1 and p53. Initially, U-87MG cells were transfected with p53
and Vpr expression plasmids alone or in combination. In this study, we
utilized the fusion Vpr expression plasmid, pCMV-Vpr-flag, which
expresses Vpr fused to amino acid flag sequences at the C-terminal
region (23). After 36 h, protein extracts were prepared and
reacted with either anti-Sp1 antibody, or anti-flag antibody that
recognizes Vpr fusion protein. The immunocomplexes were separated from
the remaining protein by centrifugation and analyzed by Western blot
technique directed for the detection of p53. As shown in Fig.
6A, the anti-Sp1
immunocomplexes from untransfected cells and cells transfected with
pCMV-p53 contained p53 protein, suggesting that p53 and Sp1 may be
associated with each other in these cells. Of note, the intensity of
the band corresponding to p53 in the transfected cell extract was
higher compared with the control, indicative of overproduction of p53 by pCMV-p53 (Fig. 6A, compare lane 2 to
1). This observation is in agreement with previous data
pointing to the interaction of endogenous Sp1 and p53 (36). The
anti-flag-directed immunocomplex obtained from the Vpr transfected
cells showed an extremely weak signal corresponding to the p53 band. In
contrast, the immunocomplex obtained from the cells with ectopic
expression of Vpr and p53 by using anti-flag antibody showed an intense
p53 band, suggesting the in vivo association of p53 and Vpr
in these cells (Fig. 6A, lane 5). Similarly,
protein extract from cells transfected with Vpr and p53 expression
plasmids showed the presence of p53 in the immunocomplex that was
pulled-down by anti-Sp1 antibody (Fig. 6A, lane
6). Fig. 6B shows that the Sp1-associated immunocomplex from cells transfected with Vpr contains the 15-kDa chimeric Vpr protein. These observations strongly suggest that the HIV-1 regulatory protein, Vpr, is in complex with cellular proteins Sp1 and p53. Of
note, results from Western blot analysis revealed an approximately 3-fold increase in the level of Sp1 in the cells transfected with the
Sp1 expression plasmid than that of the untransfected cells (data not
shown). The endogenous level of p53 in untransfected cells was almost
undetectable (Fig. 6C, lane 1). In the extract from the transfected cells, an intense band corresponding to
exogenously produced p53 was easily detected (Fig. 6C,
lane 2). Furthermore, from the intensity of the bands, it
was evident that only 30% of the protein was immunoprecipitated and
found in the pellet, whereas 70% of the overproduced p53 remained in
the supernatant (Fig. 6C, lanes 3 and
4, respectively). Results from silver staining of the
protein gel showed the presence of p53, the immunoglobulin subunits,
and one peptide with lower mobility than p53 (data not shown). In the
next series of studies, we utilized SL2 cells to investigate the
importance of Sp1 in the association of Vpr and p53. These cells were
transfected with the expression plasmids alone or in various
combinations allowing for ectopic production of Sp1, p53, and Vpr. Of
note, SL2 cells contain endogenous p53 protein, which co-migrates with
the p53 produced by the expression plasmid (Fig. 6D, compare
lanes 1 and 2). This band was not detected in the
immunocomplex pulled down with the control preimmune sera (data not
shown). Results of the immunoprecipitation/Western blot analyses
indicate that the anti-flag-mediated pulled down immunocomplex from
cell extracts transfected with Vpr and p53 contains no strong band
corresponding to the 53-kDa protein (Fig. 6E, lane
1). Of interest, ectopic expression of Sp1 in the transfected
cells resulted in the appearance of a p53 band in the immunocomplex
pulled down by anti-flag antibody (Fig. 6E, lane
4). These observations suggest that Sp1 may mediate complexation
of Vpr and p53. The immunocomplex pulled down by Sp1 from the extract
transfected with pCMV-Sp1 alone (lane 2) or pCMV-Sp1 plus
pCMV-p53 (lane 3), pCMV-Sp1 plus pCMV-p53, and pCMV-Vpr-flag
(lane 4) contains a p53 band, further suggesting the
association of Sp1 and p53 in these protein extracts.

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Fig. 6.
Association of Vpr and p53 in
vivo. Cell lysates (300 µg) were prepared from U-87MG
(panels A-C) or Drosophila SL2 cells
(panels D and E) following transfection with
various expression plasmids, alone or in combination, as indicated.
Immunoprecipitations (designated as IP) were performed using
specific antibodies that detect Sp1, p53 (pAb 421), or flag peptide.
Immunoprecipitated proteins were resolved by SDS-polyacrylamide gel
electrophoresis and analyzed by Western blot. The antibodies used for
immunoprecipitation are indicated at the bottom of the
panels. Panel B represents the levels of Vpr
immunoprecipitated with either flag (lane 1) or Sp1
(lane 2) antibody. In panel C, lanes
1-4 represent endogenous, exogenously expressed,
immunoprecipitated p53, and the amount remaining in the supernatant,
respectively, following Western blot analysis using p53 antibody.
Panel D represents the endogenous (lane 1) and
exogenous (lane 2) levels of p53 in Schneider cells (SL2) as
determined by immunoprecipitation.
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The studies presented in this communication provide evidence for the
cooperative interaction of viral (Vpr) and cellular (p53 and Sp1)
regulatory proteins and their effect on HIV-1 gene transcription. Although the mechanism of these observations remains unclear, activation of viral gene transcription by Vpr is intriguing in light of
the findings that this small protein, by associating with the capsid
proteins, incorporates into the virion and enters into the newly
infected cells. As such, it is possible that input Vpr in the newly
infected cells stimulates transcription of the viral genome at the
immediate early phase and leads to rapid production of other regulatory
proteins such as Tat and Rev that are pivotal for productive viral
lytic infection. Results from deletion mutant analysis corroborate with
earlier results and indicate that the GC-rich nucleotide sequence
positioned between nucleotides
80 to
43, which is the binding site
for Sp1, is the target for Vpr activation. We also demonstrated that in
cells expressing Vpr, Sp1, and p53, Vpr can form a complex with Sp1.
This is an interesting observation since in previous studies it was
demonstrated that in vitro association of highly purified
Vpr and Sp1 requires the presence of at least two Sp1 DNA motifs. Thus,
one can envision a model in which stable complexation of Sp1 and Vpr
can occur in the absence of Sp1-DNA motifs once the cellular protein,
p53, is present. Therefore, p53 may function as a co-factor and
facilitate interaction of Vpr and Sp1. Experiments are currently in
progress to establish functional relevance of such interactions through the use of mutant versions of these proteins that lack the ability to
interact with each other. It is also interesting to remember that p53
is a major player of host cell cycle machinery, and by modulating a
series of cellular genes such as p21, can dictate the rate of
G1, G1/S, S, and G2 phases of the
cell cycle (42). Also, earlier studies by several laboratories have
indicated that Vpr can induce cell cycle arrest of the cells in the
G2 phase by preventing activation of the mitotic
cyclin-dependent kinases and thereby prevent cells from
undergoing mitosis and proliferation (for review, see Ref. 43).
Although, the significance of these observations with respect to HIV-1
gene transcription and replication remains to be established, it is
noteworthy to mention that cooperative interaction between another
viral protein, Tat, and host-cell cycle machinery results in elongation
of the G1 phase and prevents rapid cell proliferation (14,
44). Thus, it appears that both HIV-1 regulatory proteins, Vpr and Tat,
can stall cell cycle proliferation on one hand, and stimulate viral
gene transcription on the other hand. Evidently, to exert their dual
activity on viral and host functions, these regulatory proteins require
interaction with cellular proteins such as Sp1 and p53 (for Vpr) as
presented in this communication, and NF
B and pRb for Tat (45, 46).
As such, our future efforts will rest on better understanding of the
cooperative interaction of viral and host regulatory proteins and their
effect on viral and host function.
We thank Drs. A. Srinivasan, R. J. Tjian, S. Zeichner, J. Jaynes, and R. Gartenhaus for providing
plasmids. The authors thank members of the Center for NeuroVirology and
NeuroOncology, for sharing biological reagents and DNA clones and
for insightful discussion, and C. Schriver, for editorial
assistance.