(Received for publication, May 19, 1995; and in revised form, July 17, 1995)
From the
Acquired immunodeficiency syndrome (AIDS) is a result of
replication of the human immunodeficiency virus type 1 (HIV-1)
predominantly in CD4 T lymphocytes and macrophages.
However, most of these cells in vivo are immunologically
quiescent, a condition restricting HIV-1 replication. Vpr is an HIV-1
virion protein suspected to enhance HIV-1 replication in vivo.
We demonstrate in this report that Vpr specifically activates HIV-1
long terminal repeat (LTR)-directed transcription. This effect is most
pronounced on a minimal promoter from HIV-1 LTR containing the TATA box
and binding motifs for the ubiquitous cellular transcription factor
Sp1. Evidence is presented that Vpr interacts with Sp1 when Sp1 is
bound to the Sp1 motifs within the HIV-1 LTR. Both Vpr-Sp1 interaction
and Vpr trans-activation require a central Leu/Ile-rich domain in Vpr.
Our findings suggest that Vpr trans-activation through Sp1 is most
critical for the immediate early transcription of HIV-1 when other
positive regulators, such as NF-
B, are limited or inactive, a
condition presumably present in vivo. By interacting with Sp1,
Vpr also has the potential to influence cellular gene expression and
cellular functions. Thus, therapeutic approaches directed toward
blocking the Vpr trans-activation function could prove valuable in
treating AIDS.
HIV-1 ()is the etiological agent of AIDS. The
hallmark of AIDS is the slow but progressive depletion of
CD4
-T cells, a class of T cells crucial for immune
functions. Depletion of CD4
-T cell results in
immunodeficiency and AIDS-related disorders, including encephalopathy,
dementia, and malignancies(1) . Despite tremendous efforts in
the past, the mechanism of these AIDS-related disorders has remained
unclear. However, it is clear that these are a consequence of function
of HIV-1 encoded gene products. For example, the HIV-1 envelope
glycoprotein was implicated to be involved in toxic effects on neuronal
cells(2) . Recently, the HIV-1 Vpr protein in peripheral blood
of HIV-1-infected people was shown to activate HIV-1 replication in
latently infected cells(3, 4) . This effect of Vpr was
suggested to contribute to HIV-1 pathogenesis in vivo.
The
HIV-1 genome encodes structural as well as regulatory gene
products(5, 6) . Recently, great efforts have been
made toward understanding the function of the so-called accessory
regulatory genes, namely vif, vpr, vpu, and nef.
These genes are generally non-essential for HIV-1 to replicate in
activated T cells. Yet, animal model studies with two of these genes, vpr and nef, suggested that they are required for in vivo replication and pathogenesis of the simian
immunodeficiency virus(7, 8) . The paradox between
HIV-1 replication in vitro and that in vivo suggests
that HIV-1 replication may be subjected to different modes of
regulation in vivo compared to in vitro. For example, in vitro studies have shown that HIV-1 replication is highly
dependent on cellular activation and availability of activated
NF-B transcription factor(5, 6, 9) .
However, in vivo, the majority of the susceptible cells are
immunologically quiescent and do not have a high level of NF-
B
activity to support a productive HIV-1 replication. Nevertheless, HIV-1
replication in vivo has been demonstrated to be relatively
rapid(10) .
To understand the role of HIV-1 accessory
regulatory genes during HIV-1 replication and pathogenesis, we focused
on the vpr gene product, which is a 96-aa protein produced
late in the virus life cycle and assembled into the virion through
binding to Gag(11, 12, 13) . Function of Vpr
appears to be critical for HIV-1 to replicate in
macrophages(14) . In lymphocytes, the effect of Vpr on HIV-1
replication is difficult to detect(15) . Our earlier studies
and results from others showed that Vpr has a tendency to localize in
the nucleus (16, 17) without utilizing a classical
nuclear localization signal(16) . These results are consistent
with the notion that Vpr may play a role during the nuclear migration
of the pre-integration complex (18) . However, they were also
consistent with the hypothesis that Vpr can function in the nucleus to
trans-activate HIV-1(19) . We report here that Vpr
trans-activates HIV-1 LTR through interaction with the cellular
transcription factor Sp1. Sp1 is an O-glycosylated
transcription factor which binds to decanucleotide Sp1 motifs
(consensus core sequence: GGGCGG) through three zinc finger domains (20, 21, 22) . It is ubiquitously expressed
and is involved in transcription of a variety of cellular genes
including the proto-oncogenes Ha-ras-1 (23) and
pim-1(24) . Purified Sp1 protein was shown to bind to all three
Sp1 motifs within the HIV-1 LTR(25) . We found that Vpr
trans-activation was more dramatic when the transcriptional activity of
HIV-1 LTR was lower. Our results are consistent with the notion that
Vpr function is most critical for the immediate early transcription of
HIV-1 when other positive regulators, such as NF-B, are limited or
inactive, a condition presumably present in vivo.
Figure 1:
A, templates used for in vitro transcription (see ``Materials and Methods''). Plasmids
were digested with restriction enzymes so that transcripts of expected
lengths can be generated. B, oligo sequences used for gel
mobility shift assay (Fig. 4). Oligos were synthesized as
double-stranded, and end-labeled with T4 polynucleotide kinase and
[-
P]ATP. Sp1 probe sequences are according
to the published sequence (33) and base positions are numbered
with respect to the transcription start site as ``+1''. CRE, cAMP responsive element.
Figure 4:
Vpr-Sp1 interaction. A, gel
mobility shift assay with Sp1 protein (25 ng, Promega) for lanes
1-7 and 9-14, and ATF-1 protein (25 ng) for lanes 15 and 16. Sp1 Ab is
a mouse monoclonal for Sp1 (1 µg, Santa Cruz Biotechnology). The
assay (10 µl) contained 0.1 ng of labeled probe and 5 ng of
polydI
polydC under a condition as described earlier(29) .
Human Sp1 protein was purified from HeLa cells by Promega following an
established protocol(20) . The probe sequences are listed in Fig. 1B. B, co-precipitation of the
3
Sp1 probe, but not the Sp1(I) probe, with Sp1 and Vpr-T by the
Flag antibody Affi-Gel (IBI) that recognizes the C-terminal tag in
Vpr-T. Lane 1 shows 20% of the input probe mixture without
precipitation. Radioactive bands in between the two probes and below
the Sp1(I) probe are single-stranded oligos. C, co-precipitation as in B except unlabeled 3
Sp1
oligo (100 ng) was used and the precipitated proteins examined by
Western blot with Sp1 antibody and the Flag antibody. Lane 1 was 100% of the input proteins directly
examined.
The p3Sp1-CAT plasmid was obtained by
insertion of the EcoRI-HindIII region of pUC-Sp1-LTR
plasmid into a vector containing the CAT reporter gene. The vpr(wt) gene in pET-vpr(wt) was transferred to the
RSV-HnefPFH plasmid (16) to generate
RSV-vpr(wt) plasmid. The construction of
RSV-vpr-LR-mu and RSV-S-vpr-T has been
described(16) .
Figure 2:
In vitro trans-activation of the
HIV-1 LTR by Vpr. A, in vitro transcription with nuclear
extracts prepared from different cell lines. Where indicated, HIV-1
Vpr-T protein (16) (0.5 µg) was included in the
transcription reaction. M, 1-kilobase DNA ladder (Life
Technologies, Inc.) end-labeled with P. B, quantitation of transcripts by PhosphoImager (Molecular Dynamics,
Inc.). Values were graphed as when the activity in lane 1 of A was normalized to 100. C, in vitro transcription
with templates (Fig. 1A) indicated on the right-hand
side. Upper panel, transcription with 0.5 µg each of the
LTR template and the NF-
B+Sp1 template. Lower panel,
transcription with 0.5 µg each of the LTR template and the Sp1
template. Both the Sp1+NF-
B template and the Sp1 template
yielded a 202-base transcript. Lanes 2-5 contained 0.05,
0.25, 0.5, and 1 µg of Vpr-T, respectively. D, quantitation of transcripts in C the same way as in B. Activation index was expressed as the gain in transcription
in the presence of Vpr-T divided by the control transcription without
Vpr-T.
The HIV-1 LTR contains as positive
regulatory elements two binding motifs for the transcription factor
NF-B followed by three binding motifs for the Sp1 transcription
factor(5, 6) . To decipher which region of the HIV-1
LTR mediated response to Vpr-T, three templates containing: (a) NF-
B and Sp1 motifs (NF-
B+Sp1 template), (b) Sp1 motifs (Sp1 template), or (c) neither
NF-
B nor Sp1 motifs (TATA template), were prepared (Fig. 1A). These templates produced a shorter mRNA
transcript than the full-length LTR template, and were individually
mixed with the LTR template in equal molar amounts for in vitro transcription with different amounts of Vpr-T. It was clear that
the NF-
B+Sp1 template and the Sp1 template were both
responsive to Vpr-T (Fig. 2, C and D) while
the TATA template had no detectable basal activity and no Vpr response
(data not shown). Among the three templates shown (Fig. 1A), the Sp1 template had the lowest level of
basal activity (Fig. 2C, lane 1). However, it was
activated by Vpr the most: the maximum gain in transcription reached
400% (Fig. 2D). Thus, it seemed that the Sp1 motifs in
HIV-1 LTR were sufficient to confer Vpr response. Comparison between
the results from the NF-
B+Sp1 template and the Sp1 template
suggested that presence of NF-
B in the promoter reduced Vpr
trans-activation. Thus, the NF-
B activation pathway appears not to
be targeted by Vpr. Consistent with this analysis, when the two
NF-
B motifs in HIV-1 LTR were cloned immediately upstream of the
TATA box, at position -40 of the HIV-1 LTR (refer to Fig. 1), the resulting template did not respond to Vpr (data not
shown).
Control experiments were carried out with two other Vpr-related proteins: the authentic Vpr (Vpr(wt)), and the Vpr-T mutant: Vpr-LR-mu which contains a mutation of 8 aa residues in the Leu/Ile-rich domain (LR-domain, aa numbers 60-81) of Vpr(16) . For this experiment, the Sp1 template was used alone. It was clear that the authentic Vpr(wt) activated transcription of the Sp1 template to the same extent as Vpr-T while the ability of the Vpr-LR-mu protein to trans-activate was impaired (Fig. 3, A and B). We noticed that under this condition the maximum Vpr trans-activation was lower than when the Sp1 template and the LTR template were used together (Fig. 2C, lower panel). Thus, it seemed that the LTR template competed for the basal transcription machinery much more efficiently than the Sp1 template when Vpr was absent. However, when Vpr was present, the Sp1 template gained a competitive advantage.
Figure 3: Specificity of Vpr trans-activation. A, transcription reaction was carried out with 0.25, 0.5, and 1 µg of Vpr-T (lanes 2-4), Vpr(wt) (lanes 5-7), and Vpr-LR-mu (lanes 8-10) using 1 µg of the Sp1 template. B, the highest activation indices observed for the three Vpr proteins were plotted for comparison. C, transcription reaction with 0.5 µg each of the Sp1 template and the AdML template. D, activation indices for both templates at different Vpr-T concentrations were plotted for comparison.
A template containing the TATA box basal promoter of adenovirus major late promoter (AdML) was also mixed with the Sp1 template in equal molar amounts and tested for potential activation by Vpr-T. As Fig. 3C shows, the Sp1 template gave a much higher level response to Vpr-T than the AdML template, although the latter template was also significantly activated by Vpr-T. Inspection of the AdML sequence revealed two blocks of GC-rich sequences surrounding the TATA box(26) . Preliminary studies suggested that Sp1 could bind to these sequences (data not shown). Thus, it may be possible that the observed activation of AdML promoter by Vpr also involved Sp1 binding to AdML. This may be in contrast to an earlier report which showed that Sp1 significantly activated a promoter in the absence of an identifiable Sp1 motif (27) .
In the same
assay, the authentic Vpr(wt) protein also induced a mobility shift of
the Sp1-probe complex (Fig. 4A, lane 5). The
apparently higher efficiency of Vpr(wt) was most likely due to the
higher amount of Vpr(wt) used as compared to Vpr-T, since doubling the
amount of Vpr-T also generated the same pattern. The Vpr-LR-mu protein,
which failed to trans-activate the Sp1 template (Fig. 3A), had a severely reduced activity (lane
3) and Vpr43-96 had no significant activity (lane
4). Interestingly, the SIVmac Vpr protein (S-Vpr-T) expressed and
purified the same way as HIV-1 Vpr-T(16) , was not active (lane 6), suggesting that there is a host specificity in the
Sp1 protein or a viral specificity in the Vpr protein. The potential
existence of host specificity in Sp1 was consistent with the
observation that with a HeLa nuclear extract S-Vpr-T did not
trans-activate either HIV-1 LTR or SIVmac LTR, while HIV-1 Vpr
trans-activated both templates (data not shown). With a control system,
the ATF-1 (29) bound to the cyclic AMP-responsive element (Fig. 4A, lane 15), but was not affected by Vpr-T (lane 16).
The Vpr-Sp1 interaction was further examined by
co-immunoprecipitation assays. First, the DNA-protein complex formed as
per the gel shift assay was co-precipitated by the Flag IgG-Affi-Gel
which recognizes the C-terminal tag in Vpr-T(16) . The
precipitated DNA probe was examined by gel analysis and
autoradiography. It was clear that the probe was co-precipitated only
with Vpr-T (Fig. 4B, lane 3), but not with Vpr-LR-mu (lane 4) or S-Vpr-T (lane 5). Second, the Sp1 protein
and Vpr-T protein were directly mixed and then co-precipitated with the
Flag IgG-Affi-Gel. The precipitated proteins were examined for the
presence of Vpr-T and Sp1 proteins by Western blot. Under this
condition, Sp1 and Vpr-T did not co-precipitate (data not shown).
However, when during the co-precipitation an excess of unlabeled
3Sp1 oligo was included, Sp1-Vpr-T co-precipitation was observed (Fig. 4C, lane 3). Under this condition, Sp1 did not
co-precipitate with Vpr-LR-mu (lane 4) or S-Vpr-T (lane
5).
The above results are consistent with the conclusion that
Vpr interacts with Sp1 in the context of the Sp1-DNA complex. To
decipher whether individual Sp1 motifs can support Vpr-Sp1 interaction,
the three Sp1 motifs in HIV-1 LTR were synthesized separately, and used
for the gel mobility shift assay (Fig. 1B). None of
these single Sp1 motifs supported Vpr-Sp1 interaction efficiently (Fig. 4A, lanes 9-14). In addition, during the
co-precipitation analysis, the radiolabeled Sp1(I) probe was not
co-precipitated with Vpr-T and Sp1 (Fig. 4B, lane 3).
Thus, the dependence of Vpr-Sp1 interaction on the 3Sp1 DNA, as
shown by both the gel mobility shift assay and the co-precipitation
assays (Fig. 4), suggests that Vpr forms a stable complex with
Sp1 only when Sp1 binds to multiple Sp1 motifs in the 3
Sp1 DNA.
Figure 5:
Vpr trans-activation of the minimal
promoter from HIV-1 LTR. The p3Sp1-CAT reporter plasmid (3
µg) was transfected alone (lane 1) or co-transfected with
activator plasmids (based on RSV-LTR), into CEM
174 cells by the
DEAE-dextran method. Activator plasmid amounts: 100 ng for lanes 2,
5, and 6; 200 and 400 ng for lanes 3 and 4, respectively. CAT assay was performed 2 days
post-transfection. Activation is represented as fold of percent of
converted chloramphenicol compared with lane 1, as analyzed
with a PhosphoImager (Research Dynamics).
In this report, we identified the ubiquitous cellular
transcription factor Sp1 to be a target for HIV-1 Vpr. Vpr interaction
with Sp1 was correlated with Vpr trans-activation of HIV-1 LTR. Maximum in vitro trans-activation was found with a minimal promoter
from HIV-1 LTR that contains only three Sp1 motifs and the TATA box.
This minimal promoter also had the lowest detectable level of basal
transcription activity among the templates tested. It is likely that
trans-activation of the HIV-1 LTR by Vpr plays an important role for
the immediate early transcription of the HIV-1 genome when the
alternative positive regulations, such as the one committed by
NF-B, are low. This condition apparently exists in vivo where the majority of HIV-1 infected cells are immunologically
quiescent.
Vpr interaction with Sp1 and trans-activation both
required the central Leu/Ile-rich domain (LR-domain, aa numbers
60-81), which was previously reported to be important for Vpr
interaction with a 180-kDa cellular protein RIP(16) . However,
Vpr trans-activation through Sp1 apparently is an event separable from
Vpr interaction with RIP, since a point mutation of
Arg-Ser within the LR-domain was found to abolish Vpr-RIP
interaction but not Vpr interaction with Sp1 or Vpr trans-activation. (
)It may be proposed that RIP does not participate directly
in the trans-activation process, but rather regulates Vpr stability or
participates in Vpr nuclear migration as previously
suggested(16) .
Vpr-Sp1 interaction appeared to require Sp1
binding to multiple Sp1 motifs within the HIV-1 LTR. Individual Sp1
motifs did not support Vpr-Sp1 interaction efficiently (Fig. 4).
We have recently obtained data that the two Sp1 motifs distal to the
TATA box in HIV-1 LTR are sufficient to support Vpr-Sp1 interaction
(data not shown). Based on these observations, a model may be proposed
for Vpr-Sp1 interaction (Fig. 6). In pathway a, Sp1
monomer binds to the two Sp1 motifs in the DNA and forms an unstable
dimer. This DNA-Sp1 dimer complex may dissociate during the gel
mobility shift assay, leaving predominantly a complex containing a
monomeric Sp1 (Fig. 4A). However, Vpr preferably binds
to the dimeric Sp1 and stabilizes Vpr-Sp1 dimer-DNA complex. In
addition, monomeric Sp1 bound to a single Sp1 motif may also interact
with Vpr (Fig. 6, top), resulting in a Vpr-Sp1
monomer-DNA complex. This complex is presumably unstable, but can be
strengthened by additional binding by another Sp1 monomer to the second
Sp1 motif. Since Vpr exists as an oligomer(31) , it should be
possible that one Vpr oligomer can bind to two adjacent Sp1 molecules.
We noticed that each of the three Sp1 motifs in HIV-1 LTR occupies one
-helical turn (10 base pairs) with 1 base pair space between
adjacent Sp1 motifs. Thus, two adjacently bound Sp1 molecules are most
likely on one side of the
-helical DNA. This alignment is expected
to be best for simultaneous interaction of two adjacent Sp1 molecules
with the Vpr oligomer. In pathway b, Vpr directly interacts
with free monomeric Sp1, and forms a Vpr-Sp1 monomer complex. This
complex is unstable, and additional weak interaction between the
Vpr-Sp1 monomer complex with another Sp1 monomer does not help
stabilize this complex.
Figure 6: Model for Vpr-Sp1 interaction (see ``Discussion'').
Our data also suggested that the Vpr
proteins of HIV-1 and SIVmac may be involved in host- or viral-specific
protein-protein interaction and trans-activation. The SIVmac Vpr does
not interact with the human Sp1 protein (Fig. 4) or
trans-activate with the HeLa nuclear extract (data not shown). Although
the C-terminal region of Vpr is most divergent between SIVmac Vpr and
HIV-1 Vpr, preliminary analysis suggested that this region is not
essential for Vpr-Sp1 interaction or Vpr trans-activation (data not
shown). Instead, our current study identified the LR-domain to be
important for Vpr-Sp1 interaction and Vpr trans-activation. Comparison
between the LR-domains of HIV-1 Vpr and SIVmac Vpr revealed that there
are five amino acid differences within the 22-aa
LR-domain(16) . When the LR-domain of SIVmac Vpr was
substituted for that of HIV-1 Vpr in the context of an infectious HIV-1
clone, a chimeric HIV-1 virus was obtained that showed a delayed
replication kinetics in CEM174 cells compared with the wild type
HIV-1 virus.
This observation is consistent with the
possibility that the LR-domain is involved in host- or viral-specific
functions of HIV-1 and SIVmac Vpr proteins. It also implied that Vpr
trans-activation involving the LR-domain confers an advantage to HIV-1
replication even in lymphocytes.
In summary, we provided biochemical evidence to support a specific trans-activation function associated with the HIV-1 Vpr. By targeting a ubiquitous cellular transcription factor Sp1, Vpr could affect not only HIV-1 replication, but also expression of cellular genes. Both effects of Vpr could contribute to AIDS pathogenesis. Thus, therapeutic approaches directed against the trans-activation function of Vpr should have two advantages: arresting HIV-1 replication at the very early stage of the virus life cycle, and relieving symptoms potentially caused by altered cellular gene expression induced by Vpr.