From the Laboratory of Fundamental Virology and
Immunology and
Laboratory of Medical Chemistry and Medical
Oncology, Institute of Pathology, University of Liège,
B-4000 Liège, Belgium
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ABSTRACT |
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Varicella-zoster virus open reading
frame 4-encoded protein (IE4) possesses transactivating properties for
varicella-zoster virus genes as well as for those of heterologous
viruses such as the human immunodeficiency virus type 1 (HIV-1).
Mechanisms of HIV-1 LTR (long terminal repeat) transactivation were
investigated in HeLa cells transiently transfected with an IE4
expression plasmid and a CAT reporter gene under the control of the
HIV-1 LTR. These results demonstrated that IE4-mediated transactivation
of the HIV-1 LTR in HeLa cells required transcription factor B
(NF-
B). Using the gel retardation assay, it was shown that
transfection of the IE4 expression vector in HeLa cells was not
associated with induction of NF-
B under the p50·p65 heterodimeric
form and that no direct binding of IE4 to the
B sites could be
detected. Both Western blot and immunofluorescence analyses suggested
that the ability of IE4 to activate transcription through
B motives was not connected with its capacity to override the inhibitory activities of I
B-
or p105. Finally, in vitro
protein-protein interactions involving IE4 and basal transcription
factors such as TATA-binding protein and transcription factor IIB were
carried out. A direct interaction between IE4 and TATA-binding protein or transcription factor IIB components of the basal complex of transcription was evidenced, as well as binding to the p50 and p65
NF-
B subunits. Mutagenesis analysis of IE4 indicated that the
COOH-terminal cysteine-rich and arginine-rich regions (residues 82-182) were critical for transactivation, whereas the first 81 amino
acids appeared dispensable. Moreover, the arginine-rich region is
required for the in vitro binding activity, whereas the
COOH-terminal end did not appear essential.
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INTRODUCTION |
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Varicella-zoster virus
(VZV)1 is an -herpesvirus,
which causes two distinct diseases in man: chicken pox and shingles.
Shortly after entry into the infected cells, VZV genes are expressed in a temporal cascade. The immediate-early genes are expressed first; these stimulate early gene expression, providing most of the proteins necessary for viral DNA replication. After DNA synthesis has occurred, genes of the late class, which mainly encode structural proteins, are
expressed. This orderly pattern of expression has been proposed mainly
by comparison with herpes simplex virus type 1 (HSV-1) (1), another
-herpesvirus that has been intensively studied. The use of transient
transfection assays has clearly shown that several VZV proteins,
i.e. those encoded by ORFs (open reading frames) 4, 10, 29, 61, 62, and 63, possess regulatory properties (2-8). Three of these
polypeptides, encoded by ORFs 4, 62, and 63, are expressed during the
immediate-early phase of lytic infection (9-11), and are thus referred
to as IE4, IE62, and IE63. Hence, VZV immediate-early proteins
contribute to the control of the viral cycle progression as in other
herpesviruses.
The IE4 protein is a transactivator of gene expression whose regulatory
properties are not yet fully understood (2, 4, 11-14). IE4 stimulates
VZV gene expression regardless of the cell type envisaged,
i.e. monkey fibroblasts or human T lymphocytes (2, 4, 11,
12). It also appears that IE4 is capable of heterologous
transactivation (11-13). The available data suggest that IE4 could
exert its functions through transcriptional and post-transcriptional
mechanisms (11-13). Evidence for transcriptionally mediated activation
was provided by the characterization of critical cis-elements necessary for the IE4 inducibility of
heterologous viral promoters (13). The presence of a TATA box alone is
apparently not sufficient to convey transactivation by IE4, because
transactivation of a minimal promoter containing only a TATA box is not
possible (13). Two different factor-binding sites, the Sp1 and nuclear factor B (NF-
B)-responsive elements, have been proposed as
critical for IE4-mediated transactivation (13).
Protein-encoding genes in eukaryotes are transcribed by RNA polymerase
II associated with general transcription factors. Control of their
expression requires specific regulatory factors with DNA binding and
transactivation domains that interact with the general transcription
factors. These interactions influence RNA polymerase II recruitment to
the promoter and stabilize and/or modify polymerase II holoenzyme
activity (15). The ubiquitous Sp1 factor is a transcription factor that
participates in the basal activity of the transcription machinery by
binding to specific sites that have been found in numerous core
promoters. Sp1 possesses a zinc finger DNA-binding domain as well as a
glutamine-rich transactivation domain, and its activity seems to be
controlled by its phosphorylation status (16-18). NF-B consists of
a group of dimeric complexes composed of varied combinations of
polypeptides of the Rel/NF-
B family (19). These factors are
ubiquitous and have been involved in transcription regulation of
cellular genes important in the inflammatory response and oxidative
stress (19-21). The activation of NF-
B is dependent on the binding
of these complexes to cis-sequences found in the promoter
regions of target genes (20, 22). The prototypic form of NF-
B is a
heterodimer containing the p50 and p65 proteins (19, 21). In most
cells, NF-
B activity is kept under control through cytoplasmic
sequestration by members of a family of inhibitory proteins, including
I
B-
and p105, the latter being the cytoplasmic precursor of p50
(23). The majority of induction signals lead to the rapid
phosphorylation of I
B-
at serine 32 and serine 36 (24), which
targets it to the ubiquitin-proteasome pathway (25). Subsequent
proteolytic degradation of I
B-
allows the nuclear translocation
of the active NF-
B dimer. A large number of stimuli such as
mitogens, cytokines, stress, and viruses have been shown to induce
NF-
B (19, 21).
Because viruses are intracellular parasites, they appropriate the
cellular machinery for their own benefit. NF-B belongs to the host
transcription factors used by a number of viruses to induce their own
expression or that of specific host genes (19). Inducible human
immunodeficiency virus type 1 (HIV-1) gene expression is generally
mediated by the binding of NF-
B to the enhancer
B-binding sites
in the long terminal repeat (22, 26, 27). Molecular interactions among
herpesviruses and HIV-1 have been frequently investigated, as the most
common opportunistic viral infections in individuals with AIDS are
caused by herpesviruses (28-30). Herpesviruses could act as
co-factors in enhancing HIV-1 replication. Direct effects of
herpesvirus proteins on HIV-1 LTR activity have been detected in the
case of HSV-1 (31-33), cytomegalovirus (CMV) (34, 35), and
Epstein-Barr virus (EBV) (36). Some of these effects could be
associated with NF-
B induction (27, 33, 36-38), although some
controversy exists as to the identity of the responsible proteins in
HSV-1 (32, 37, 39, 40) and CMV (34, 35, 41). Such discrepancies might
possibly be attributed to the different cell lines used in the various studies (31, 34, 35). The mechanisms of NF-
B induction by the latent
membrane protein (LMP1) of EBV, for example, are post-translational and
involve I
B-
degradation (38). Little is known about the mechanism
of LTR induction by VZV, but it was shown that VZV infection of HeLa
cells could stimulate HIV-1 LTR activity (42). It has also been
demonstrated that a DNA fragment carrying ORFs 61, 62, and 63 could
transactivate the HIV-1 LTR in transient transfection (32). We and
others have shown that IE4 could also stimulate the LTR in CAT assays
(12, 13, 43). This transactivation seems to occur transcriptionally
(11, 13).
The purpose of this report was to clarify the molecular mechanisms of
IE4-mediated transactivation of the HIV-1 LTR. Our results indicate
that B-binding sites are essential for LTR transactivation by IE4.
However, under our experimental conditions, neither significant NF-
B
induction nor degradation of I
B-
or p105 could be detected. In vitro protein interaction experiments showed that IE4
could interact with TBP, TFIIB, p50, and p65, which suggests that IE4 could enhance the efficiency of the NF-
B complexes bound to their sites in the LTR through interactions with the activators and the
general transcription machinery. Mutational analysis was performed in
an attempt to identify the critical domains of IE4 involved in these
protein-protein interactions.
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MATERIALS AND METHODS |
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Cells and Transfections-- The HeLa human cervical epithelioid carcinoma cell line was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfections were performed using the LipofectAMINE reagent (Life Technologies, Inc.). The total amount of DNA was kept constant under the different conditions by addition of sonicated herring sperm DNA or pCMV expression vector without insert. Whole cell extracts were prepared by the freeze-thaw method, and CAT assays were performed as described previously (12). CAT assay data were collected from at least four independent transfection experiments.
Plasmids--
Plasmid pCMV-4, which carries VZV ORF4 under
the control of the powerful CMV promoter-enhancer, was a gift from Dr.
P. Kinchington (University of Pittsburgh, Pittsburgh, PA). In pGi4, IE4
expression is directed from the cognate ORF4 promoter (2). Plasmid
pCMV-Tax directed HUMAN t CELL LEUKEMIA VIRUS (HTLV-1) Tax
expression from the CMV promoter/enhancer (44). IB-
was expressed
from pRSV-I
B in which the I
B-
gene was under the control of
the Rous sarcoma virus LTR (a gift from Dr. U. Siebenlist, National
Institutes of Health, Bethesda, MD). Plasmids pHIV-CAT and pHIV*-CAT
have been described previously (28). In these constructs, the wild-type LTR of HIV-1 and an LTR mutated in both
B sites, respectively, are
cloned upstream of the CAT gene. p
B-CAT contains the HIV-1
B
enhancer element upstream of the c-fos TATA box (45, 46). The expression plasmids used for eukaryotic expression of p50 and p65
were those employed previously (45), and p105 was similarly cloned at
the EcoRI site of the pMT2T vector (a gift from Dr. U. Siebenlist).
Gel Retardation Assays--
Nuclear proteins from
untreated, phorbol myristate acetate-treated (100 ng/ml), or
transfected cells were prepared by high salt extraction (49). Five
micrograms of nuclear extracts were incubated for 20 min at room
temperature with 0.2 ng of 32P-labeled B oligonucleotide
(45) in a volume of 10 µl containing 20 mM Hepes-KOH, pH
7.9, 75 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5 mM MgCl2, 1 mg of bovine serum albumin, 1.3 µg of poly(dI-dC), and 1 mM dithiothreitol. DNA-protein
complexes were analyzed on a low ionic strength 4% or 6%
polyacrylamide gel. Following electrophoresis, gels were dried and
submitted to autoradiography overnight at
80 °C. To quantify
DNA-protein complexes, gels were further analyzed with a PhosphorImager
scanner equipped with Image Quant software (Molecular Dynamics,
Sunnyvale, CA). The specificity of the complexes was confirmed by
competition experiments with a 50-fold molar excess of cold wild-type
or mutant oligonucleotide (45). To characterize proteins in the
electrophoretically retarded complexes, nuclear extracts were incubated
for 15 min on ice with specific antibodies to p50, p65 (50), p52, c-Rel
(51), and IE4 (52) before the 32P-oligonucleotide was
added.
Western Blot Analysis--
Cells were rinsed three times in
phosphate-buffered saline, then scraped in phosphate-buffered saline
and lysed either in 1% (w/v) sodium dodecyl sulfate (SDS) buffer to
extract total proteins or in high salt buffers to extract nuclear and
cytoplasmic proteins (49). Ten micrograms of proteins were resolved by
12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to
polyvinylidene difluoride membranes (Millipore, Inc., Bedford, MA).
Membranes were blocked with 5% (w/v) milk powder in TBS-T buffer (20 mM Tris-HCl, pH 7.6, 500 mM NaCl, 0.1% (v/v)
Tween 20). Incubation with the primary antibodies was performed for 1 h at room temperature under agitation in TBS-T buffer with 5% (v/v) milk blocking reagent (Amersham Pharmacia Biotech, Brussels, Belgium). The rabbit antisera used were a peptide-specific antiserum for either p50 (first 13 amino acids) (50) or p65 (amino acids 531-550, Santa Cruz Biotechnology, CA) and an antiserum directed against a fusion protein between GST and the full-length IB-
(a
gift from Dr. U. Siebenlist). Immunoreactive proteins were detected by
using anti-rabbit immunoglobulin G from pig conjugated to horseradish
peroxidase (DAKO). After several washes, blots were prepared for
enhanced chemiluminescence (ECL) detection as prescribed by the
manufacturer (Amersham Pharmacia Biotech, Brussels, Belgium). Band
intensities on the impressed films were quantified by photodensitometry
(LKB, Sweden). To perform protein synthesis inhibition experiments,
cells were exposed to cycloheximide (50 µg/ml; Sigma).
In Vitro Analysis of Protein-Protein Interactions--
GST,
GST-IE4, GST-TK, GST-p50 (50), and GST-TFIIB (Dr. M. Müller,
University of Liège, Belgium) were expressed in bacteria following classical induction with 0.1 M
isopropyl-1-thio--D-galactopyranoside) for 1-3 h at
37 °C. Lysates were prepared using the anionic detergent N-laurylsarcosine (Sarcosyl) 1.5% (v/v) in STE (10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA). Following three cycles of sonication, bacterial
debris were removed by centrifugation. Proteins were then purified on
glutathione-Sepharose 4B affinity beads (Amersham Pharmacia Biotech) in
STE-Triton 4% (v/v). Protein-coupled Sepharose beads were washed eight
times in phosphate-buffered saline before the interactions were
performed. Proteins were labeled with [35S]methionine
(ICN, Brussels, Belgium) using the in vitro coupled transcription/translation system from wheat germ extracts or
reticulocyte lysates (Promega Inc., Madison, WI). A control for
in vitro transcription is provided by production of the
firefly luciferase (61 kDa). Equal amounts of 35S-labeled
proteins were incubated with 50 µl of protein-coupled Sepharose beads
in 400 µl of NETN (20 mM Tris-HCl, pH 8, 100 mM NaCl, 1 mM EDTA, 1.5% (v/v) Nonidet P-40).
Binding reactions were allowed to take place for 1 h at room
temperature, and the beads were then washed six times in NETN. Bound
proteins were eluted by boiling for 2 min in 1× SDS sample buffer,
followed by loading on 12% SDS-PAGE. Gels were subsequently dried and
autoradiographed. To ensure optimal washing conditions, 5 µl of
35S-labeled luciferase were incorporated into each
interaction reaction and disappearance of the corresponding band was
followed.
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RESULTS |
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NF-B Binding Motifs Are Required for HIV-1 LTR Activation by
IE4--
Transactivation of the HIV-1 LTR by the IE4 protein has been
detected in Vero cells and human A3.01 T-cells (2, 12, 13). In the
A3.01 cells, IE4 inducibility was dependent on the presence of Sp1
and/or NF-
B binding motifs. We set out to analyze LTR transactivation by IE4 in HeLa cells, because it has been shown that
VZV infection induces LTR activation (42) and because NF-
B induction
is well documented in these cells (53). To characterize the role of
NF-
B, a full-length LTR-CAT construct (pHIV-CAT) or a promoter
harboring point mutations in both
B elements (pHIV*-CAT) (Fig.
1A) was transfected in the
presence or absence of an IE4-expressing vector, pGi4. Transfection of
the pHIV-CAT construct resulted in a basal level of CAT activity,
whereas pHIV*-CAT elicited no detectable levels of CAT activity under
the same conditions (data not shown). In the presence of IE4, pHIV-CAT
was transactivated up to 58-fold, but no increase in CAT activity was
seen with pHIV*-CAT (Fig. 1B). To further analyze the role
of NF-
B, a minimal construct p
B-CAT containing both HIV-1
B
sites upstream of the c-fos TATA box was used (Fig.
1A). The activity of this minimal promoter has been shown to
be entirely associated with NF-
B (46, 50). p
B-CAT displays a
higher basal activity than pHIV*-CAT. Transactivation of this promoter
by IE4 reached a 10-fold activation, lower than with the full-length
LTR (Fig. 1B). These experiments were also performed in Vero
cells. In these cells, pHIV-CAT and p
B-CAT were stimulated by IE4
(1.5 µg and 2.5 µg of expressing vector) as in HeLa cells but a
clear activation was also seen with pHIV*-CAT (data not shown),
suggesting that a NF-
B-independent mechanism co-existed with the
NF-
B-dependent mechanism.
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Influence of IE4 on NF-B Induction in HeLa Cells--
Having
established that the NF-
B-responsive elements are essential to
IE4-driven transactivation of the HIV-1 LTR in HeLa cells, we wanted to
determine whether IE4 could induce NF-
B in this cell line.
Therefore, HeLa cells were transiently co-transfected by the
IE4-expressing vector (pCMV-4) or by a vector without insert (pCMV) and
pHIV-CAT. Because the HTLV-1 Tax protein has the capacity to activate
members of the NF-
B family (54, 55), the effect of a Tax-expressing
vector (pCMV-Tax) was tested in parallel. Whole cell and nuclear
protein extracts were prepared from these cells. Fig.
2A shows the
dose-dependent stimulation of the HIV-1 LTR by both IE4 and
Tax. -Fold stimulation of CAT activity was of the same order of
magnitude in the presence of the two proteins. To detect NF-
B
activation, gel retardation assays were performed using a probe
encompassing the
B sites present in the HIV-1 LTR. Incubation of
nuclear extracts from non-transfected cells (used as an uninduced
control) with the
B probe exhibited a very low level of the specific
NF-
B band (Fig. 2B, lane 1). Transfection of
pCMV or pCMV-4 had little effect on the amount of the NF-
B complex
(Fig. 2B, lanes 2 and 3), whereas a
strong induction was seen with pCMV-Tax (Fig. 2B, lane
4). The electrophoretic mobility of these DNA-protein complexes
was identical to that induced by phorbol myristate acetate, a powerful
NF-
B inducer (19). These complexes specifically competed with an
excess of unlabeled
B oligonucleotides, but an excess of mutant
oligonucleotides, which are devoid of NF-
B binding activity, had no
effect (data not shown). The immunological characterization of these
complexes demonstrated the presence of p50 and p65 and the absence of
p52, c-Rel, and IE4 (data not shown). Therefore, IE4 does not bind to
the
B sites. Under these experimental conditions, expression of IE4
is not associated with a nuclear translocation of the p50·p65 NF-
B
complex, whereas expression of Tax induces such a translocation.
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IE4 Has No Effect on the Cytoplasmic Retention of Ectopic p50 and
p65 by the IB-
and p105 Inhibitors--
To ascertain the absence
of NF-
B induction, we looked at the degradation of the cytoplasmic
inhibitors I
B-
and p105 in cells co-expressing various members of
the Rel/NF-
B family (i.e. p50, p105, p65, and I
B-
)
with IE4. This methodology was applied previously in the molecular
characterization of NF-
B activation (54). Multiple transient
co-transfections involving p50 and p65; p50, p65 and I
B-
; and
p50, p65, I
B-
, and IE4 were therefore conducted. Every
transfection condition was analyzed by Western blotting and
immunofluorescence to determine the intracellular repartition and
expression levels of each protein. To conduct the immunoblotting
experiments, nuclear and cytoplasmic proteins were first extracted from
transfected cells prior to resolution on SDS-PAGE. As shown in Fig.
3, the anti-p50 antibody detected the
intrinsic p50 and p105 proteins as well as a third band on the gel
which corresponds to the ectopic p50. The molecular weight difference
between transfected and endogenous p50 is a consequence of the
pMT2T-p50 construction (45). Ectopic p50 is as predominantly nuclear as
the intrinsic p50; upon expression of I
B-
, a significant reduction in nuclear levels of ectopic p50 could be observed (Fig. 3).
No clear effect of IE4 on these levels could be evidenced when the
IE4-expressing vector was included. Similarly, IE4 had no influence on
the nuclear p65 levels in the presence of I
B-
(Fig. 3). These
observations were connected with the analysis of I
B-
levels. As
expected, ectopic I
B-
was predominantly cytoplasmic (Fig. 3).
Presence of IE4 in the transfected cells did not lead to a modification
in either cytoplasmic or nuclear levels of I
B-
. The second
pathway of investigation, which was to follow intracellular
localization of the NF-
B/Rel proteins by immunofluorescence,
confirmed that expression of IE4 had no significant effect on the
intracellular repartition of p50 and p65 (data not shown).
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IE4 Interacts with Several Transcription Factors in Vitro--
The
results obtained from the gel shift assays and the Western blot
analysis strongly suggest that the NF-B-dependent
activation of the LTR by IE4 must depend on other mechanisms than the
translocation of NF-
B to the nucleus. Stimulation of transcription
by activators results from multiple direct and indirect interactions
between these activators, the co-activators and components of the basal transcription complex (57). Thus, p65, which possesses an acidic transactivation domain, was shown to interact with the general transcription factors TBP and TFIIB (58, 59) as well as with the p300
co-activator (60). Due to the lack of information concerning the
molecular properties of IE4, its capacity to bind in vitro to TBP and TFIIB was investigated. A fusion protein between GST and IE4
was expressed in E. coli and purified on
glutathione-Sepharose beads. A GST-TK protein fusion that carries VZV
thymidine kinase was purified according to the same procedure and used
as a negative control in addition to GST alone. p50 fused to GST also
served as a ligand in this experiment. Equal amounts of in
vitro translated [35S]methionine-labeled TBP and
TFIIB were incubated with GST, GST-TK, GST-p50, and GST-IE4-coupled
Sepharose beads. A definite amount of 35S-labeled
luciferase protein was also included in each interaction reaction.
After extensive washing, which was monitored by disappearance of the
luciferase signal, bound proteins were eluted in 1× SDS sample buffer
and analyzed by SDS-PAGE. Most of the TBP and TFIIB interacted
specifically with GST-IE4 (Fig. 4,
A and B, lanes 3 and 4),
whereas they were not retained by the control GST and GST-TK-coupled
Sepharose beads (Fig. 4, lanes 1 and 2 in both A and B). No interaction of TBP and TFIIB with
GST-p50 could be evidenced (Fig. 4B, lane 3; data
not shown). VZV IE63 (in a GST-IE63 fusion protein), another VZV
regulatory protein, was also tested for its ability to bind to TBP and
TFIIB under the same conditions; no definite interactions could be
demonstrated (data not shown). Because NF-
B is required for the
IE4-mediated stimulation of the HIV LTR, we also assessed the binding
of IE4 to p50 and p65. As shown in Fig. 4 (C and
D), GST-IE4 could associate in vitro with p65 and
p50 (lanes 3 and 4, respectively). No interaction was detected with the GST or GST-TK controls (lanes 2 and
4 and lanes 1 and 2, respectively). As
expected, radiolabeled p65 or p50 interacted with GST-p50 (Fig.
4C, lane 1; Fig. 4D, lane
3). To ensure that interactions were not an artifact due to the
GST-IE4 construct, reciprocal experiments involving GST-p50 and
GST-TFIIB fusion proteins and in vitro translated IE4 were
conducted. The GST-p50 and GST-TFIIB ligands were able to bind
efficiently to the [35S]methionine-labeled IE4, because
the IE4 protein was detected in the GST-p50 and GST-TFIIB eluates but
not in that from the GST and GST-TK immobilized on
glutathione-Sepharose beads (Fig. 5A). Binding was detected when
the GST-IE4 construct was used as a ligand for IE4, suggesting that the
IE4 protein could oligomerize in vitro.
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DISCUSSION |
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The molecular mechanisms underlying VZV IE4 regulatory properties are still greatly misunderstood, although it now seems clear that IE4 is a strong transactivator whether expressed alone or in synergy with VZV IE62 (2, 4, 5, 12, 43). In the past, interesting evidence has been presented concerning the identification of specific upstream elements involved in IE4 inducibility (13). Moreover, the same study established that IE4 could act transcriptionally in A3.01 cells using the HIV-1 LTR as a model system for investigation. Our previous work based on mRNA steady-state level analysis had led us to conclude that IE4 could fulfill its functions through transcriptional and post-transcriptional mechanisms (11, 12). Because the transcriptional effect was also studied using the HIV-1 LTR as experimental system, the present work was focused on the pathway of LTR activation.
The first main observation from the transfection assays is that the
B-responsive elements are essential to IE4-mediated activation in
HeLa cells, inasmuch as an LTR mutated at both
B sites is refractory
to the action of IE4. Interestingly, this mutant construct was still
transactivated in Vero cells, indicating that the other cis-acting sequences present in the LTR contribute to IE4
inducibility in these cells. Co-expression of the cytoplasmic inhibitor
I
B-
prevented transactivation of the HIV LTR by IE4, which
clearly confirmed that NF-
B is required in HeLa cells. Despite some
controversy as to the nature of the HSV-1 IE proteins implicated in the
transactivation of the LTR by HSV-1, it is currently thought that
ICP27, the IE4 homolog in HSV-1, has no effect on HIV-1 LTR expression
(32, 39). Therefore, our study demonstrates another difference in the
properties of IE4 and ICP27, inasmuch as IE4 is able to activate the
HIV-1 LTR.
Several viral proteins (LMP1, Tax, IE1, etc.) are capable of triggering
NF-B induction (35, 36, 38, 61). Transactivation of the HIV-1 LTR by
LMP1 of EBV or IE1 of CMV is associated with an increase in NF-
B
binding to the
B-responsive elements (35, 36). In HeLa cells, no
significant NF-
B induction was observed after transient transfection
of the IE4 expression vector, whereas an important induction was seen
with Tax. It should be mentioned here that LTR activity was stimulated
to similar if not higher levels by IE4 than by Tax. Moreover, the
strong NF-
B induction seen with Tax indicates that our experimental
conditions allow for detection of such an effect and that the poor
signal seen with IE4 is not an artifact due to a low transfection
efficiency. Therefore, IE4 is not a strong inducer of NF-
B, unlike
Tax, LMP, or IE1. Recent lines of evidence indicate that Tax promotes
translocation of NF-
B from the cytoplasm into the nucleus by
liberating NF-
B from several distinct cytoplasmic complexes (54, 55,
61, 62). We detected the I
B-
degradation induced by Tax but,
under the same experimental conditions, IE4 was not capable of
triggering I
B-
proteolysis. Moreover, the Western blot analysis
of transfected I
B-
levels in the presence of IE4 corroborated the
CAT assays, illustrating the absence of IE4 transactivation in the
presence of ectopic I
B-
. It should be noted that experiments with
transiently transfected Tax have previously allowed detection of a
variation in levels or repartition of NF-
B/Rel proteins by Western
blot or immunofluorescence (54, 56). The degradation of I
B-
, another isoform of I
B, is frequently associated with agents that elicit a persistent NF-
B response, including bacterial
lipopolysaccharide and interleukin-1 (63). Similarly, the constitutive
activation of NF-
B in Tax-expressing cells could possibly be
maintained by the chronic down-regulation of I
B-
protein
expression (55). I
B-
turnover in IE4-transfected cells could also
be investigated in the future; however, this hypothesis seems unlikely
as no NF-
B induction is observed with IE4.
Our in vitro binding experiments shed new light on the
mechanism by which IE4 could cooperate with NF-B, thereby activating transcription of the HIV-1 LTR. An interaction between IE4 and the TBP
or TFIIB components of the basal complex of transcription was evidenced
in our GST pull-down experiment. A significant binding of IE4 to itself
and to the p50 and p65 NF-
B subunits was also observed. The use of
mutant proteins provided interesting data on the properties of IE4.
Deletion of the COOH-terminal region rich in His/Cys residues led to a
total loss of transactivation, although the capacity to interact with
itself or with p50 or TFIIB was not totally diminished. Therefore, the
59 COOH-terminal amino acids are not sufficient in themselves to
mediate the physical interaction between IE4 and the GST ligands. The
point mutation at residue 426 showed that this Cys is not a critical
residue for the binding of IE4 in vitro but is very critical
for the transactivation of the HIV-1 LTR. Two NH2-terminal
truncated mutants that are fully functional in vivo are
still capable of interacting with IE4, TFIIB, and p50. The
amino-terminal acidic region is thus not required for the
transactivation of the HIV-1 LTR. This observation is in accordance
with a previous report proposing a role for this region in the
transactivation of reporter genes carrying minimal poly(A) signals
(14). Removal of the arginine stretch from amino acids 70 to 82 did not
influence either the binding or transactivating capacities of IE4. In
contrast, the
182 mutant, which had lost the property to interact
with IE4, TFIIB, and p50, displayed an abrogated transactivation
capacity. The region between amino acids 82 and 182, which contains
arginine- and serine-rich clusters, appears crucial to the regulatory
functions of IE4. Although this mutant protein turns out to be as
stable as IE4, we cannot exclude that the lack of biological activity
of this mutant reflects a major structural alteration. Deletion or
mutation of the COOH-terminal region prevents transactivation without
completely abolishing binding, possibly by disrupting a critical
contact point. Despite the COOH-terminal modifications, the interaction
could still be mediated by the amino-terminal region. Indeed, a
pull-down experiment carried out between a GST-IE4 (1-402) and an
in vitro translated IE4 containing a stop codon at position
393 demonstrates that these molecules can still dimerize (data not
shown), suggesting that the amino-terminal part of IE4 plays an
important role in dimerization. This dimerization hypothesis will be
tested in the future with the analysis of mutants in a two-hybrid
system. Meanwhile, computer analysis of the amino-terminal part of IE4
reveals the presence of a bZIP-basic domain at position 120, within the
arginine-rich region. Because this domain has been shown to be required
for the dimerization of many transcription factors (64), this
observation reinforces the potential importance of the arginine-rich
region in dimerization. The arginine-rich motives that have been
identified in ICP27 have been implicated in the properties of the
protein (65). Mutant proteins deleted in these regions cannot
complement infection with an HSV-1 virus deficient in ICP27. The
arginine residues are required for correct HSV-1 late gene expression. They could be important in the post-transcriptional functions of ICP27,
given that they are involved in RNA binding activity, as shown for RGG
motifs found in numerous RNA-binding proteins (66). Our present data
suggest that the arginine regions of IE4 could be involved in physical
protein-protein interactions. Deleting this region either removes a
critical domain or disrupts the overall structure of the protein in
such a way that binding is prevented. Experiments are currently being
conducted to investigate the role of these regions in the
transcriptional and post-transcriptional properties of IE4.
It has been shown that a GAL4-IE4 chimera transactivates a promoter
carrying GAL4-binding sites, suggesting that once IE4 is brought to the
vicinity of the promoter it can activate transcription (13). The
interactions between IE4 and p50·p65 on the one hand and between IE4
and TBP and TFIIB on the other suggest that IE4 could be targeted to
the vicinity of the basal transcription complex through protein-protein
interactions with DNA-binding proteins tethered to their sites and
could therefore stabilize or enhance processivity of the multimeric
transcription complex. This activity is reminiscent of a co-activator,
which stimulates activator-dependent transcription (67),
and of Bcl-3 activity, which could tightly associate to p52 homodimers
to transactivate through the B motif (51). p65 also binds to TBP and
TFIIB, which contact next to each other the promoter site (58, 59). It
could therefore be possible that the redundant binding of IE4 to both
general transcription factors and NF-
B increases initiation of
transcription. There is accumulating evidence that interactions with
multiple transcription factors, activators, and co-activators exist for viral transactivators such as Tax of HTLV-1 or E1A of adenovirus. E1A
has been shown to bind to TBP and p65 to promote LTR transactivation. E1A is also capable of binding to p300, a co-activator recruited to a
number of unrelated promoters by protein-protein interactions, although
this interaction is not involved in LTR transactivation (68). Recently,
Tsukada et al. (69) have established that Tax stimulates
human pro-interleukin-1
promoter by direct interactions with the
transcription factors NF-IL6 and Sp-1. It is therefore possible that
IE4 represents a critical co-activator used by VZV to divert cellular
transcription factors and enhance viral-specific gene expression. If
so, IE4 could act as an adaptator/bridging factor for the VZV IE62
protein. It is interesting that a co-activating fraction termed USA
(upstream factor stimulatory activity) purified from HeLa cells is
required for NF-
B activation of the HIV-1 LTR in cell-free
transcription systems (70). A PC1 co-factor originating from this USA
fraction was shown to enhance p65-dependent activation in
this cell-free reconstruction of transcription (59). This USA fraction
is also required for activation by USF (upstream stimulatory factor),
which is a helix-loop-helix regulatory factor (70). Now, this USF
cooperates with IE62 to activate expression of VZV genes 28 and 29 (71). We know that IE4 and IE62 act synergistically to stimulate
expression of genes unresponsive to IE4 alone (2, 4, 12, 43), whereas
IE62 induces nuclear translocation of IE4 in transfected cells (52).
This suggests very strongly that the increase in IE62-mediated
transactivation by IE4 could potentially involve a collaboration of IE4
with this USA and USF/IE62, which might be involved in the LTR
transactivation. Various experiments are currently under way in our
laboratory to further define the interplay between IE4, IE62,
activators, co-activators, and transcription factors both in
vitro and in vivo. Direct effects of IE4 on
transcription will be envisaged using in vitro transcription
assays. These should help to elucidate the molecular mechanisms
implicated in transcriptional activation by the multifunctional
IE4.
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ACKNOWLEDGEMENTS |
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We thank P. Kinchington, J. M. Ostrove, and M. Müller for providing plasmids. We are very grateful to U. Siebenlist for the different plasmids and antibodies. We extend our gratitude to E. Janssens for technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants from the Belgian National Fund for Scientific Research (Brussels, Belgium), the VZV Research Foundation (New York), the Concerted Action Program (Communauté Française de Belgique, Brussels, Belgium), SmithKline Beecham Biologicals (Rixensart, Belgium), and the SIDACTION Program (Paris, France).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a FNRS-Télévie grant and a VZV Research Foundation fellowship.
¶ Supported by the Concerted Action Program.
** Research Associate at the Belgian National Fund for Scientific Research.
Research director at the Belgian National Fund for Scientific
Research. To whom correspondence should be addressed: Laboratory of
Fundamental Virology and Immunology, Institute of Pathology B23,
University of Liège, B-4000 Liège, Belgium. Tel.:
32-4-366-24-42; Fax: 32-4-366-24-33; E-mail: jpiette{at}ulg.ac.be.
1
The abbreviations used are: VZV,
varicella-zoster virus; LTR, long terminal repeat; HIV-1, human
immunodeficiency virus type 1; GST, glutathione
S-transferase; PCR, polymerase chain reaction; HSV, herpes
simplex virus; ORF, open reading frame; IE, immediate-early; NF-B,
nuclear factor-
B; USA, upstream factor stimulatory activity; USF,
upstream stimulatory factor; CMV, cytomegalovirus; EBV, Epstein-Barr virus; WT, wild-type; CAT, chloramphenicol acetyltransferase; TBP,
TATA-binding protein; TFIIB, transcription factor IIB; PAGE, polyacrylamide gel electrophoresis; HTLV, human T cell leukemia virus.
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REFERENCES |
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