(Received for publication, November 28, 1995; and in revised form, February 7, 1996)
From the
VP16 activates transcription by stimulating initiation, and for
this function the aromatic residue at position 442 within its
activation domain is critical. Recent studies have suggested that VP16
also stimulates transcriptional elongation. It has been shown that VP16
can activate transcription tethered downstream of the transcription
start site to RNA. Here, we analyze the synergistic activation features
of hybrid VP16 fusion proteins when tethered simultaneously to RNA
downstream of the start site and to DNA upstream of a promoter in order
to investigate its role in postinitiation control of transcription.
Upon targeting the VP16 activation domain simultaneously to both DNA
and RNA, high levels of transcriptional synergism is observed.
Importantly, a transcription-defective VP16 minimal activation domain
(amino acids 413-453) mutated at critical residue 442
(phenylalanine proline) maintained synergism, when bound to RNA,
with the DNA-bound wild-type VP16 minimal activation domain. Targeting
of this ``functionally defective'' VP16 minimal activation
domain via RNA and an intact activation domain via DNA allowed us to
uncover a postinitiation activity for VP16 not previously detected in
DNA targeting studies. We suggest that, in addition to stimulating
initiation, VP16 also acts at a postinitiation step involving residues
other than the critical residue at position 442 within the same
41-amino acid minimal activation domain (amino acids 413-453)
required for initiation.
VP16 protein is a component of the herpes simplex virus virion
and in addition to its structural role selectively activates
transcription of the viral immediate early genes during lytic infection
by interacting with host cellular proteins that bind to specific
enhancer elements located upstream of each immediate early gene
(reviewed in Thompson and McKnight(1992)). The C-terminal 78-amino acid
region of VP16 (amino acids 413-490) is highly acidic, and when
fused to the DNA binding domain of the heterologous yeast protein GAL4
can activate transcription potently from target promoters containing
GAL4-binding sites (Sadowski et al., 1988; Cousens et
al., 1989). Mutational analysis of the C-terminal activation
domain of VP16 suggests that this domain can be subdivided into the
proximal subdomain extending from amino acids 413-456 and the
most C-terminal distal subdomain spanning amino acids 457-490. It
is concluded, from studies using mammalian cells, that both subdomains
are required for overall efficient transcriptional activation by VP16
(Regier et al., 1993; Walker et al., 1993). However,
the proximal subdomain (amino acids 413-456) of VP16 constitutes
the minimal activation domain and, when fused to GAL4 DNA binding
domain, can activate transcription from a minimal promoter, retaining
about 50% of the activity of the full-length domain (Triezenberg et
al., 1988; Greaves and O'Hare, 1989). Studies have shown
that the aromatic residue phenylalanine at position 442
(Phe) is critical for the function of this truncated
activation domain rather than its net negative charge (Cress and
Triezenberg, 1991; Regier et al., 1993). Mutation of
Phe
to proline (F442P) within this minimal activation
domain abolished transcriptional activity in both in vivo and in vitro transcription assays even when tethered to multiple
DNA-binding sites highlighting the critical role of this residue and
subdomain in the activation process (Cress and Triezenberg, 1991;
Regier et al., 1993; Walker et al., 1993; Jiang et al., 1994). This region of VP16 interacts in vitro with components of the basal transcription complex, TFIID (
)(Stringer et al., 1990) and TFIIB (Lin and Green,
1991; Lin et al., 1991). Mutation of phenylalanine to proline
(F442P) disrupted the interaction with both TFIIB and TBP in vitro (Ingles et al., 1991; Lin and Green, 1991; Lin et
al., 1991). It is, therefore, believed that VP16 stimulates
transcription by interactions with TFIID and/or TFIIB to increase
assembly of the preinitiation complex (Choy and Green, 1993; Chi et
al., 1995). VP16 has also been shown in vitro to bind
basal transcription factors, TFIIH (Xiao et al., 1994) and
human positive coactivator 4 (PC4) (Ge and Roeder, 1994), and mutation
at position 442 (F442P) substantially reduced these interactions.
The distal most C-terminal 39-amino acid region has been shown to
constitute an independent domain and to have the ability to activate
transcription, but poorly compared to the proximal region (Regier et al., 1993; Walker et al., 1993). Interestingly,
this region of VP16 was found in vitro to directly bind
dTAF40 and hTAF
32, subunits of Drosophila and human TFIID, respectively, both of which in turn bind to
TFIIB, suggesting a ternary interaction among the activator VP16, the
coactivator TAF, and the basal transcription initiation factor TFIIB
(Goodrich et al., 1993; Klemm et al., 1995). Thus,
this subdomain may also contribute to VP16's transcriptional
activity by stimulating initiation of transcription. Recent studies
have suggested that, in addition to stimulating transcription
initiation, VP16 also plays a role in transcription elongation
(Yankulov et al., 1994; Krumm et al., 1995).
Here, in order to investigate VP16's role in postinitiation control of transcription, we have taken an independent in vivo functional approach and have analyzed the synergistic activation features of VP16 when tethered simultaneously upstream of a promoter to DNA and downstream of the transcription start site to RNA. Cullen and colleagues reported earlier that the VP16 activation domain could activate transcription from the human immunodeficiency virus-1 long terminal repeat (HIV-1 LTR) when tethered to a promoter-proximal RNA element (Tiley et al., 1992). We have constructed a modified HIV-1 LTR reporter plasmid, pG6RRE (see Fig. 1A) which allowed us to target fusion protein derivatives of VP16 simultaneously to RNA and DNA in transfection experiments in mammalian cells. We find that RNA-bound VP16 can cooperate with DNA-bound VP16 to activate transcription synergistically. Furthermore, the minimal activation domain of VP16 (amino acids 413-453) was found to be sufficient for synergistic activation of transcription via RNA and DNA. Activation was abolished when the DNA-bound VP16 minimal activation domain was mutated at critical residue 442 (F442P). However, the RNA-bound VP16 minimal activation domain mutated at critical residue 442 maintained synergism with DNA-bound VP16. Our studies suggest that the 41-amino acid minimal activation domain of VP16 (amino acids 413-453), which plays an important role in transcription initiation, also acts at a postinitiation step to increase rates of transcription. Furthermore, this postinitiation activity of the minimal activation domain does not involve the critical residue at position 442 required for initiation. Thus, VP16 activates transcription by a sequence of at least two major events. The first event involves stimulation of initiation upon interaction of both the proximal and distal subdomains of VP16 activation domain with components of the basal transcription initiation machinery at the promoter. Next, distinct determinant(s) within the proximal 41-amino acid activation region of VP16 activation domain act at a postinitiation step to enhance rates of transcription leading to the overall potent activation characteristic of VP16.
Figure 1:
VP16 tethered
simultaneously to RNA and DNA act synergistically in trans-activating the HIV-1 LTR. A, schematic drawing
of the reporter and effector plasmids used in cotransfections. In the
pG6RRE reporter, HIV-1 LTR (-119 to +80 relative to
transcription start site) is linked to the reporter gene CAT and has
six synthetic GAL4-binding sites introduced upstream of NF-B
sequences and downstream the apical region of TAR element (+30 to
+35) is replaced by the SLIIB subdomain of the HIV-1 RRE. The TAR
element is rendered nonfunctional while the SLIIB RRE sequence forms an in vivo RNA target for the HIV-1 Rev RNA-binding protein
(Tiley et al., 1992). GAL4-VP16 directs the synthesis of a
hybrid protein containing the DNA-binding domain of GAL4 (amino acids
1-147) fused to the C-terminal acidic activation domain of VP16
(amino acids 413-490), while Rev-VP16 expresses the 113-amino
acid Rev protein fused in-frame with the C-terminal acidic activation
domain of VP16 (amino acids 413-490). B, Jurkat cells
were transfected with pG6RRE and either GAL4-VP16 or Rev-VP16, or both.
GAL4-VP16 fusion targets VP16 to GAL4 DNA and Rev-VP16 targets VP16 to
RRE (SLIIB) RNA. At 48 h after transfection, cells were harvested, cell
lysates were prepared, and the level of CAT enzyme activity was
determined by the solvent partition method of Neumann et
al.(1987) where CAT activity is expressed as the rate of formation
of
H-labeled acetylated chloramphenicol (cpm/min) and is
normalized to the amount of protein in the lysate. Trans-activation of the HIV-1 LTR reporter was monitored by
CAT enzyme activity, and the data are reported as
fold-trans-activation, where levels of CAT activity in the
presence of the effector are divided by the basal activity level of the
reporter in the absence of the effector and in the presence of carrier
DNA (pGEM-7Zf+ vector DNA, Promega). When Rev-VP16 and GAL4-VP16
were cotransfected alone, GAL4 and Rev, respectively, or vector DNA
were used as carrier. The basal activity of the pG6RRE reporter plasmid
as measured by CAT activity is 1.85 cpm/min. Synergistic activation was
obtained when GAL4-VP16 and Rev-VP16 were cotransfected. GAL4 and Rev
did not have any effects on activation indicating that the synergism
obtained with the hybrid fusions is specifically due to the activation
domains of VP16. The data presented are representative of three
transfections, each done in duplicate. Transfection efficiency was
examined by cotransfection of RSV-luciferase reporter plasmid. Less
than 10% variation in luciferase activity from the mean was detected
among the different transfections.
Jurkat cells were transfected with plasmid DNA using
the DEAE-dextran method (Sambrook et al., 1989). Briefly, 1
10
cells were washed with phosphate-buffered saline
and then incubated with 10 µg of target reporter and 5 µg of
effector plasmids in 2 ml of serum-free RPMI 1640 medium containing 300
µg of DEAE-dextran/ml for 2 h at 37 °C. Carrier DNA
(pGem7Zf+; Promega) was added where necessary to maintain the same
total amount of DNA in every transfection. Subsequently cells were
pelleted and resuspended in 10% dimethyl sulfoxide (made in
phosphate-buffered saline) and incubated for 2 min. Cells were then
washed twice with phosphate-buffered saline, and RPMI supplemented with
10% fetal calf serum was added. Cells were harvested 48 h after
transfection, cell lysates prepared, and chloramphenicol
acetyltransferase (CAT) assays performed on 25 µg of lysate.
Transfection of Hela and CV1 cells were performed using the
DEAE-dextran method as described earlier (Ghosh et al., 1993).
CAT assays were performed by the solvent partition method (Neumann et al., 1987) where CAT activity is expressed as the rate of
formation of H-labeled acetylated chloramphenicol and is
normalized to the amount of protein in the lysates. Protein
concentrations were determined by the Bio-Rad protein assay kit
(Bio-Rad). Transfection efficiency was examined by cotransfection of
RSV-luciferase reporter plasmid, and luciferase assays were performed
using the luciferase assay systems kit (Promega).
The effector plasmids GAL4-VP16N and GAL4-VP16Nm442 have been described previously as SW3 and SW5, respectively, and so has SW22 (Walker et al., 1993). GAL4 and GAL4-VP16 have also been reported earlier (Lin and Green, 1991). Rev, Rev-VP16, and Rev-VP16m442 have been described earlier as pcRev, pcRev/VP16, and pcRev/deltaVP16, respectively (Tiley et al., 1992). Rev-VP16N and Rev-VP16Nm442 were created by introducing translational termination sequence at the SmaI site of pcRev/VP16 and pcRev/deltaVP16, respectively, which resulted in the expression of a truncated VP16 fusion protein. Rev-VP16C and GAL4-VP16C were constructed by fusing in-frame a polymerase chain reaction (PCR) fragment containing sequences coding for the most C-terminal 39-amino acid activation domain of VP16 to Rev and GAL4(1-147), respectively. All sequences were verified by double-stranded DNA sequencing (U. S. Biochemical Corp. Sequenase kit).
Figure 2: The minimal activation domain of VP16 targeted simultaneously to RNA and DNA is sufficient for synergistic activation of transcription. A, schematic drawing of the effector plasmids coding for truncated hybrid fusions of VP16 with either GAL4(1-147) or Rev used in cotransfections with the reporter, pG6RRE. The activation domain of VP16 (amino acids 413-490) has been divided into two independent activation domains, VP16N (proximal amino acids 413-452) and VP16C (distal amino acids 453-490). GAL4-VP16N and GAL4-VP16C are fusion's expressing the GAL4(1-147) DNA-binding domain fused to VP16N and VP16C regions of VP16, respectively, while Rev-VP16N and Rev-VP16C fusion's express the Rev RNA-binding protein fused to VP16N and VP16C, respectively. B, trans-activation resulting from targeting truncated VP16 fusion derivatives simultaneously to RNA and DNA in pG6RRE reporter in Jurkat cells. The basal activity of pG6RRE as measured by CAT activity is 2.55 cpm/min. Reporter CAT activity data expressed as fold-trans-activation indicates that RNA-bound VP16N (Rev-VP16N) can synergize with DNA-bound VP16N (GAL4-VP16N) and that the N region contains all the determinants for synergism. RNA and DNA-bound VP16C (GAL4-VP16C and Rev-VP16C) did not synergize indicating that the synergism seen for VP16N is a specific event. Similar results were also obtained with CV1 and Hela cell lines. The data presented above are representative of three transfections, each done in duplicate.
Figure 3:
Transcription-defective VP16 activation
domain mutated at critical residue 442 (phenylalanine proline)
maintains synergism when tethered to RNA with wild-type VP16 activation
domain tethered to DNA. A, amino acid sequence of the minimal
activation domain of VP16 (VP16N; amino acids 413-453). The asterisk at position 442 highlights the aromatic residue
phenylalanine shown to be critical for transcription activation in
vivo and for interaction with basal transcription factors TFIIB,
TFIID, TFIIH, and human coactivator PC4, in vitro. B,
structure of the hybrid VP16 fusion plasmids and mutated derivatives
used in cotransfections with the pG6RRE reporter. The phenylalanine
residue at position 442 is mutated to proline in the GAL4-VP16N,
Rev-VP16, and Rev-VP16N fusions to give GAL4-VP16Nm442, Rev-VP16m442,
and Rev-VP16Nm442, respectively. C, CAT activity data from
transfections in Jurkat cells show that the N region of VP16 when
mutated at the critical residue 442 fails to activate transcription
from the promoter when tethered to DNA, yet can maintain synergism when
bound to RNA with a functional GAL4-VP16N fusion but not with the
mutated GAL4-VP16Nm442 fusion tethered to upstream DNA. Basal activity
of the pG6RRE reporter is 3.69 cpm/min for transfections shown in the top-most panel, and 2.55 cpm/min for the rest of the
transfections shown. Levels of synergistic activation were always lower
when the mutated Rev-VP16Nm442 fusion was cotransfected as compared to
the unmutated Rev-VP16N. Also, the full-length VP16 activation domain
mutated at Phe
and fused to Rev (Rev-VP16m442) synergized
more effectively than the truncated and mutated Rev-VP16Nm442. We
suggest that the Phe
Pro mutation at 442 within region N of VP16
may potentially disturb the structure of this region, resulting in
lowered levels of activity. Higher levels of synergistic activation
shown by Rev-VP16m442, containing both the mutated N and C region of
VP16, may be due to the stabilization effect of the C region on the N
domain that contains the potentially disrupting mutation at
442.
Our analysis indicates that VP16 when tethered to RNA can
synergize with upstream DNA-bound VP16 to activate transcription
potently. Tethering the ``functionally defective'' minimal
activation domain of VP16 mutated at critical residue 442 (F442P) via
RNA and a wild-type minimal activation domain via DNA allowed us to
uncover a second pathway for VP16 function, mediated by this minimal
activation domain, hitherto unknown. This feature is distinct from its
known function in initiation in that it does not require the
participation of the critical residue Phe. Our studies
suggest that VP16 activates transcription by a sequence of at least two
events. As shown in the model in Fig. 4, the first event
involves both the proximal(N) and distal (C) regions of VP16 activation
domain interacting with components of the basal transcription
initiation machinery at the promoter to stimulate rates of initiation.
The N region may stimulate initiation by contacts with TFIIB and TFIID,
and for which the participation of the aromatic residue at 442 is
critical (Lin and Green, 1991, Ingles et al., 1991), while the
C region may interact with TAF(s) in the TFIID complex (Goodrich et
al., 1993; Klemm et al., 1995), leading to a stable
initiation complex. VP16 has also been reported to bind in vitro to the human coactivator PC4 and the critical residue at 442 was
found to be important for this interaction. Since PC4 directly binds
TFIIA (or a TBP
TFIIA complex), it is suggested that PC4 mediates
functional interactions between VP16 and TFIIA (or a TBP
TFIIA
complex) (Ge and Roeder, 1991). Next, we hypothesize, once a stable
initiation complex is assembled, the N region of VP16 activation domain
may interact either directly or indirectly with target(s) in the
transcription complex, modifying the complex to increase its
processivity and for this function the aromatic residue at 442, found
to be critical for initiation, is dispensable. The dispensability of
this critical residue may suggest that the targets mediating this
additional activity of VP16 may not include TFIID, TFIIB, or PC4, since
interaction of these components with VP16 is disrupted by this
mutation. The ability of VP16 to contact both general transcription
factors involved in stimulating initiation and factor(s) modulating the
processivity of chain elongation by RNA polymerase II would result in
the potent activation characteristic of VP16. We suggest that the
reason this postinitiation activity could not be detected in DNA
targeting studies is that mutation of Phe
abolishes
VP16's initiation function and consequently assembly of stable
initiation complexes upon which VP16 could act to increase
processivity. Tethering VP16 to RNA resulted in a large induction of
transcription from the HIV-1 promoter even though multiple VP16
activation domains were tethered to DNA upstream of the promoter. We
suggest that tethering VP16 to the 5` end of nascent RNA in close
spatial proximity to the transcription start site may have allowed for
a more efficient targeting of VP16 to the nascent elongating polymerase
complexes initiated from the promoter and consequently for VP16 to
exert its postinitiation processive activity leading to high overall
rates of transcription. Interestingly, the HIV-1 Tat protein which is
an extremely potent viral trans-activator activates
transcription from the HIV-1 promoter via interaction with the nascent
TAR RNA element located at the 5` end of all HIV transcripts, and it
has been shown by both in vivo and in vitro studies
that Tat acts as an elongation factor and can also stimulate
transcription initiation (see Zhou and Sharp(1995) and references
therein). However, the mutated VP16 activation domain tethered to RNA
did not have any effects on the basal activity of pG6RRE. Rev-VP16m442
or Rev-VP16Nm442 fusions did not alter the basal expression of pG6RRE,
individually but synergized with an intact VP16 activation domain
targeted to upstream DNA (Fig. 3C). The low levels of
basal activity obtained with our modified HIV-1 LTR reporter, pG6RRE,
is consistent with previous findings from several laboratories that the
HIV-1 LTR promoter, in the absence of activator, is characterized by
the production of low levels of productive full-length RNA. Phillip
Sharp and colleagues have proposed that these RNA species are generated
from the more processive class of polymerase elongation complexes that
initiate from the HIV promoter (Marciniak and Sharp, 1991). We suggest
that these more processive elongating polymerase complexes derived from
the HIV promoter in the absence of activator may not be responsive any
further to the processive activity of VP16. The processive function of
VP16 specifically acts on the transcription initiation complexes
stimulated from the promoter by the intact VP16 activation domain
requiring critical residue 442, modifying it to increase its
processivity resulting in increased rates of transcription.
Figure 4:
Model
for transcriptional activation by VP16. Analysis of synergistic
activation features of VP16 acidic activation domain when targeted
simultaneously to RNA downstream of the start site and to DNA upstream
of the promoter suggest that VP16 in addition to stimulating rates of
transcription initiation also plays a role in postinitiation control of
transcriptional activation. We hypothesize that VP16 activates
transcription by a sequence of at least two events. First, the
activation domain of VP16 interacts with basal components of the
transcription initiation machinery via both its proximal N region
(amino acids 413-453) and the distal C region (amino acids
454-490) to stimulate rates of transcription initiation from the
promoter. This function of VP16 involves the participation of the
critical aromatic residue at position 442 within the N region of its
activation domain. Mutation of Phe to proline (m442)
drastically affects the initiation step and in context of the minimal
activation domain (N region) abolishes transcription. Next, distinct
determinant(s) within the same 41-amino acid N region of VP16
activation domain required for initiation, may interact with target(s)
in the transcription complex at a postinitiation step to increase rates
of transcription resulting in potent activation characteristic of VP16.
Phe
critical for initiation is dispensable for this
postinitiation function.
It is generally believed that transcriptional synergy exhibited by VP16 on DNA may be a result of it interacting with more than one target affecting multiple steps involved in assembly of preinitiation complex. This hypothesis is supported by in vitro studies demonstrating GAL4-VP16 interactions with multiple basal transcription factors. However, transcriptional activation by RNA polymerase II is a multistep process, where each step may serve as a potential control point for activators and for transcriptional synergism. Transcriptional activation begins with assembly of a preinitiation complex followed by ATP-dependent events including melting of the DNA at the transcription start site to give an open complex and phosphorylation of the CTD (C-terminal domain) of RNA polymerase II. Next is the promoter clearance step resulting in the transition of the initiation complex to elongation complex and synthesis of phosphodiester bonds of the nascent RNA transcript followed by chain elongation (reviewed in Herschlag and Johnson(1993)). It has been shown using HeLa cell nuclear extracts that binding of GAL4-VP16 to DNA was required for efficient open complex formation at the adenovirus E4 promoter, i.e. melting of the DNA around the transcription start site using energy derived from ATP hydrolysis to expose the template strand for subsequent events leading to the synthesis of RNA (Wang et al., 1992). Gralla and colleagues have shown that mutation of critical residue 442 within the VP16 activation domain which drastically affects transcriptional activity in vivo led to a defect in open complex formation in in vitro studies (Jiang et al., 1994). This finding is consistent with the previous studies showing that mutation at residue 442 (F442P) disrupts VP16's interaction with general transcription factors TBP, TFIIB, and PC4, all of which act at steps preceding open complex formation. Since the additional transcriptional activity of VP16 which we have uncovered in our synergism assay does not require the participation of the critical residue at 442, we suggest that this new activity of VP16 acts at a step that follows open complex formation. For example, VP16 could facilitate the steps leading to CTD phosphorylation which allows uncoupling of polymerase II from the promoter (reviewed in Eick et al.(1994)). Alternatively, this postinitiation activity of VP16 might act at the promoter clearance stage. It has been shown that TFIIE and TFIIH are not required for initiation, but are necessary for promoter clearance (Maxon et al., 1994; Goodrich and Tijan, 1994) and may be potential targets for the postinitiation activity of VP16. Of the two, TFIIE, may be a more likely candidate since interaction of VP16 with TFIIH has been shown to require residue 442 (Xiao et al., 1994). We suggest that the transcriptional synergism obtained when VP16 is tethered to multiple DNA-binding sites is a result of both the action of VP16 on multiple steps leading to preinitiation complex formation, via interactions with multiple targets, as well as on a postinitiation step(s) leading to overall potent levels of mRNA synthesis. However, our experimental approach has limitations and cannot determine the exact stage of the transcription process at which the proposed postinitiation activity of VP16 functions. We are currently examining the residue(s) within the N region of the VP16 activation domain which is(are) responsible for the postinitiation activity detected in our in vivo analysis of VP16 transcriptional activation, the stage in transcription at which it acts, and the cellular targets with which this region interacts using biochemical approaches. Our studies may lead to an understanding of the mechanism by which the herpes simplex virus VP16 protein triggers activation of the viral immediate early genes upon interaction with host factors, Oct-1, and host cell factor protein (reviewed in Thompson and McKnight(1992)). Finally, a dual role in both transcription initiation and postinitiation may be a general property of other potent viral and cellular activators.