(Received for publication, August 22, 1995; and in revised form, October 3, 1995)
From the
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. A number of distinct properties of VP16 have been implicated in the assembly of the VP16-induced complex (VIC). These include its independent association with VCAF-1 and, under appropriate conditions, its ability to bind to DNA or to DNA-bound Oct-1 in the absence of VCAF-1. In order to probe the requirements of these individual interactions in the functional asembly of VIC, we mutated selected charged amino acids in two subdomains of VP16 previously shown to be important in protein-DNA complex formation. Purified VP16 proteins were analyzed for their ability to direct protein-DNA complex formation and to interact directly with VCAF-1. Several classes of mutants that were differentially compromised in VCAF-1 interaction, direct DNA binding, and/or association with DNA-bound Oct-1 were obtained. Interestingly, all of the derivatives were still capable of generating the VIC complex in vitro and activating transcription in vivo. Our findings indicate that the cooperative assembly of functional VP16-containing complexes can occur by pathways that do not necessarily require the prior interaction of VP16 with VCAF-1 or the ability of VP16 to bind directly to DNA or associate with DNA-bound Oct-1.
Transcriptional regulation of the herpes simplex virus immediate
early (IE) ()genes by the viral transactivator VP16 (also
called Vmw65 or
TIF) has provided a valuable model system to
investigate how multicomponent protein-protein and protein-DNA
assemblies orchestrate specific gene regulatory patterns (reviewed in (1, 2, 3) ). VP16 is an abundant 490-amino
acid-long structural phosphoprotein which, when delivered into the host
cell by the infecting virus particle, strongly stimulates the
transcription of the viral IE genes through recognition of cis-regulatory TAATGARAT (R = purine) target elements
that are present in one or more copies in the upstream regions of
responsive genes. VP16, however, has only weak intrinsic DNA binding
activity and efficient binding to target sites requires the assembly of
VP16 into a multicomponent complex along with at least two cellular
factors, the ubiquitously expressed POU homeodomain protein Oct-1, and
an additional cellular factor variously called VCAF-1, HCF, C1, or CCF ((4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) ;
referred to as VCAF-1 in this paper). The fully assembled functional
complex, referred to as the VP16-induced complex (VIC), is thought to
position the strong carboxyl-terminal acidic activation domain of VP16
in the correct spatial arrangement for functional interaction with
downstream target proteins, among which include several basal
transcription factors and adapter proteins(15, 16) .
The ordered assembly of VP16-containing protein-DNA complexes involves cooperative sequence-specific protein-DNA interactions as well as specific and selective protein-protein associations with Oct-1 and VCAF-1. Oct-1 binds independently to TAATGARAT elements and directs the recruitment of VP16 via determinants present in the POU homeodomain (17, 18, 19, 20, 21) . The prior binding of Oct-1 to TAATGARAT elements is a prerequisite to the formation of VIC; however, the efficient incorporation of VP16 into the complex requires the auxiliary component VCAF-1, a large cellular factor which can interact directly with VP16 in the absence of Oct-1 and DNA(5, 7, 8, 13, 14, 17) . There is no evidence for the formation of Oct-1-VP16 or Oct-1-VCAF-1-VP16 heteromeric complexes in the absence of DNA; however, it has been demonstrated that VP16 is able to bind directly to DNA or to generate a complex with DNA-bound Oct-1 in the absence of VCAF-1 when high concentrations of VP16 are used in binding assays(17, 19, 22) .
VP16 is a modular protein and contains separable domains that are important for complex formation and transactivation(19, 23, 24, 25, 26, 27, 28, 29) . The carboxyl-terminal acidic activation domain is essential for transcriptional activation but not for complex assembly. The residual 400 amino-terminal amino acids are necessary and sufficient for VIC formation as well as for interaction with VCAF-1 and binding to DNA (14, 24, 25, 27) . More recently, it has been demonstrated that this region also contains determinants for interaction with the virion host shutoff protein (Vhs), a viral structural protein that is responsible for the cessation of host protein synthesis following viral infection (30) . At least two subregions within the amino-terminal domain are involved in complex assembly with mammalian factors in vitro; region 1, spanning residues 140-250, and region 2, encompassing residues 335-390(19, 24, 25, 27) . Both subregions are involved in VIC formation, binding to VCAF-1, interaction with DNA-bound Oct-1, and contribute to the weak DNA binding activity associated with VP16(19, 24, 27, 31, 32, 33) .
Despite a large number of mutational studies, it is not known whether the individual interactions attributed to VP16 are essential for the assembly of transcriptionally active complexes. For instance, while VCAF-1 is essential for VIC formation, it is not known whether the intrinsic ability of VP16 to bind independently to VCAF-1 is a prerequisite for multicomponent complex assembly. Also, the weak DNA binding activity associated with VP16, or VCAF-1-independent interaction with DNA-bound Oct-1, is only observed when very high concentrations of VP16 are used in binding assays(17, 19) ; thus, the physiological significance of these interactions is not clear. Most mutational studies of VP16 have been carried out using large deletions or linker insertions and these might be expected to cause conformational changes in VP16 that could have secondary effects on complex assembly and protein-protein interactions. The only point mutational analysis so far described assessed the overall effects of specific point mutants on VIC formation and transactivation(25) . In order to determine if individual protein-protein and protein-DNA interactions involving VP16 can be uncoupled from VP16-induced complex formation and transactivation, we converted selected charged residues in regions 1 and 2 into alanine residues by site-directed mutagenesis. Mutant proteins were analyzed in vitro for protein-DNA complex formation with Oct-1 and interaction with VCAF-1 and in vivo for transactivation of a VP16-responsive reporter gene. Among the mutants generated were those that were defective in direct VCAF-1 interaction and DNA binding but were still capable of generating the VIC complex and directing transcriptional activation in vivo. Our findings indicate that the assembly of functional VP16-containing multicomponent complexes does not necessarily require the prior interaction of VP16 with VCAF-1 or the capability of VP16 to bind directly to DNA or associate with DNA-bound Oct-1, suggesting that the cooperative assembly of VP16-dependent complexes can occur by different pathways.
On-line formulae not verified for accuracy
The altered amino acid codons are underlined. The notation refers to the type and position of the wild type amino acid (single letter code) targeted for alteration to an alanine residue. Purified phage DNA containing the desired mutations were screened by DNA sequence analysis and the replicative form DNA was prepared(34) .
Previous studies have shown that amino acids 140-250 and 335-390 of VP16 contain determinants that are important for VIC formation and VCAF-1 interaction(19, 24, 25, 27, 31) . Both regions 1 and 2 are enriched in positively charged residues, in particular arginine residues, relative to the whole protein. Since positively charged amino acids are important in both protein-protein and protein-DNA interactions, we decided to alter selected arginine residues to alanine residues by site-directed mutagenesis. Alanine residues were chosen, since these would not be expected to alter the conformation of the polypeptide backbone(36) . The introduced mutations are shown in Fig. 1A. These include alanine substitutions of arginines at positions 155, 162, 164, 169, 360, 366, and 368. Cys-176 was also mutated to examine the role of this sulfhydryl group. Also, Lys-379 was mutated, since the region surrounding this amino acid has been shown to be surface exposed and to be involved in protein-DNA complex formation(32) .
Figure 1: Amino acid substitutions in VP16. A, map of VP16 showing two subdomains important for protein-DNA complex assembly (Region 1 and Region 2). AAD (amino acids 420-490) represents the carboxyl-terminal acidic activation domain. The indicated amino acid residues were altered to alanine (A) residues by site-directed mutagenesis. The nomenclature refers the amino acid residue and its position in the primary sequence. B, expression of MBP-VP16 fusion proteins in E. coli. The above mutant derivatives were cloned into the MBP expression vector pMal-C, expressed in E. coli, and purified by affinity chromatography. Shown is a Coomassie Blue-stained gel of the different purified MBP-VP16 fusion proteins (lanes c-l). Lane b is MBP purified from induced cultures in an identical manner. WT in this and subsequent figures (unless indicated otherwise) denotes the MBP-VP16 fusion protein containing wild type amino acids 5-411 of VP16. Molecular size markers, in kilodaltons, are shown in lane a.
VP16 and the various mutant derivatives were cloned into an MBP expression vector, and the proteins were purified by affinity chromatography on amylose resin (Fig. 1B; all MBP-VP16 derivatives encoded residues 4-411 and are thus missing the acidic activation domain). The purified proteins were used to monitor the following properties of VP16: direct DNA binding to the TAATGARAT elements, interaction with DNA-bound Oct-1, direct interaction with VCAF-1, VIC formation, and association with Vhs.
Figure 2: Interaction of VP16 mutant derivatives with DNA and with DNA-bound Oct-1. A, purified MBP-VP16 fusion proteins (10 µg) were incubated with labeled probe and analyzed by gel retardation. In lane a, purified PA-VP16 (wild type VP16 fused to protein A) was used. PA-VP16 is larger than MBP-VP16, and thus the protein-DNA complex has a slower mobility. The probe used in this and subsequent experiments was the promoter proximal TAATGARAT element from the ICP0 promoter. B, gel retardation experiments were carried out with the indicated fusion proteins (6 µg) in the presence of purified GST-Oct-1 POU homeodomain fusion protein (0.05 µg; referred to as Oct-1 in all figures). Lane a is GST-Oct-1 alone. The positions of the VP16 DNA complex and the VP16-Oct-1 DNA complex are indicated.
Figure 3: VP16-induced complex assembly with VP16 mutants. The various MBP-VP16 mutants were incubated alone (6 µg), in the presence of GST-Oct-1 (0.05 µg), or in the presence of GST-Oct-1 and VCAF-1 (2 µl), as indicated at the top of the figure. VIC indicates the position of the VP16-dependent ternary DNA complex that contains Oct-1, VP16, and VCAF-1. Control experiments carried out with MBP alone or in the presence of GST-Oct-1 and VCAF-1 are shown in lanes 1-3. VP16 was used at a 10-fold excess (compared with what is normally sufficient to form VIC under these conditions) in order to also visualize the VP16 and VP16-Oct-1 complexes, as indicated.
Figure 4: Interaction of VP16 mutant derivatives with VCAF-1. VCAF-1 was incubated with MBP or the different MBP-VP16 fusion proteins immobilized on amylose resin as indicated in the figure. Equivalent amounts of nonbound (A) and bound (B) fractions recovered from the supernatant and beads, respectively, were assayed for VCAF-1 activity in the presence of 0.05 µg of GST-Oct-1 and 0.5 µg of wild type MBP-VP16. Lane a in A and B is a control containing GST-Oct-1 (0.05 µg), MBP-VP16 (0.5 µg), and purified VCAF-1 (3 µl). The position of the VCAF-1-dependent VIC complex is indicated.
Figure 5:
Interaction of VP16 mutants with the
virion host shutoff protein (Vhs).
[S]Methionine-labeled Vhs protein was
transcribed and translated in vitro (lane a) and
incubated with the indicated immobilized MBP-VP16 fusion proteins.
Bound material was eluted from the beads and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Lane a is the
total Vhs translation product. Lanes b and h are
controls in which Vhs protein was incubated with beads complexed with
MBP (lane b) or beads alone (lane
h).
Figure 6: Transactivation by VP16 mutants in vivo. The various VP16 mutants were cloned into a mammalian expression vector that restored the acidic activation domain and transfected, at the indicated concentrations, into Vero cells along with the VP16-responsive p175cat reporter gene. CAT activity was monitored 48 h post-transfection. The values represent the average (±S.D.) from at least three separate transfections carried out in duplicate and normalized in each case to the value obtained from transfection of the reporter gene alone (control), which was taken as 1. WT represents wild type full-length VP16.
In this study, we have analyzed the properties of specific point mutants of VP16 to examine the contributions of individual protein-protein and protein-DNA interactions of VP16 to the ordered assembly of VP16-containing complexes and VP16-mediated transcriptional activation. We showed that specific point mutations in two regions of VP16 selectively affected the ability of VP16 to directly bind to DNA, to associate with DNA-bound Oct-1, and to form a complex with VCAF-1 (summarized in Table 1). All of the mutants tested remained capable of forming VIC and transactivating gene expression in vivo, demonstrating that these individual interactions are not essential for the formation of a transcriptionally active VP16-containing protein-DNA complex.
VP16 derivatives in which arginines at position 162, 360, and 366 were altered failed to bind directly to DNA. This is consistent with previous mutational studies which showed that deletion of either region 1 or 2 destroyed direct DNA binding activity of VP16(19) . The putative DNA binding domain of VP16 has not been defined; however, Stern and Herr (19) showed that a synthetic peptide spanning a part of region 1 (amino acids residues 170-202) bound to DNA, albeit in a nonsequence-specific manner. All of the region 1 mutants were capable of forming a weak complex with DNA-bound Oct-1; however, the region 2 mutants were defective. This is consistent with previous findings that region 2 contains determinants for direct interaction of VP16 with DNA bound-Oct-1(19, 24, 31) . Our findings that the R368A and K370A mutants cannot recognize DNA-bound Oct-1, yet are still able to bind to DNA indicate that direct DNA binding activity of VP16, is not required for this interaction.
The functional relevance
of direct DNA binding activity of VP16 or interaction with DNA-bound
Oct-1 is not clear, since there is no evidence that the VP16-Oct-1
binary complex or VP16 on its own is transcriptionally active in
vivo. As demonstrated previously by others (17, 19) and also shown here, the amount of VP16
required to observe these properties is significantly greater
(10-100-fold) than that which is sufficient to generate the VIC
complex. The high concentration of VP16 necessary to observe these
interactions is probably not physiologically significant. Indeed,
transactivation of viral IE genes is detrimentally affected by large
amounts of VP16(24) . Moreover, the source of VP16 may have an
effect on the efficiency of these interactions. For instance, PA-VP16
has a higher affinity for DNA compared with MBP-VP16 (Fig. 2A), ()whereas GST-VP16 has only very
weak intrinsic DNA binding activity(22) . Thus, the carrier
protein may have some influence on binding properties, perhaps by
masking or exposing cryptic binding interfaces(17) . It has
been proposed that VP16 binds to the GARAT portion of the target site
which, while not required for Oct-1 binding, is necessary for VIC
assembly(5, 17) . More recent evidence indicates that
the GARAT subregion serves to alter the conformation of the POU
homeodomain and that this is necessary for subsequent recognition by
VP16(22) . Our findings directly demonstrate that the
independent DNA binding activity and interaction with DNA-bound Oct-1
is not absolutely essential for VIC assembly or for transcriptional
activation in vivo (see below).
Region 2 is essential for VIC formation and also contains critical determinants for direct interaction with VCAF-1(19, 24, 31) . We, and others, have shown that the requirements for interaction with VCAF-1 can be uncoupled from VIC formation(19, 31) . Thus, VIC formation requires residues up to amino acid 388, whereas VCAF-1 can still interact with VP16 that is truncated to amino acid 379(24, 31) . In addition, a linker insertion at position 379 abolished VIC formation but not interaction with VCAF-1(19) . Hayes and O'Hare (32) have shown previously, using limited proteolysis of VP16 and of VP16-containing protein-DNA complexes, that the region surrounding Lys-370 is exposed on the surface of the protein and thus likely to be important for protein-protein interactions. Moreover, using peptide competition, these authors showed that a synthetic peptide encompassing amino acids 360-373 (REHAYSRARTKNNY) or a truncated variant containing amino acids 360-367 was able to inhibit VIC formation. The simplest conclusion was that this peptide interfered with the ability of VP16 to bind to VCAF-1, although this was not directly demonstrated. Our findings that mutation of arginine residues 366, 368, and lysine 370 disrupt VCAF-1 interaction is consistent with the conclusion that this subregion represents an important interface for VCAF-1 interaction. Furthermore, the finding that mutation of arginine 169 (this work) or a linker insertion at amino acid 177 (19) also disrupts association with VCAF-1 indicates that region 1 contributes to this interaction, further emphasizing that the overall conformation of VP16 is important. More significantly, all of the VP16 derivatives were capable of generating VIC in vitro and of transactivating expression of a VP16-responsive CAT reporter gene in vivo. Thus, the direct and independent interaction of VP16 with VCAF-1 is not absolutely required for the assembly of functionally active VIC complexes, suggesting that complex assembly can occur by different pathways. It is possible that multiple and additional layers of cooperativity occurring between VCAF-1, VP16, and Oct-1 in the full complex may compensate for deficiencies in individual interactions. The fact that determinants important for VP16 interaction with VCAF-1 and Oct-1 overlap, or are in close proximity to each other, suggests that VCAF-1 also contacts Oct-1, as implied previously by mutation and peptide competition studies(19, 32) . Similarly, the observation that the intrinsic DNA binding activity of VP16 is dispensable for VIC formation does not necessarily mean that this interaction does not occur in the fully assembled complex.
In summary, our findings suggest versatility and flexibility in the assembly of VP16-containing complexes, whereby complexes can assemble by different pathways. This is compatible with recent findings that demonstrate that the conformation of Oct-1 POU homeodomain, and possibly the VP16-induced complex itself, is different on distinct TAATGARAT elements and that diverse response elements may have different functional properties in vivo(22, 38, 40, 41, 42) . This flexibility could provide a mechanism by which VP16 function can adapt to different physiological conditions in the host cell and thus modulate progression of the lytic cycle. For instance, the surprising observation that direct interaction of VCAF-1 with VP16 can be uncoupled from subsequent higher order protein-DNA complex assembly and transactivation does not necessarily mean that prior interaction between VCAF-1 and VP16 is dispensable for VIC formation under all conditions. Thus, in cells where VCAF-1 concentration is low, prior assembly of the VCAF-1-VP16 binary complex may be a necessary prerequisite for efficient VIC formation and subsequent transcriptional activation. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.