Transactivation of a Late Herpes Simplex Virus Promoter

Transcription

1 MOLECULAR AND CELLULAR BIOLOGY, Mar. 1984, p /84/3544-8$2./ Copyright 1984, American Society for Microbiology Vol. 4, No. 3 Transactivation of a Late Herpes Simplex Virus Promoter DOUGLAS DENNIS AND JAMES R. SMILEY* Department of Pathology, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 Received 1 August 1983/Accepted 9 December 1983 We have asked whether the promoter for the gene encoding the major capsid protein (VP5) of herpes simplex virus functions in uninfected mouse cells. Our experimental strategy was to first fuse the VP5 promoter to the herpes simplex virus thymidine kinase () structural sequence and then to use the resulting hybrid gene to transform - cells to +. The recombinant gene transferred at an extremely low frequency by comparison with the wild-type gene, and the transcripts present within the resulting rare transformants initiated within the structural gene, rather than in the vicinity of the VP5 promoter. However, after infection with herpes simplex virus, large amounts of RNA driven from the VP5 promoter accumulated. We conclude that the VP5 promoter does not function in uninfected cells but is efficiently activated by virally coded factors, most likely one or more immediate-early proteins. During lytic infections of mammalian cells with herpes simplex virus (HSV), at least three differentially regulated classes of viral genes are expressed (19). Immediately after infection, the viral DNA is transported to the nucleus where the five or so immediate-early (or a) viral genes are transcribed from separate promoters by the host cell RNA polymerase II (1, 8, 23, 35, 42). These five transcripts are the only ones made in the absence of viral protein synthesis. Although the immediate-early viral promoters are recognized by the uninfected cell transcription apparatus (24-26, 34), their efficient use appears to also require one or more factors supplied as components of the infecting virus particle (3, 34). In turn, the regulatory immediate-early protein ICP4, a product of this first wave of transcription, is required continuously throughout infection to allow transcription of the remaining delayed-early (or 3) and late (or y) viral genes (21, 35, 41). At present, it is not clear whether additional immediate-early proteins are also required for this activation. The mechanism by which ICP4 activates transcription of the delayed-early and late HSV genes remains unclear, but it is likely to be of considerable interest as this process appears to involve both the trans-acting immediate-early proteins and cis-acting DNA sequences located in the promoter regions of the target genes (39, 45). Since HSV genes are, as a rule, each transcribed from a separate promoter, ICP4 therefore seems to exert its effects at many different cis-acting sites. Delayed-early viral genes have been defined as those which are maximally expressed before the onset of viral DNA replication, and late genes have been defined as those maximally expressed after replication has begun (19). This current classification may be somewhat oversimplified, because some late genes (e.g., that for VP5) are detectably expressed before replication (12), whereas others are not (18). In the former case, (the so-called 3--y class) it seems possible that the observed pattern of transcription can be accounted for by two distinct processes: first, the 13-y promoters are activated by ICP4 with delayed-early kinetics and second, template amplification, the result of DNA replication, boosts the level of expression further. By contrast, the latter class of late genes (the true -y genes) may require additional regulatory factors, DNA replication, or both for their expression. * Corresponding author. Certain aspects of the regulatory events outlined above are preserved when purified HSV genes are introduced into uninfected cultured cells. For example, while several immediate-early promoters have been shown to drive low-level transcription in uninfected cells (24, 26, 34), expression from these promoters is boosted substantially by factors present as part of superinfecting virus particles (3, 34). Similarly, although the delayed-early thymidine kinase () gene is expressed at a low level in transfected mouse cells (44), this level is increased up to 3-fold by the immediate-early proteins supplied by superinfection of the cells with deficient HSV type 1 (HSV-1) (2, 22, 39, 45). In both of these cases, DNA sequences upstream from the transcription initiation site appear to be necessary for the response to superinfection (26, 39, 45). These results suggest that HSVcoded regulators somehow increase the efficiency with which the cellular transcription machinery uses the constitutively weak viral promoters. Relatively little attention has been paid to date to the factors which control the expression of the late class of HSV genes. Recently, Frink et al. (12) have reported that the promoter for the gene encoding the major capsid protein, VP5 (a delayed-early/late or 1-ry gene) was not recognized by an in vitro transcription system prepared from uninfected cells. This result was of interest to us as it was in marked contrast to the results obtained by Read and Summers (36) with the delayed-early promoter and the promoter of another late gene, both of which are recognized in vitro by extracts from uninfected cells. This raised the possibility that some HSV promoters are not recognized by uninfected cells. This in turn would imply that HSV regulators act to alter the specificity of the cellular transcription machinery not only to increase the efficiency of constitutively weak promoters (e.g., ), but also to allow the use of otherwise silent promoters. However, as the results obtained in in vitro transcription systems do not always parallel the in vivo situation, we wished to determine directly whether the VP5 promoter functions in uninfected cells in vivo. The results presented in this communication suggest that it does not, but that it is efficiently activated by superinfecting HSV, most likely by immediate-early proteins. 544 MATERIALS AND METHODS Virus and cells. HSV-1 wild type was strain KOS; the deficient mutants B26 (1) and d2 (38) were derived from

2 VOL. 4, 1984 strain CL.11 (1). Virus was propagated on Vero cells as previously described (38). Vero cells and Ltk- cells (44) were propagated in a- minimal essential medium (GIBCO Laboratories) supplemented with 1% fetal bovine serum. Transformation of Ltk- to +. Transformations were performed by using a modification (13) of the calcium phosphate coprecipitation technique of Graham and van der Eb (14). Briefly, subconfluent monolayers of Ltk- cells growing in tissue culture dishes (6 mm; Coming Glass Works) were exposed to a coprecipitate formed with the indicated amounts of plasmid DNA and 1,ug of highmolecular-weight Ltk- cellular DNA per ml. The precipitate was added directly to the growth medium (13). After 16 h the medium was changed, and 24 h later, HAT selection (44) was applied. a-hat medium was replaced every 3 to 4 days, and transformed colonies were counted after 2 weeks. Isolated clones were picked and expanded into cell lines, which were maintained in a-hat plus 1% fetal bovine serum. The cell lines transformed by the VP5- hybrids were analyzed for transforming DNA content by Southern blot hybridization; in every case, at least 1 complete, unrearranged copies of the appropriate recombinant gene were found per cell (data not shown). Plasmids. pxl (11) contains the 3.5-kilobase -bearing HI Q fragment of HSV-1 DNA cloned at the HI site of pbr322. pjs1 contains the larger BglII subfragment of HI Q in the HI site of pbr322. pibn1 contains the 5.5-kilobase BglII N fragment of HSV-1 DNA at the BglII site of pkc7. A 6-base HI-BglII fragment containing the VP5 promoter was purified from pibn1, and cloned into the BglII site of pxl, generating recombinants N12, N6, and N17 (see Fig. 4). The promoter was removed from N17 by cleavage with Sall and BglII followed by Si nuclease and ligation. The resulting plasmid was designated N17BS3. Induction of enzymatic activity in transformed cells. Subconfluent monolayers of transformed cells growing in tissue cultured flasks (75 cm2; Corning) were infected at the indicated multiplicities of infection with HSV-1 B26. At appropriate times, the cells were harvested and assayed for enzymatic activity with 14C-labeled thymidine as previously described (39). Si nuclease mapping. The methods of Berk and Sharp (6), TRANSACTIVATION OF A LATE HSV PROMOTER 545 as modified by Weaver and Weissman (43), were used. Cytoplasmic RNA was prepared from uninfected or infected cells by the method of Berk and Sharp (6). DNA probes were 5'-end-labeled with polynucleotide kinase (Bethesda Research Laboratories) and [y-32p]atp (New England Nuclear Corp. or Amersham Corp.). In some cases, the complementary strands were separated by gel electrophoresis; in other cases, a double-stranded uniquely end-labeled probe was prepared by secondary cleavage. For hybridization, 5 x 14 to 1 x 15 dpm of the probe DNA were mixed with 1,ug of RNA and precipitated with ethanol. The pellet was dissolved in 3,ul of.4 M NaCl-4 mm piperazine-n,n'-bis(2- ethanesulfonic acid) (ph 6.4)-i mm EDTA containing either 8% (duplex probes) or 5% (single-stranded probes) recrystalized, deionized formamide. After denaturation at 8 C for 5 min, the mixture was hybridized for 4 h at 61 C (duplex probes) or 16 h at 42 C (single-stranded probes). After hybridization,.3 ml of ice-cold 15 mm NaCl-5 mm Na acetate-5 mm ZnSO4 (ph 4.6) containing 3.2 x 14 U of S1 nuclease (Boehringer Mannheim) was added. Digestion was for 1 h at 37 C; titration experiments revealed that lower nuclease concentrations resulted in incomplete digestion of the single-stranded portions of RNA-DNA hybrids (data not shown). After digestion, 5,u1 of 4 M NH4 acetate and 15,ul of 2 mm EDTA were added, and the mixture was extracted once with phenol-chloroform (1:1) and then ethanol precipitated along with 1,ug of trna carrier. The digestion products were analyzed on 8 or 6% polyacrylamide sequencing gels; DNA sequence analysis was by the method of Maxam and Gilbert (27). RESULTS Mapping the 5' end of VP5 mrna. Frink et al. (12) have shown that the unspliced 6.-kilobase mrna encoding VP5 is transcribed from right to left on the prototype arrangement of HSV-1 DNA and that the 5' end of the transcript is located close to the HI cleavage site separating fragments F' and B' (Fig. 1). Although an initial report placed the initiation site ca. 7 nucleotides to the right of this HI site, more recent results from this same group have demonstrated that the major species in fact initiates at ca. 15 bases to the left of the HI site (R. Costa and E. K. Wagner, personal communication). We have independently confirmed this VP HI B' HI FBgl E 1. kb 32p *.. Si probe Rsal Hhal FIG. 1. Location of the VP5 and genes of HSV-1. The positions and polarities of the VP5 and genes are indicated on the map of the prototype arrangement of HSV-1 DNA. In addition, an expanded map of the region encoding the 5' end of VP5 mrna is given in the lower portion of the figure. S1 probe, the RsaI-HhaI fragment used as a hybridization probe in the experiments of Fig. 2.

3 546 DENNIS AND SMILEY revised assignment, as indicated by the Si mapping experiment (Fig. 2). Infected Vero cell cytoplasmic RNA was hybridized with a double-stranded RsaI-HhaI fragment endlabeled with 32P at its RsaI site (Fig. 1). After Si nuclease digestion, the protected portion of the probe was sized on a 6% polyacrylamide sequencing gel, along with markers generated by the thymine plus cytosine (T+ sequencing reaction, and by cleaving the probe with HI and SmaI. As is evident in Fig. 2, the major fragment protected by the infected cellular RNA comigrated with the SmaI cleavage product, indicating that the 5' end of the transcript is located close to the residue labeled + 1 in Fig. 3. Appreciable amounts of the VP5 transcript were present even when viral DNA replication was blocked by the presence of 15,ug of phosphonoacetic acid per ml (Fig. 2), in accord with the previous classification of this gene as a member of the B-y class (12). The nucleotide sequence upstream from the VP5 initiation site had some features similar to those thought to Q q~ FIG. 2. Mapping the 5' end of VP5 mrna. The double-stranded RsaI-HhaI fragment indicated in Fig. 1 was hybridized to infected Vero cell RNA prepared 12 h postinfection (multiplicity of infection, 5 PFU/cell). After S1 nuclease treatment, the products were sized on a 6% polyacrylamide sequencing gel. Lane 1, C+T sequencing reaction products of the probe; lane 2, probe hybridized to infected Vero cell RNA (infection in the presence of 15,ug of phosphonoacetic acid) per ml; lane 3, HI and SmaI cleavage products of the probe; lane 4, probe hybridized to infected Vero cell RNA (no phosphonoacetic acid). Uninfected Vero cell RNA gave no hybridization signal (not shown). MOL. CELL. BIOL. be important for RNA polymerase II promoter function (Fig. 3). Specifically, the sequence TATATAA starting at -28 resemble the Goldberg-Hogness box (9) located at an analogous position in many promoters, and sequences at -11 and -8 resemble the CAAT consensus sequence (5) found at similar sites in some promoters. (The CAAT consensus at -11, not shown in Fig. 3, has the sequence 5'- GGCCAATTC-3'.) Fusion of the VP5 promoter to the structural gene. To investigate the factors which control the activity of the promoter for the VP5 gene in vivo, we wished to introduce DNA fragments bearing the presumed regulator sequences into uninfected cultured cells. The strategy we chose to accomplish this end was to first place the HSV structural gene under the control of the VP5 promoter and then to use the resulting recombinant gene to transform deficient Ltk- cells to +. A comparable strategy has been used in studies of other promoters, including those for several HSV immediate-early genes (24, 34), the SV4 early region (33), and the mouse metallothionen gene (7). In all of these cases, the resulting recombinant genes transformed cells to + with relatively high efficiencies. In the present case, however, it was not clear at the outset whether the VP5 promoter would function in uninfected cells. If it did not, one might then expect that the recombinant gene, being functionally promoterless, would not transfer efficiently. We were encouraged to proceed, however, by the observation that even a promoterless structural gene was able to transform cells to +, albeit with a very low efficiency (this paper; 37). The resulting transformants generally have acquired many copies of the promoterless gene and in some cases, express from weak internal promoter sites located within the gene (37). These observations have suggested that even if the VP5 promoter was completely inactive in uninfected cells, it would still be possible to select for the acquisition of the hybrid gene by this alternative expression pathway. Inspection of the nucleotide sequence in the vicinity of the VP5 promoter suggested that the simplest way of placing under the control of the VP5 promoter was to fuse the HI site at -15 in the VP5 sequence to the BglII site in the nontranslated leader for (position +53 in ) (Fig. 3). Based on analogies with other eucaryote promoters, one would then expect that transcripts driven from the VP5 Goldberg-Hogness sequence would initiate within the sequences that ordinarily encode the nontranslated leader sequence of mrna. If this were the case, then the resulting transcripts should direct the synthesis of fully active thymidine kinase, provided that the truncated leader did not inhibit translation. Accordingly, several recombinants were constructed by inserting a 6-nucleotide-long BglII-HI fragment containing the VP5 promoter into the unique BglII site of the -containing plasmid pxl (Fig. 4). Recombinants N12 and N17 bear the VP5 promoter in the correct orientation with respect to, whereas N6 bears the promoter in the opposite orientation. In addition, a mutant lacking the wild-type promoter was derived from N17 (N17BS3). Transformation of - cells. Each of the recombinants was used to transform deficient Ltk- cells to + by the calcium phosphate coprecipitation technique of Graham et al. (13, 14). As controls, we also transformed cells with the wild-type gene (px1) and a promoterless structural sequence (pjs1). As is evident in Table 1, all plasmids bearing the VP5 promoter insert transformed cells ca. 1- to 1,-fold less efficiently than pxl. This low efficiency

4 VOL. 4, r 51 ed3 cl I c z 3 I TRANSACTIVATION OF A LATE HSV PROMOTER n. ^, o R3 I * n ~ CD. op o o > o) H *CD > CO t-a z C- > -' H No 17V- I 2 C > ; ~~~~ ~~~~ ~~~~ ~~~~ -4 r, D O> ~~~~ C D ~~~~ CM ~~~~~~~ -. _ P 2. _ 2 -C. -3 o 31-4 fd=. 9 C D E3 - C. t J CA -)C CO) a -4o O o~uc C C -4> >~~~~~~~~~~ _ v ' 3 < a 9 o -4-4 was independent on the presence or absence of the promoter and of the orientation of the VP5 insert. Comparably low efficiencies were obtained with the promoterless pjs1. These results are consistent with the idea that the VP5 promoter does not function efficiently in uninfected cells, but do not constitute proof of this, as the low efficiency could also be accounted for by a number of other factors. For example, the VP5 promoter may contain essential sequence elements located downstream from -1, which are not present in the recombinant (e.g., the VP5 cap site); or the novel nucleotide sequences placed downstream from the VP5 Goldberg-Hogness sequence by the construction may be inconsistent with efficient initiation of transcription. Alternatively, the VP promoter in the recombinants may function well but give rise to unstable or inefficiently translated transcripts. These alternative explanations for the low transforming activity of the VP5- recombinants appeared to be excluded in light of the data presented below. Transactivation of activity by superinfecting HSV. If the activity of the VP5 promoter is controlled by virus positive regulators, as appears to be the case for other HSV promoters, then one would predict that the enzymatic activity would increase substantially in cells transformed by the N12, N17 and N17BS3 recombinants after superinfection with -deficient HSV-1. Such an increase would not be expected in cells transformed by the N6 recombinant, in which the putative target of the regulatory signal is in the incorrect transcriptional orientation with respect to the coding sequence. Figure 5A shows that these predictions were verified experimentally: when transformed cells were infected with -deficient HSV-1 B26 and assayed 12 h later, a marked multiplicity-dependent increase in activity was observed, except, as expected, in the case of cells transformed by N6. These data are consistent with the idea that the VP5 promoter is activated in trans by the superinfecting virus. N17BS3 cells reproducibly displayed a lower induction of compared with N12 and N17. It is not yet clear whether this difference simply reflects the clone-to-clone variability in inducibility that we have observed earlier (39) or instead results from the fact that the N12 and N17 constructs retain the wild-type promoter as well as the VP5 promoter, whereas N17BS3 contains only the VP5 promoter. The kinetics of induction of activity in N 12, N17, and N17BS3 cells were compared with those observed with px1/a cells in which is induced from the promoter (39). Within the limits of error of the experiment, the kinetics were identical in these four cell lines (Fig. 5B), suggesting that the N17 recombinant gene is induced with delayed-early (1) kinetics. This finding is in agreement with the previous classification of the VP5 gene as a 13-y gene (reference 12 and discussed further below). Superinfection activates the VP5 promoter. The results presented in the preceding sections are consistent with the hypothesis that the VP5 promoter is essentially inactive in uninfected cells but is activated by superinfection with HSV-1. To further test this hypothesis, we examined the transcripts derived from the resident recombinant gene present in transformed cells before and after superinfection with HSV-1. In these experiments, we used cells transformed with N17BS3, which lacks the wild-type promoter. To detect specifically transcripts originating from the resident gene without interference from transcripts derived from the superinfecting viral genome, we chose to use a deletion mutant (d2 [38]) as the superinfecting virus. This mutant virus lacks sequences extending from +12 to

5 548 DENNIS AND SMILEY Bgl 1I ~~P I PP ATG P Bgl H I ATG Baam/BgI H pxi MOL. CELL. BIOL. N17, N12 /Bgl H P PVP5 Bgl II N6 ATG tpvp lbgl E1 N17 BS3 A P Bgl II/ FIG. 4. Structure of the hybrid genes used in this study. The structures of the viral inserts carried by the various plasmids are indicated. The constructions are described in the text. P refers to the promoter, whereas PVP5 refers to the presumed VP5 promoter. The transcriptional orientation of the VP5 promoter is indicated for each recombinant gene. pjsl +852 in the wild-type transcript. Therefore, transcripts present in N17BS3 cells can be specifically detected using hybridization probes which overlap the deleted portion of the superinfecting d2 genome. In the first of these experiments, cytoplasmic RNA prepared from uninfected and d2-infected N17BS3 cells was hybridized to a single-stranded recombinant DNA probe which was 5' end labeled at an RsaI site located at +118 in the gene and which extended to an RsaI site upstream of the VP5 promoter (Fig. 6A). As a control, a portion of the probe was also hybridized to cytoplasmic RNA isolated from Vero cells lytically infected with wild-type HSV-1. After hybridization and treatment with S1 nuclease, the protected portions of the probe were sized on an 8% polyacrylamide sequencing gel, with reference to markers generated by the adenine plus guanine (A+G) and cytosine plus thymine sequencing reactions carried out on the same probe. We were unable to detect any transcripts that hybridized to this probe in uninfected N17BS3 cells (Fig. 6) even after prolonged exposures (lane 1), whereas a strong hybridization signal was observed after infection with d2 (lane 2). The length of the DNA fragment protected by superinfected cell RNA indicated that the transcript induced from the N17BS3 gene initiates ca. 26 nucleotides downstream from the VP5 Goldberg-Hogness sequence, within sequences that comprise the nontranslated leader of wild-type mrna (Fig. 3). Thus, the induced transcript corresponds to that predicted to arise by activation of the VP5 promoter. As expected, wild-type mrna from lytically infected Vero cells protected the probe up to the site of fusion to the VP5 promoter (lane 4), at which point the RNA ceases to be complementary to the probe. Incidentally, this lane also shows that even under our stringent conditions of S1 digestion, not all of the probe is degraded to precisely the predicted length. A signal is observed at the position of the first divergent nucleotide (indicated by the arrow), but signals are also seen one, two, seven, and eight residues further upstream. This result shows that heterogeneity in the length of the protected fragment does not necessarily indicate a multiplicity of initiation sites in this type of experiment, but instead may reflect incomplete digestion of the DNA portion of the hybrid. Uninfected N17BS3 cells express low levels of viral and therefore must contain transcripts, yet we were unable to detect transcripts with the RsaI probe. Roberts and Axel (37) have suggested that when a promoterless fragment is expressed in transformed cells, the transcripts initiate at sites located within the structural gene. Accordingly, we tested for such downstream initiation events by using a probe derived from the wild-type gene (Fig. 6B). This was a single-stranded fragment 5' end labeled at the AvaI site located at position +351 in the wild-type mrna and extending to the upstream PvuII site (-2). With the same RNA preparations as in the experiments of Fig. 6A, we detected a transcript in uninfected cells which initiates at about residue +2 in the wild-type sequence (lane 1). After superinfection, this transcript was present in somewhat decreased amounts, while much larger amounts of RNA initiating from the VP5 promoter were detected (lane 2). Lane 3 shows that no signal was observed with RNA prepared from HSV-d2 infected Ltk- cells. This experiment demonstrated that uninfected N17BS3 cells contained small amounts of RNA initiating within the structural sequence and did not contain any detectable RNA arising from the VP5 promoter. By contrast, after superinfection, large amounts of RNA initiated from the VP5 promoter accumulated. In the remainder of this paper, we refer to the shorter RNA found in uninfected cells as the constitutive transcript, and the longer RNA observed after superinfection as the induced transcript. TABLE 1. Transformation of - cells by various plasmidsa Plasmid + colonies per plate None...,,,, pxl , 296, 148, 1, 24 pjs1...,,, 1 N6... 5,, 2 N17..., 1,, 1 N17BS3b...,,,, N12..., 1,, a A 1-ng amount of plasmid DNA was used per plate; colonies were scored after 3 weeks. b One cell line transformed by N17BS3 was isolated in another experiment.

6 VOL. 4, a: E 1 1 TRANSACTIVATION OF A LATE HSV PROMOTER 549 DISCUSSION The major conclusion that we have drawn from the work presented in this communication is that the HSV-1 VP5 promoter did not function at a detectable level in uninfected mouse cells but was efficiently activated after superinfection with HSV-1. The evidence that the VP5 promoter was inactive before infection is twofold. First, when the coding sequence was attached to the VP5 promoter, the resulting hybrid gene transformed Ltk- cells to + at an extremely low frequency. This low frequency of transformation was in marked contrast to the efficiencies that have been B~~~~~~~N A AG CT i-f AG CT It~~~~~~~~~~~~~~~~~~~~~~~~~ B AG CT C Ni 2 N6 3 Wa~~~~~~~~~~~~1 -U in wo -p.-.. w op I-s 2 LL 'E HOULTPICTOFT INFECTION ~~~~~~~~N176S a *B - as 8 a -pg- - -p _O 6 df HOURS AFTER INFECTION FIG. 5. Induction of enzymatic activity in several cell lines. (A) The indicated cell lines were infected with HSV-1 B26 and harvested 12 h later. Cell extracts were then assayed for activity (see text). The numbers express the fold increase above the basal level of activity found in uninfected cells. (B) The indicated cell lines were infected at a multiplicity of infection of 5 PFU per cell and were assayed for activity at various times. The results are expressed as in (A). I _ a -= _I. _ saw a adw am _-ur -U-. mu A_ d The constitutive transcript cannot encode a full length polypeptide, as it lacks the first ATG codon for the protein (28, 4); however, it can encode a shorter protein lacking the first 44 residues of the wild-type enzyme. The transcript initiates 44 nucleotides upstream from what is ordinarily the second in-phase methionyl codon of mrna. We have evidence from another study showing that a mutant virus which synthesizes a similar RNA (initiating ca. +198) induces 4% of the wild-type levels of enzymatic activity (M. E. Halpern and J. R. Smiley, unpublished data). Therefore, the constitutive transcript found in N17BS3 cells should encode a catalytically active, although truncated, enzyme. For this reason, we conclude that the basal-level enzymatic activity present in uninfected N17BS3 cells is encoded by the constitutive transcript. After superinfection, the level of the constitutive RNA drops somewhat, whereas the induced transcript accumulates. This in turn suggests that the increase in enzymatic activity observed after infection results from translation of the induced RNA. FIG. 6. Induction of transcripts from the VP5 promoter after infection of transformed cells. Cytoplasmic RNA prepared from cells before and 12 h after infection (multiplicity of infection, 2 PFU per cell) was hybridized to the indicated single-stranded DNA probe. After Si digestion, the products were sized by gel electrophoresis. (A) Hybridization to an RsaI fragment prepared from N17BS3 plasmid DNA (see text). The products are displayed on an 8% polyacrylamide sequencing gel. AG and CT, products of the A+G and C+T sequencing reactions carried out on the probe; lane 1, uninfected N17BS3 cell RNA; lane 2, HSV d2-infected N17BS3 cell RNA; lane 3, uninfected Vero cell RNA; lane 4, HSV wild-type infected Vero cell RNA. (B) Hybridization to an AvaI-PvuII fragment prepared from the wild-type gene (see text). The products are displayed on a 6% polyacrylamide sequencing gel. G, AG, CT, and C, products of the corresponding sequencing reactions carried out on the probe; lane 1, uninfected N17BS3 cell RNA; lane 2, HSV d2-infected N17BS3 cell RNA; and lane 3, HSV d2-infected Ltkcell RNA.

7 55 DENNIS AND SMILEY obtained in other studies, with a variety of promoters, but was similar to that obtained with a promoterless fragment. Second, when one of the resulting rare + transformants was analyzed, it was found that the transcripts present in this cell line initiated within the protein coding sequence rather than in the vicinity of the VP5 promoter. We take this as indicating that there is a weak promoter within the gene just upstream from +2, which accounts for the constitutive transcript. Roberts and Axel (37) have reached similar conclusions. Although it is not clear which sequences comprise this weak promoter site, it is interesting that the sequence CATAGCAA, which resembles the Goldberg-Hogness sequence (CATAT- TAA), is found 3 nucleotides upstream from the 5' end of the constitutive transcript, and the sequence GCCATCAAC, which resembles a CAAT homology, is found beginning at -77. Perhaps these two sequences contribute to the weak promoter function that generates the constitutive RNA. The inactivity of the VP5 promoter in N17BS3 cells before infection probably cannot be accounted for by unsuspected defects in our constructions (for example, the lack of the normal VP5 mrna cap site), as the same promoter is efficiently used in these cells after infection with HSV. Consequently, we believe that its inactivity indicates that uninfected cells lack factors necessary for its activity as a promoter. We assume that this also is the case with the unmodified VP5 promoter. This conclusion is in agreement with the data of Frink et al. (12), who demonstrated that the unmodified VP5 promoter is not recognized by an in vitro transcription system prepared from uninfected HeLa cells. It is not obvious what features account for the inactivity of the VP5 promoter in uninfected cells. As indicated above, it displays two of the elements characteristic of many eucaryotic polymerase II promoters: a Goldberg-Hogness sequence at -28 and CAAT homologies at -8 and -12. The presence of these conserved sequences is therefore an insufficient condition for HSV promoter function. A variety of studies with other genes have shown that, although the Goldberg-Hogness and CAAT sequences play a role in transcription initiation (4, 9, 15, 17, 29-31), additional sequences are also required for efficient expression in vivo (2, 4, 16, 29-31). These additional sequences are located at various positions upstream from the transcription initiation site, and in the case of viral enhancers (2), exert their potentiating effects on transcription over relatively large distances in both directions. With other activators, bidirectional, long-range effects have not been documented (e.g., HSV ). In the case of the well-characterized HSV promoter, two separate upstream sequences, one on either side of the CAAT homology, are required for expression in uninfected cells (29-31). Interestingly, sequences on either side of the CAAT homology are also required for the increase in expression effected by immediate-early viral proteins (39, 45). Although the locations of the necessary sequences have not yet been precisely determined, the data are consistent with the idea that they overlap the bipartite upstream constitutive promoter element. It therefore seems possible that the constitutive -activator sequences are also somehow involved in the control of transcription exerted by viral regulators. One explanation of the behavior of the VP5 promoter is that the VP5 upstream region contains an analogous essential activator sequence which functions only in the presence of viral regulatory proteins. Thus, in the absence of the regulator, the VP5 promoter is silent. By this model, the VP5 upstream region differs from that of the HSV gene in that in uninfected cells the activator functions at a low level, MOL. CELL. BIOL. whereas the VP5 activator does not function at all. In both cases, however, superinfection dramatically increases the activity of the activator. By this model, the viral regulators are postulated to exert positive control of transcription. Another possibility is that HSV regulators act by overcoming negative control of transcription. For example, HSV promoters may be repressed by cellular regulators, which are then inactivated by HSV-coded proteins. A similar model has been proposed to account for the action of the adenovirus ElA transcriptional activator function (32). To accommodate the differences between the behavior of the and VP5 promoters in uninfected cells within this scheme, one need only postulate that the VP5 promoter has a much higher affinity for a putative cellular repressor than the promoter does. To the best of our knowledge, none of the data in the literature allows one to distinguish between these two different models for the action of HSV transcriptional regulators. Which viral proteins are responsible for the activation of the VP5 promoter? Although we have no certain evidence on this point, the kinetics of induction of enzymatic activity from the VP5 promoter in superinfected transformed cells offer a clue. These kinetics are similar to those of induction from the wild-type promoter (Fig. 5B), suggesting that the VP5 promoter is activated in this situation with delayed early kinetics. This is in contrast to the kinetics observed during the course of normal lytic infections, in which the VP5 gene is expressed at a relatively low level before the onset of viral DNA replication and at a substantially higher level after replication has begun. We believe that this difference can be explained by postulating that the rate of production of VP5 mrna during lytic infection reflects two separate processes. We propose that the promoter is first activated with delayed-early kinetics, by immediate-early proteins. Second, viral DNA replication results in an increase in gene dosage, whereas the transcription rate per gene remains relatively constant, yielding an increased net rate of RNA synthesis. By contrast, the VP5- hybrid present in transformed cells is presumably not induced to replicate after superinfection, and consequently only the promoter-activation phase is monitored in this case. If this explanation is correct then it follows that only immediateearly viral proteins are needed to activate the VP5 promoter. We are currently testing this prediction. We believe that one of the most important points raised by this study is that HSV infection of mammalian cells somehow allows the efficient use of a promoter which is otherwise inactive. As this situation parallels some aspects of the developmental control of cellular genes, it seems likely that identifying the viral factors involved and determining their modes of action may shed some light on the control of cellular transcription as well. ACKNOWLEDGMENTS This work was supported by the National Cancer Institute of Canada and the Medical Research Council of Canada. J.R.S. is a Research Scholar of the National Cancer Institute of Canada. The expert technical assistance of H. Rudzroga is gratefully acknowledged. LITERATURE CITED 1. Anderson, K. P., R. H. Costa, L. E. Holland, and E. K. Wagner Characterization of herpes simplex virus type 1 RNA present in the absence of de novo protein synthesis. J. Virol. 34: Banerji, J., S. Rusconi, and W. Shaffner Expression of a p-globin gene is enhanced by remote SV4 DNA sequences.

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