Gene 271 (2001) 117±130
Regulation of herpes simplex virus gene expression Jerry P. Weir* Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA Received 2 February 2001; received in revised form 19 April 2001; accepted 4 May 2001 Received by A.J. van Wijnen
Abstract Expression of the more than 80 individual genes of herpes simplex virus 1 (HSV-1) takes place in a tightly regulated sequential manner that was ®rst described over 20 years ago. Investigations since that time have focused on understanding the mechanisms that regulate this orderly and ef®cient expression of viral genes. This review examines recent ®ndings that have shed light on how this process is regulated during productive infection of the cell. Although the story is still not complete, several aspects of HSV gene expression are now clearer as a result of these ®ndings. In particular, several new functions have recently been ascribed to some of the known viral regulatory proteins. The results indicate that the viral gene expression is regulated through transcriptional as well as post-transcriptional mechanisms. In addition, it has become increasingly clear that the virus has evolved speci®c functions to interact with the host cell in order to divert and redirect critical host functions for its own needs. Understanding the interactions of HSV and the host cell during infection will be essential for a complete understanding of how viral gene expression is regulated. Future challenges in the ®eld will be to develop a complete understanding of the mechanisms that temporally regulate virus gene expression, and to identify and characterize the relevant interactions between the virus and the distinctive cell types normally infected by the virus. Published by Elsevier Science B.V. Keywords: Herpesviruses; Gene regulation; Virus±host cell interactions; Transcription
1. Introduction Herpes simplex virus (HSV) has played a major historical role in the development of our current understanding of eukaryotic gene regulation. Like other DNA viruses which use the host cell RNA polymerase for transcription of mRNA, HSV was at one time considered a good model system for investigating the complexities of gene expression. The studies in the early 1980s which de®ned and characterized the cis-acting regulatory elements of a `typical' eukaryotic gene, the HSV-1 thymidine kinase gene, are considered classics in the ®eld of eukaryotic gene regulation as well as in the herpesvirus research community (McKnight et al., 1981, 1984; McKnight, 1982; McKnight and Kingsbury, 1982). Now, however, HSV and other DNA viruses for that matter are more likely to be studied by virologists interested in understanding the intricacies of Abbreviations: cdk, cyclin-dependent kinase; HSV, herpes simplex virus; ICP, infected cell protein; IE, immediate-early; PKR, protein kinase R; RNA PolII, RNA polymerase II; TAF, TATA-associated factor; tk, thymidine kinase; UL, unique long region; US, unique short region * Division of Viral Products, HFM-457, Center for Biologics Evaluation and Research, 1401 Rockville Pike, Rockville, MD 20852, USA. Tel.: 11301-827-2935; fax: 11-301-480-6124. E-mail address: [email protected]
a.gov (J.P. Weir). 0378-1119/01/$ - see front matter. Published by Elsevier Science B.V. PII: S 0378-111 9(01)00512-1
the virus itself, rather than as a model for eukaryotic gene regulation. One reason for this change in focus is the remarkable advances, not the least of which are technological, that have been made over the last two decades in directly studying the eukaryotic genome. In addition, it has become increasing clear that the regulation of expression of a `eukaryotic' gene differs depending on whether it is present in the genome of the cell or the genome of the virus. Thus, there are still formidable challenges to developing a complete picture of viral gene expression. In an interesting role reversal, it is now the ability to study the host eukaryotic cell in ®ner detail that provides the opportunity to address the crucial host cell±virus interactions that function to regulate the orderly and ef®cient expression of HSV genes during infection. There have been numerous excellent reviews on the regulation of HSV gene expression, including several within the last few years (Hay and Ruyechan, 1992; Ward and Roizman, 1994; Wagner et al., 1995; Roizman and Sears, 1996). Exhaustive reviews covering all aspects of HSV biology can be found in Fields Virology (Roizman, 1996; Roizman and Sears, 1996; Whitley, 1996). The aim of the present review is not to duplicate these works (and in fact they will be referenced quite extensively), but rather to focus on ®ndings that have been reported fairly recently. In particular, this
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review will examine how these observations are changing some of our views regarding the regulation of viral gene expression and the involvement of the host cell in this process. While one of the most fascinating aspects of HSV biology is the virus's ability to establish a state of latency in the neuron as part of its biological life cycle, this review will focus only on the regulation of viral gene expression during productive or lytic infection. This is not due to a lack of interest in the subject of latency, but to the enormity of the task when coupled with the subject of productive infection. The double-stranded DNA genome of HSV type 1 is approximately 152,000 bp, consisting of two segments of unique DNA, referred to as the unique long (UL) and unique short (US) regions. These unique segments are ¯anked by inverted repeats of DNA (e.g. RL and RS for repeats ¯anking the UL and US, respectively), as shown in the schematic diagram in Fig. 1 (for review, see Roizman and Sears, 1996). More than 80 different genes are distributed throughout the genome on both strands; genes located in the inverted repeat regions are present in two copies. In general, each gene has its own promoter to direct transcription, although some transcripts share 3 0 ends. The nomenclatures used by herpesvirologists over the years to designate HSV genes have been somewhat confusing since many genes and proteins were given different names by different investigators. Since the published sequence of the genome became available (McGeoch et al., 1985, 1988), it has become commonplace to refer to genes by their location in the genome, e.g. UL1-56, US1-11. Encoded proteins, even if assigned multiple names, are usually referenced to their respective genomic location, e.g. the UL48 gene encodes a viral transactivator referred to variously as VP16 (Virion Protein 16), aTIF (a gene Trans-Inducing Factor), ICP25 (Infected Cell Protein 25) or VMW65 (Virion Molecular Weight 65 kDa). A complete description of the HSV-1 genome, genes, coding regions, and function of viral proteins can now be found at www.stdgen.lanl.gov. 2. The general scheme of viral gene expression 2.1. Overview The general pattern of HSV gene expression in produc-
Fig. 1. Schematic representation of the HSV genome. The approximately 152,000 bp genome is divided into a UL and US region. The unique sequences are separated by inverted repeats (shown as boxes and designated as RL and RS). Detailed descriptions of the genome and the location of individual transcripts can be found in Roizman and Sears (1996) and Wagner et al. (1995) and at www.stdgen.lanl.gov.
tively infected cells was ®rst described over 20 years ago (for review, see Roizman and Sears, 1996). Extensive investigations since that time have revealed layers of complexity to the pattern, but the overall picture as ®rst portrayed remains remarkably the same. The scheme of viral gene expression that was established revealed that three groups of virus-speci®c polypeptides, designated as a (immediateearly, IE), b (early) and g (late), are synthesized during infection in a coordinated and sequential fashion (Honess and Roizman, 1974). It was subsequently demonstrated that in general the pattern of viral protein synthesis re¯ects the pattern of viral gene transcription and the level of viral mRNA accumulation in the infected cell (Jones and Roizman, 1979). Methodical mapping and characterization of viral transcripts using a variety of techniques (e.g. Northern blotting, hybrid selection of RNA, primer extension, RNase protection, etc.) has resulted in assignment of almost all transcripts to a temporal class (for review, see Wagner et al., 1995). Most recently, the use of oligonucleotide-based DNA microarrays has con®rmed the general classi®cation of transcripts and illustrated how the host cell used in such analyses contributes to the expression and regulation of individual transcripts (Stingley et al., 2000). This technology will become invaluable for our future understanding of virus±host cell interactions and the role of speci®c viral proteins in gene expression and replication. Numerous investigations also have described the general features of genes of each temporal class, but a de®nitive understanding of the mechanisms involved in the regulation of the cascade remains elusive. 2.2. IE gene expression The regulation of HSV IE gene expression, as well as the characterization of the IE genes and their encoded proteins, has probably been studied to a greater extent than the regulation of either early or late gene expression. Most likely this is due not to the fact that these are the ®rst viral genes transcribed during infection but rather to the essential roles that the encoded proteins play in the regulation of viral gene expression. IE genes were originally de®ned as genes whose expression occurs in the absence of de novo protein synthesis, and ®ve such genes were identi®ed: the genes encoding ICP4 (RS1), ICP0 (RL2), ICP27 (UL54), ICP22 (US1), and ICP47 (US12). Expression of the IE genes is regulated by various cis-acting regulatory elements including TATA and CAAT elements, binding sites for the transcription factor SP1, and in some cases, binding sites for ICP4 (Fig. 2A). The most distinctive facet of IE gene regulation, however, is that expression of these genes is controlled by the action of a virion trans-activator protein (for review, see O'Hare, 1993). This virion protein, VP16, encoded by the UL48 gene, forms a complex with cellular proteins and mediates IE gene activation through a common regulatory element (often designated as TAATGARAT for the most well conserved portion of the extended consensus sequence
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Fig. 2. Schematic representation of IE, early, and late HSV promoters. The general arrangement and composition of cis-acting regulatory elements for generic IE, early and late promoters is shown. The actual number and arrangement of elements varies among promoters of each class. (A) In addition to the TATA element, IE promoters have TAATGARAT elements in the upstream region through which the viral VP16 protein mediates transcriptional activation. Binding sites for eukaryotic transcription factors such as SP1 are also present upstream from the TATA element, and in at least some IE promoters, binding sites for ICP4. (B) Early promoters have binding sites for eukaryotic transcription factors upstream of the TATA element but no identi®ed cis-acting regulatory elements further downstream. (C) Late promoters have an initiator element (Inr) at the start of transcription, and in at least some late promoters, a downstream activation site.
involved, GYATGNTAATGARATTCYTTGNGGG) in the upstream region of each promoter (Fig. 2A). There are reports of at least two other viral transcripts (UL39 and the transcript encoding ORF P) (Lagunoff and Roizman, 1995) that are expressed to some degree in the presence of cycloheximide (which blocks de novo protein synthesis). Nevertheless, the ®ve genes listed above that are directly regulated by VP16 and expressed during productive infection are commonly considered to be the true HSV IE genes. Activation of the HSV IE genes is initiated soon after virus infection by the formation of a multi-protein complex containing the virion protein VP16 and two host cell proteins, Oct-1 and HCF (Host Cell Factor); the complex binds to the TAATGARAT elements present in each IE promoter. Binding speci®city is conferred by Oct-1, a member of a subclass of homeodomain proteins referred to as POU proteins (O'Hare, 1993). Transcriptional activation is a function of the highly acidic carboxy terminus of VP16. Indeed, several studies have shown that the VP16 acidic domain can be tethered to various DNA binding domains to activate transcription, indicating that the target for VP16 activation is most likely a component of the basal transcriptional machinery of the cell (for review, see Flint and Shenk, 1997). Recent studies have now shed light on the
role of HCF during HSV infection as well as its normal role in the cell. HCF is required for the expression of several cellular genes necessary for progression through the cell cycle (Goto et al., 1997). Expression of these cell cycle genes is likely regulated through HCF interaction with another cellular protein, an interaction mimicked by HCFVP16 (Freiman and Herr, 1997). During HSV infection, HCF binds to VP16 to promote stable interaction with Oct-1 (Hughes et al., 1999) and may act as a nuclear import factor for VP16, particularly in the early stage of infection (La Boissiere et al., 1999). In addition, HSV infection alters the subcellular distribution of HCF (La Boissiere and O'Hare, 2000). Interestingly, recent studies have shown that HCF remains in the cytoplasm of sensory neurons, but is rapidly relocated to the nucleus under experimental conditions that induce reactivation of HSV from latency. These results suggest a possible role for this cellular protein in the switch between latency and productive infection (Kristie et al., 1999). Four of the ®ve IE proteins play critical roles in the regulation of HSV gene expression. ICP4 and ICP27 are essential regulatory proteins in all experimental systems (in vitro and in vivo); ICP0 and ICP22 are dispensable in at least some systems but the evidence indicates that each plays an important regulatory role in viral gene regulation. A complete understanding of how each of these proteins functions in regulating the cascade of viral gene expression is not yet available, but recent studies have unraveled at least part of their distinctive yet interrelated roles. ICP4 is perhaps the most extensively studied HSV protein (for review, see Roizman and Sears, 1996). This approximately 175 kDa protein is encoded by the RS1 gene which is present in two copies in the viral genome. It was established many years ago that functional ICP4 is necessary for the activation of both early and late gene expression (Watson and Clements, 1980); it was subsequently shown that ICP4 also functions to repress the expression of some viral genes, including its own gene (DeLuca and Schaffer, 1985; O'Hare and Hayward, 1985). Neither the mechanism of activation nor the mechanism of repression is completely understood. Nevertheless, a combination of biochemical and genetic analyses have successfully de®ned multiple functional domains of the protein and provided clues as to at least some of the functions and mechanisms of action of ICP4. For example, functional domains for transactivation, dimerization, nuclear localization, repression, and DNA binding have been described. The ability of ICP4 to repress expression of its own mRNA expression and of the gene encoding ORF P apparently requires direct ICP4 binding to speci®c DNA binding sites near the start of transcription (Michael and Roizman, 1993; Lagunoff and Roizman, 1995). However, the mechanism responsible for repression does not appear to be a simple steric block to transcription, but rather involves speci®c ICP4 interactions with the general transcription factors TATA-binding protein (TBP) and TFIIB (Gu et al., 1995; Kuddus et al., 1995). On the other
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hand, even though numerous ICP4 binding sites have been reported throughout the viral genome, the role of these sites in ICP4 transactivation is still not clear. Speci®c ICP4 DNA binding sites do not appear to be necessary for ICP4mediated transactivation, although the DNA binding domain of ICP4 is required. In fact, the exact mechanism(s) by which ICP4 activates gene expression is still not known, although it appears that transactivation is mediated through ICP4 interaction with the TBP-containing general transcription factor TFIID (Gu and DeLuca, 1994); this interaction occurs through the TBP-associated factor (TAF) 250 (Carrozza and DeLuca, 1996). Studies designed to detect additional factors involved in ICP4-mediated activation identi®ed the cellular high mobility group factor 1 (HMG1) protein as another cofactor, and it was proposed that HMG1 might facilitate ICP4 interaction with TFIID by means of its ability to bend DNA (Carrozza and DeLuca, 1998). Most recently, it was reported that ICP4 may promote transcription by facilitating the formation of preinitiation complexes through recruitment of TFIID. These studies demonstrated that ICP4 was able to recruit TFIID in the absence of other proteins and suggested that the mechanism by which ICP4 activates gene expression occurs at the very earliest stages of transcription (Grondin and DeLuca, 2000). The question of how ICP4 speci®cally targets the viral genome for transactivation of gene expression remains to be answered. The 63 kDa ICP27 phosphoprotein is the second IE protein that is essential for lytic replication of HSV. As for ICP4, multiple functions have been attributed to this protein, including roles in both the transcriptional and post-transcriptional regulation of viral gene expression (for review, see Phelan and Clements, 1998). Thus, ICP27 has been implicated in the switch from early to late gene expression during virus replication, polyadenylation site selection and 3 0 RNA processing, inhibition of mRNA splicing, RNA binding, and in transport of intronless viral mRNA out of the nucleus. Furthermore, as might be expected for a multi-functional protein, multiple functional domains have been identi®ed. Identi®ed domains include activation and repression domains, nuclear localization signals, RNA binding domains, and a nuclear export signal. To what extent the varied functions ascribed to ICP27 are interrelated is not yet completely clear. Nevertheless, some of the functions of ICP27, particularly some of its posttranscriptional functions, have become clearer over the last few years as a result of numerous investigations. For example, the inhibition of mRNA splicing that is observed in HSV-infected cells requires ICP27 (Hardy and SandriGoldin, 1994). Since only four of the more than 80 viral transcripts expressed during HSV infection contain introns, splicing inhibition would seem to confer an advantage to viral transcripts over most cellular transcripts. Accordingly, three of the four spliced HSV transcripts are expressed from IE genes before maximal inhibition of splicing takes place. The fourth spliced viral transcript, that of the UL15 gene, is
expressed late in infection. By the time of UL15 expression, splicing may no longer be inhibited by ICP27 or the transcript may be spliced by an alternative mechanism that is not inhibited by ICP27. Recent work has now shown that ICP27 interacts with several cellular proteins, including at least one that is known to inhibit splicing (Bryant et al., 2000), suggesting a possible mechanism for ICP27mediated inhibition. A second post-transcriptional function of ICP27 that has been extensively explored in recent years is the potential role of this regulatory protein in RNA binding and transport. As already noted, ICP27 binds RNA; an RGG RNA binding motif and KH-like RNA binding motifs have been identi®ed (Mears and Rice, 1996; Soliman and Silverstein, 2000). In addition, several recent reports have demonstrated that ICP27 shuttles from the nucleus to the cytoplasm by means of a leucine-rich nuclear export signal (Phelan and Clements, 1997; Soliman et al., 1997; Mears and Rice, 1998; Sandri-Goldin, 1998). Since shuttling occurs late in infection and ICP27 appears to bind preferentially to intronless viral RNA in both the nucleus and the cytoplasm, a model has emerged that strongly suggests that ICP27 mediates the export of intronless viral mRNA to the cytoplasm (Sandri-Goldin, 1998; Soliman et al., 1997). Taken together, these ®ndings suggest that ICP27 functions in a post-transcriptional manner to promote the expression of viral mRNA by inhibiting early in infection the splicing of spliced mRNAs, the majority of which are of cellular origin, and by selectively increasing late in infection the amount of un-spliced viral mRNAs in the cytoplasm. Thus, at least part of the mechanism by which ICP27 regulates the expression of viral genes has become clearer. However, there are still unanswered questions regarding the mechanism of action for some of the other reported functions of ICP27, including the extent of its role in regulating the switch from early to late gene expression. Several studies have now shown that ICP27 is necessary for normal expression levels of several, but not all, early genes (Samaniego et al., 1995; Uprichard and Knipe, 1996). Since some of the early genes that require ICP27 code for proteins necessary for viral DNA replication, these ®ndings probably explain the requirement of ICP27 for normal viral DNA synthesis. It may be that the function of ICP27 in facilitating viral mRNA transport becomes progressively more important during the course of infection as normal host cell functions, such as RNA export, become increasingly compromised. The function of ICP22 in viral gene regulation is less clear than that of either the ICP4 or ICP27 proteins. This is partly due to the fact that ICP22 is not required for virus replication in many cell culture systems. In certain rodent cell lines and primary human cells, however, ICP22 is necessary for ef®cient virus replication and for the expression of both ICP0 and a subset of viral late genes (Sears et al., 1985). The carboxy terminus of ICP22 is also encoded by a second overlapping transcript that has been designated as US1.5 (Carter and Roizman, 1996). Recent analysis of
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ICP22 and US1.5 has shown that virus containing a deletion of the amino terminal portion of ICP22, i.e. the domains unique to ICP22, is avirulent in experimental animal systems (Ogle and Roizman, 1999). Other domains in the amino terminus have also been identi®ed, including a putative nuclear localization signal and a domain that interacts with a cellular protein, p78. Domains in the carboxy portion of ICP22 common to both ICP22 and US1.5 are necessary for optimal expression of a subset of late genes, and virus mutants lacking these domains are also avirulent in animal models of infection. Interestingly, the role of ICP22/US1.5 is intimately tied to that of the UL13 protein kinase (Purves et al., 1993). The UL13 kinase, along with the US3 kinase, phosphorylates ICP22/US1.5, and HSV mutants lacking functional UL13 have similar phenotypes as ICP22 mutants. Recent work has shown that ICP22 is responsible for an altered phosphorylation of the large subunit of RNA polymerase II (RNA PolII) (Rice et al., 1995; Long et al., 1999), although the functional signi®cance of this interaction is not yet clear since ICP22-de®cient virus will replicate in some cells such as Vero without observable altered PolII phosphorylation. In addition, ICP22 has been reported to interact with several cellular proteins including cell cycle regulated proteins (Bruni and Roizman, 1998; Bruni et al., 1999). One such interaction, however, that has become better understood is the ICP22/UL13-mediated activation of the cellular cdc2 protein kinase (cdk1). Experiments have now shown that cdc2 kinase is activated in HSV-infected cells and is dependent upon functional ICP22 and UL13 (Advani et al., 2000a). Speci®c inhibition of this cellular kinase results in inhibition of expression of the same set of late genes inhibited by the inactivation of ICP22/UL13 (Advani et al., 2000c). How cdc2 actually regulates the expression of this subset of late genes, whether this regulation is direct or indirect or involves the reported modi®cations to RNA PolII, and why a subset of late genes would be regulated differently from other late genes are questions still to be answered. The exact role of the IE ICP0 protein in regulating viral gene expression also has been dif®cult to establish. Although not absolutely required in tissue culture systems, ICP0-negative HSV is impaired for growth at low multiplicities of infection. It has also been implicated in the establishment and reactivation of the virus from latency, but it has been dif®cult to separate the role of the protein in these processes from its effects on virus growth. ICP0 has long been referred to as a `promiscuous' transactivator, a description of its ability in transient assays to transactivate a variety of unrelated genes either by itself or in concert with ICP4. There is no evidence that ICP0 binds to DNA, either speci®cally or non-speci®cally, but ICP0 localizes to the nucleus soon after infection, where presumably its role in transactivation is manifested. Nevertheless, ICP0 is found in the cytoplasm of primary cells within a few hours after infection (Kawaguchi et al., 1997a), suggesting that its role in transactivation is more complex than originally
thought or that the protein has multi-functional roles. Several ®ndings reported in the last few years, seemingly unrelated at the time, have begun to shed light on one mechanism in which this protein might function in the infected cell. The story so far suggests a fascinating and complicated interaction between the virus and the host cell that still is only partially understood (for recent reviews, see Everett, 1999, 2000a). One set of ®ndings demonstrated that ICP0 interacted with multiple cellular proteins, including cyclin D3 (Kawaguchi et al., 1997b), translation elongation factor EF-1d (Kawaguchi et al., 1997a), a ubiquitinspeci®c protease (Everett et al., 1999b), and the transcription factor BMAL1 (Kawaguchi et al., 2001). Second, ICP0 was shown to contain a so-called ring ®nger domain in its amino terminus. Although originally thought to be similar to zinc ®nger DNA binding domains, ring ®nger domains have now been shown to play critical roles in mediating the transfer of ubiquitin to substrates targeted for degradation by the proteasome (for review, see Joazeiro and Weissman, 2000). Third, early in infection ICP0 was observed to localize in the nucleus at pre-existing nuclear sub-structures known as ND10 (Nuclear Domain 10). Within a few hours these structures are disrupted, and this disruption required the ring ®nger domain of ICP0 and could be inhibited by proteasome inhibitor drugs. The view converging from these observations is that ICP0 induces the degradation of speci®c cellular targets, and that this degradation is an integral component of the mechanism by which ICP0 activates viral gene expression during productive infection (Everett et al., 1999a; Parkinson et al., 1999; Everett, 2000b). Recently, it was reported that ICP0 is poorly expressed in primary neurons in culture and in a differentiated neuronal cell line (Chen et al., 2000). This result suggests that the lack of ICP0 accumulation may favor establishment of the latent state in neurons by precluding the normal cascade of viral gene expression necessary for lytic infection. One proposed model to explain the role of ICP0 and proteasome-mediated proteolysis in HSV infection suggests that a cellular repressor of the viral lytic pathway is a target of ICP0-induced proteolysis. Thus, viruses de®cient in ICP0 can replicate only when there are a large number of input viral genomes; further, in certain cells such as neurons, in which ICP0 expression is de®cient, latency would be more likely than productive infection (Everett, 1999). An alternative view of this process would not require a speci®c repressor of HSV replication, but rather a combination of positive and negative forces acting to redirect critical cellular functions to the incoming extrachromosomal viral genome. Thus, VP16 would target the IE viral genes for expression, followed by ICP4-induced activation of viral genes. These positively acting mechanisms would be complemented by ICP0mediated degradation of speci®c cellular proteins, a process which would be necessary before other critical cellular components could be re-targeted to the HSV template. In any case, the next challenge will be to identify all of the substrates targeted by ICP0 for degradation in the infected
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cell, and to determine the exact way in which this contributes to viral gene expression in the cell types relevant for HSV replication. 2.3. Early gene expression As noted above, the HSV early gene encoding thymidine kinase (UL23) was one of the ®rst viral genes to be studied in detail. Although the original work that mapped and identi®ed cis-acting regulatory elements of this gene was done in non-viral systems (e.g. micro-injection of the tk gene into Xenopus oocytes), that work was soon followed by studies of the gene in the context of the viral genome (Coen et al., 1986). Interestingly, the same elements required for expression as an isolated eukaryotic gene, i.e. a TATA element, two binding sites for the transcription factor SP1, and a CAAT element (Fig. 2B), were required for expression during HSV infection. No additional cis-acting elements necessary for expression during infection were identi®ed. This presented a paradox that has still not been completely resolved. A viral gene such as tk, which apparently contains all of the cis-acting regulatory elements suf®cient for expression in the eukaryotic cell, is not expressed when present in the viral genome until HSV IE proteins are synthesized. Similarly perplexing, expression of such a gene is turned off within a few hours of infection, while other viral genes (late genes) are apparently being transcribed by the same host cell transcription apparatus. It has become clear from subsequent studies that not only is ICP4 necessary for expression of the tk gene in the viral genome, but also that this transactivation is mediated through the TATA element of the tk promoter. In fact, the tk TATA element alone can direct modest transcription in the absence of the upstream regulatory elements, but only in the presence of functional ICP4 (Imbalzano et al., 1991). Nevertheless, there is no indication that there are distinctive features to the TATA elements that are speci®c to early HSV promoters. Instead, the TATA element appears to in¯uence the strength of expression rather than the timing (Cook et al., 1995). While the studies on the tk gene were instrumental in de®ning the regulatory elements of a typical HSV early gene, it is only recently that other early genes have undergone similar detailed analysis. The results obtained from a detailed analysis of both the UL37 and UL50 early promoters have revealed that individual HSV early promoters contain a variety of cis-acting regulatory elements commonly found in eukaryotic genes transcribed by RNA PolII (Pande et al., 1998). Taken together with the earlier studies on the tk gene, the results demonstrate that the composition and arrangement of these elements differ considerably among HSV early promoters, but all are found upstream from an essential TATA element. Further, there is no evidence to date of cis-acting regulatory elements that directly interact with virus-speci®c transcription factors.
2.4. Late gene expression The synthesis of viral DNA heralds a major switch in the program of HSV gene expression (for review, see Wagner et al., 1995). Concurrently with DNA replication, viral late genes begin to be expressed while early gene expression begins to wane. These processes are intimately linked, since inhibition of DNA replication also inhibits late gene expression and prolongs the expression of early genes. As the majority of late genes encode structural proteins, virus replication is undoubtedly more ef®cient as a result of this integration of DNA replication and late gene expression. This is, however, an economic argument for the conservation of scarce cellular resources and does not explain the mechanistic basis for the interlinkage of the processes. Efforts to understand the difference between HSV late genes and early genes have led to a detailed analysis of the composition of late promoters. Like IE and early promoters, late promoters require a TATA element, and there seems to be no difference between late promoter TATA elements and other viral or eukaryotic TATA elements that account for temporal expression (Steffy and Weir, 1991a; Imbalzano and DeLuca, 1992). As noted earlier, ICP4 is necessary for the activation of late gene expression, and, as for early promoters, its action is mediated through the TATA element. Unlike IE and early promoters, however, true late promoters do not possess cis-acting regulatory elements upstream from the TATA element. Instead, late promoters contain other elements downstream from the TATA (Homa et al., 1986; Mavromara-Nazos and Roizman, 1989; Kibler et al., 1991; Guzowski and Wagner, 1993), including an initiator element at the start of transcription (Steffy and Weir, 1991b; Woerner and Weir, 1998), and in at least some late promoters another regulatory element further downstream in the 5 0 non-translated portion of the gene that is necessary for maximal expression (Guzowski et al., 1994; Petroski and Wagner, 1998) (Fig. 2C). Similar to the situation for early promoters, all of the regulatory elements so far identi®ed as necessary for ef®cient late promoter activity are common eukaryotic regulatory elements. The key feature that distinguishes early and late promoters from one another is the arrangement and composition of these regulatory elements. One model that would at least partly explain the temporal control of viral gene expression is that the transcription complex that recognizes the distinctive arrangement of late promoter elements and promotes late gene expression is fundamentally different from that present before DNA replication ensues (for review, see Wagner et al., 1995). The mechanism that would control the formation of such hypothetical transcription complexes, or the controlled targeting of them to the variety of HSV genes to effect temporal expression, is not known. True HSV late genes, often referred to as g2 genes, require DNA replication for any appreciable accumulation of their mRNAs. Some late genes, however, are expressed to
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a degree in the absence of DNA replication but require DNA synthesis for maximal expression. These genes are often referred to as leaky-late or g1 genes. Analysis of the regulatory regions of these viral promoters reveals that such promoters contain various cis-acting regulatory sequences upstream of the TATA element similar to early promoters, as well as an initiator element at the start of transcription similar to strict late promoters (Huang and Wagner, 1994; Lieu and Wagner, 2000b). Thus, these promoters seem to possess regulatory features of both early and late promoters, and indeed, studies have shown that promoters constructed by fusing the upstream regions from an early promoter to the downstream region of a late promoter are expressed like g1 genes (Mavromara-Nazos and Roizman, 1989). It might be assumed that g1 regulation of a particular viral gene signi®es the necessity of the encoded protein prior to viral DNA replication as well as late in infection. However, in a recent report, expression of the g1 VP5 gene as either an early or a strict late gene had little effect on virus replication, at least in the cell culture models that were examined (Lieu and Wagner, 2000a). Although the structure of late promoters has been de®ned, there has been no major advance in our understanding of the link between late gene expression and DNA replication or in the mechanism of late gene activation. As discussed above, the observed structural differences between early and late promoters suggest that the transcriptional machinery of the cell distinguishes early from late genes during the course of infection. Even if correct, such a model does not easily predict how transcriptional activation would be linked to DNA replication. One dif®culty, of course, is that it has been almost impossible to separate the two processes. In spite of the dif®culties, some of the factors necessary for ef®cient late gene expression are now known. The effects of ICP22, UL13, and cellular cdc2 on the expression of a subclass of late genes represented by US11 have already been noted. It is not known how this subclass of HSV late genes differs from other late genes such as that encoding gC (UL44). Also as mentioned earlier, functional ICP27 is required for late gene expression, and its effects on late gene expression may be manifested in multiple ways. In addition, ICP4 is necessary for activation of late gene as well as early gene transcription. Clearly though, the presence of ICP4 is not suf®cient for late gene expression since late genes are silent before DNA replication, even though ICP4 is present to activate early genes. Nevertheless, an understanding of the interactions between ICP4 and other transcription factors may shed light on whether ICP4 interacts with fundamentally distinct transcription complexes in the course of temporally regulating viral gene expression. As already noted, such in vitro studies using the late gC promoter as a template have identi®ed speci®c ICP4 interactions with HMG1 (Carrozza and DeLuca, 1998) and the cellular transcription factor TAF250 (Carrozza and DeLuca, 1996); it is not yet known whether these interactions are speci®c for late gene expression. Interestingly, it was
recently shown that HSV late gene expression is preferentially inhibited in a temperature-sensitive TAF250 cell line at the non-permissive temperature even in the presence of signi®cant viral DNA replication (Dhar and Weir, 2000). However, it is dif®cult to determine whether this effect on transcription is directly mediated at the late promoter or indirectly by the effect of the mutant TAF250 on other cellular proteins. It has also been reported that the DNA binding protein ICP8 is required for late gene expression. The role of ICP8 in late gene expression has been dif®cult to separate from its role in DNA replication, since it is one of the viral proteins absolutely required for replication. However, certain mutants of ICP8 inhibit late gene expression to a far greater extent than they inhibit viral DNA replication, suggesting a more direct role in late gene expression (Chen and Knipe, 1996; McNamee et al., 2000). Another possible explanation to explain such observations might be that the rate of late gene expression depends upon the rate of viral DNA replication as opposed to the absolute amount of DNA that has been synthesized at any one time point. If so, then the mutant's effect on late gene expression might be greater than predicted from its effect on total replicated DNA. Regardless, a model which predicts a dual role for viral protein(s) in DNA replication and late gene activation is attractive; such a model would be similar to the situation described in the replication of phage T4, where proteins required for DNA replication are also required for transcription of phage late genes (Herendeen et al., 1989, 1992; Tinker et al., 1994). The dif®culty in extrapolating such a model to the HSV system is exempli®ed by the inability to isolate mutants of HSV which are capable of normal DNA replication but which do not exhibit late gene activation. A ®nal observation concerning the link between late HSV gene expression and viral DNA replication pertains to the visualization of nuclear compartments that serve as sites for viral DNA replication and transcription. Upon infection, input viral DNA localizes to pre-existing nuclear ND10 sub-structures (Maul et al., 1996), structures which, as already noted, are subsequently degraded through the action of ICP0. Several studies have now reported the formation of pre-replicative sites adjacent to the original ND10 sites (Uprichard and Knipe, 1997; Burkham et al., 1998), and the eventual formation of replication compartments throughout the nucleus (de Bruyn Kops and Knipe, 1988). Other studies have shown that sites of transcription and replication overlap late in infection, although there is some disagreement as to whether all replication sites are transcription sites (Phelan et al., 1997; de Bruyn Kops et al., 1998). Further, it has been shown that the viral regulatory proteins ICP4 and ICP22 co-localize with RNA PolII and viral DNA replication proteins late in infection (Rice et al., 1994; Leopardi et al., 1997). More recently, a report describes two types of replication compartments in cells expressing a dominant-negative form of ICP8 (McNamee et al., 2000). In cells which formed large replication
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compartments, both DNA replication and late viral gene expression could be observed. Late gene expression was not observed in cells containing only small replication compartments, and the authors postulate that an unknown stimulus allows the formation of the visually large replication sites that correlate with high levels of DNA replication and functional late gene transcription. Although the source and identity of such a stimulus is presently unknown, the results are intriguing because they reinforce the emerging notion that there may be a spatial control element to the temporal regulation of viral gene expression. 2.5. Conclusions Several lines of investigation have now dramatically increased our understanding of the temporal regulation of HSV gene expression. There is a clearer understanding of the differences between each class of temporally expressed viral gene, as well as a better understanding of the multifunctional viral regulatory proteins that govern their expression. Nevertheless, a complete understanding of the mechanisms that turn on and turn off speci®c genes at the required times necessary for ef®cient virus replication is lacking. As the results summarized so far indicate, virus± cell interactions may play a major role in viral gene regulation by diversion and redirection of key cellular factors. We may yet ®nd that temporal control of viral gene expression is a function of space as well as time. 3. HSV±host cell interactions and the effect on viral gene expression 3.1. Overview In vivo, HSV replicates primarily in two very distinct cell types: epithelial cells at the site of initial infection, and postmitotic sensory neurons that innervate that site. It is reasonable to assume that the virus has evolved speci®c functions to facilitate productive reproduction in these two distinctive milieu. While it is obvious that productive infection has a profound effect on these cells, the unique characteristics of each cell type undoubtedly exert a substantial in¯uence on the replication of the virus and the expression of viral genes. Some of the HSV interactions with the cell which have become better understood in the past few years have already been described above; these interactions include the ICP0induced degradation of speci®c cellular proteins and the ICP22/UL13 interactions with RNA PolII and cellular cdc2 protein kinase. There certainly are many more examples of virus±host cell interactions that in¯uence the expression of viral genes, and some of these are described in more detail below. Understanding the role of the cell in the regulation of HSV gene expression will require a thorough analysis of such interactions. The application of DNA microarray analysis will be particularly useful for describing the interactions between the
virus and the cell, for determining the changes unique to distinctive cell types, and for distinguishing those interactions that have direct relevance to viral gene expression. Preliminary studies involving a limited number of cellular probes have now been described. In one study, the expression of 588 cellular genes was compared between HSV-1infected and mock-infected cells (Khodarev et al., 1999). Although there was a decrease in the level of RNA for most transcripts in infected cells, a signi®cant accumulation was noted for a small subset of the genes analyzed. Interestingly, several of the accumulated transcripts coded for transcription factors that could have either positive or negative effects on gene expression. For example, AP-2a, a member of the AP-2 family of genes, is involved in both positive and negative regulation of developmental and differentiation genes. Another up-regulated gene in infected cells was that of gadd45, one of several genes induced upon cellular DNA damage. Microarray analysis has also been used to study the cellular genes that are induced upon infection with HSV containing mutations in speci®c viral genes. For example, infection with a mutant virus that expresses only ICP0 induced the expression of only a small number of the genes examined (Hobbs and DeLuca, 1999). Among those genes induced, however, were gadd45 (above) and mdm2, a gene associated with cell cycle regulation. Another study that examined a relatively small set of cellular transcripts also revealed the accumulation of at least one known transcription factor, and revealed differences in the transcription pattern between wild-type infected cells and cells infected with a mutant virus missing ICP27 (Stingley et al., 2000). Undoubtedly more extensive analyses of the cellular transcripts induced in different types of infected cells will be forthcoming and will aid our understanding of the interactions between the virus and various cellular hosts. 3.2. Host cell shut-off One of the earliest and best studied changes in the host cell resulting from HSV infection was the shut-off of host cell protein synthesis. This so-called virion host shut-off (vhs) function was mapped to the UL41 gene, and the encoded protein was shown to be responsible for the degradation of host cell mRNA. It was subsequently shown that the vhs protein was responsible for destabilization of viral mRNAs as well as host mRNAs. Since this protein is packaged in the virion, host cell mRNA is degraded rapidly upon infection. The end result is that soon after infection, a relatively high percentage of the mRNA in the cell is of viral origin. Furthermore, the presence and indiscriminate action of vhs likely facilitates a fairly rapid transition of viral mRNAs from one temporal class to the next. Indeed, viral mutants in vhs fail to shut down host cell protein synthesis and exhibit prolonged synthesis of IE and early proteins. Interestingly, mutation or deletion of the vhs function has little effect on virus replication in tissue culture, but results
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in attenuation of the virus in many animal models of pathogenesis (Becker et al., 1993; Strelow and Leib, 1995). Although the mechanism of attenuation is unknown, it may be due to extended expression of speci®c viral antigens, or to prolonged synthesis of host cell MHC class I molecules and/or cytokines in the mutant virus-infected cell (Geiss et al., 2000; Suzutani et al., 2000). Recent work investigating the mechanism by which vhs destabilizes mRNA has shown that vhs induces endoribonuclease activity, and this activity apparently requires one or more mammalian factors (Elgadi et al., 1999; Lu et al., 2001). Further, mRNA degradation does not require either a 5 0 cap or a poly-A 3 0 tail, but rather appears to begin at internal sites in the targeted RNA, probably near the 5 0 end of the mRNA and possibly in¯uenced by RNA secondary structure or a component of the translational machinery (Elgadi and Smiley, 1999; Karr and Read, 1999). A second HSV function involved in the shut-off of host cell protein synthesis is the viral protein designated as g34.5. This protein, expressed from a gene in the repeat sequences ¯anking the UL region and thus present in two copies in the genome, is not required for virus replication in many tissue culture models such as Vero cells. However, viruses with mutations in the g34.5 gene are restricted for replication, particularly in cells of neuronal origin and in some primary human cells. Furthermore, such mutants are extremely attenuated for replication in vivo, especially in the nervous system. In contrast to the function of the vhs protein to shut-down protein synthesis early in infection by destabilizing mRNA, the g34.5 protein prevents the shutdown of protein synthesis by the host late in infection. The mechanism by which g34.5 performs this function is now known in some detail (He et al., 1997b). Speci®cally, g34.5 binds to protein phosphatase 1a, which leads to dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) and subsequently allows protein synthesis to proceed. Phosphorylation of eIF-2a by activated double-stranded RNA-dependent protein kinase R (PKR) is a common cellular response to many virus infections. One interesting aspect of the g34.5-mediated shut-off of protein synthesis is its cell-type speci®city. The fact that HSV replication in nonneuronal cells is seemingly unaffected by the absence of g34.5 suggests that the virus has other means to evade this host cell response in non-neuronal cells. Alternatively, the host cell process of protein synthesis shut-down in response to HSV infection may not be as ef®cient in non-neuronal cells. A ®nal addendum to the g34.5 story is the recent isolation of g34.5-de®cient viruses with a revertant phenotype, i.e. viruses that are capable of sustained protein synthesis in neuronal cells in the absence of g34.5 (Mohr and Gluzman, 1996; He et al., 1997a). These mutants have a genetic rearrangement that results in the expression of the late US11 gene earlier during infection than in wild-type virus (Cassady et al., 1998a; Mulvey et al., 1999). The US11 protein binds to PKR to prevent the phosphorylation of
eIF-2a; if expressed early enough in infection before the activation of PKR, protein synthesis in the infected cell proceeds (Cassady et al., 1998b). It has been speculated that the original function of the US11 protein was to prevent the shut-off in protein synthesis, but that during evolution of the virus, a second gene, g34.5, capable of preventing shutoff was acquired from the cell. This gene, which is related to the cellular GADD34 gene, may have been more ef®cient at preventing protein shut-off; as a result, the US11 protein evolved other functions (Cassady et al., 1998a,b). Thus, the function of US11 expressed under normal conditions during virus infection is not completely clear. It is known to bind mRNA in a sequence-speci®c fashion and associate with ribosomes (Roller and Roizman, 1992), and it has been reported to complement the Rev function of human immunode®ciency virus (Diaz et al., 1996). 3.3. Virus effects on the cell cycle HSV replicates in cells at various stages of the cell cycle, including non-dividing cells such as neurons that are irreversibly post-mitotic. Nevertheless, recent reports document that there are complex changes that take place in the cell cycle upon virus infection. Some of these changes may be part of the host cell defense response, but some modi®cations are likely virus-induced changes designed to optimize replication of the virus, such as the activation of cdc2 protein kinase (described earlier). Numerous other HSV interactions with cellular cell cycle regulatory components have been described recently. For example, several related reports have shown that inhibitors of some cellular cyclindependent kinases (cdk) block replication of HSV (Schang et al., 1998). Such drugs, for example the purine derivative roscovitine, have been shown to inhibit the cellular enzymes cdk1, cdk2, cdk5, and possibly other cellular kinases such as cdk7 and cdk3. Treatment of HSV-1 infected cells with roscovitine inhibits transcription of both IE and early viral genes (Schang et al., 1999). Since the steady-state levels of at least some cellular genes, such as GAPDH, are apparently unaffected by the cdk inhibitor, the results suggest that cellular cdk activity and possibly cell cycle progression are required for HSV gene expression. Interestingly, it appears that the cdk inhibitors also block viral DNA synthesis even in the presence of IE and early proteins (Schang et al., 2000). Because multiple cyclin-dependent kinases are known to be inhibited by these drugs, it has been suggested that different cellular cdks might be involved in viral transcription and viral DNA replication. It has also been suggested that cdk inhibition may affect both transcription and DNA replication because both processes are so tightly linked during virus replication (Schang et al., 2000). On the other hand, because of the multiple targets of these drugs, both known and unknown, it is very dif®cult to determine how any particular cellular kinase might be involved in viral transcription or viral DNA replication. In addition, it remains dif®cult to distinguish direct effects of cdk inhibi-
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tion on viral transcription or DNA replication from more indirect or general effects of the drugs on other basic cellular processes. For example, cdk7 is a component of transcription factor IIH and is known to catalyze the phosphorylation of RNA PolII (Serizawa et al., 1995). Thus, inhibition of cdks may have a more general effect on cellular transcription than appreciated. While the experiments with cdk inhibitors suggest the importance of essential components of the cell cycle process in HSV replication, it also has been reported that HSV infection induces a block in progression of the cell cycle (de Bruyn Kops and Knipe, 1988). HSV infection blocks phosphorylation of pRb protein, necessary for progression into the S phase, and prevents increases in cyclin D1 and D3 and the G1-speci®c forms of cdk4 and cdk2; thus, the block occurs in the early to mid-G1 phase (Ehmann et al., 2000; Song et al., 2000). In addition, analysis of infected and stimulated quiescent cells showed that HSV infection prevented re-entry into the cell cycle, indicating that virus infection also blocked cell cycle progression in the S phase (Ehmann et al., 2000). Finally, E2F proteins, which normally regulate the transcription of S phase genes, are post-translationally modi®ed during the course of infection, and the af®nity of E2F for its DNA binding site is decreased (Olgiate et al., 1999; Advani et al., 2000b). Taken together, these results suggest that virus infection blocks the progression of the cell cycle by affecting several key players in cell cycle regulation. The results do not rule out the possibility that the virus also requires some of these same cellular components, or other components of the cell cycle regulatory network, for the expression of its own genes and the orderly progression of its own life cycle. In fact, it has been suggested that the virus might actually activate the S phase early in infection, only to later shut off cell cycle progression (Advani et al., 2000b). A more global picture of cellular transcription patterns at different stages of the cell cycle and the effect of virus infection on individual transcripts may produce a clearer understanding of some of these interactions and their role in virus replication. 3.4. Conclusions While the complete story from all of these lines of investigation is not yet known, it is clear that there are fundamental changes taking place within the cell during infection. Some of these effects may yet turn out to be indirect consequences of viral infection. But it is also quite likely that many of the changes that take place in the infected cell are a direct result of deliberate processes that the virus has evolved to ensure the ef®cient expression of its genes and replication of its genome. The fact that HSV replicates in multiple distinct cellular environments only complicates the challenge of understanding virus±host cell interactions that are speci®c for the relevant cells normally infected by the virus. A corollary challenge will be to distinguish these
interactions from those that are non-speci®c or that have indirect effects on virus gene expression.
4. Conclusions and perspectives The last few years have witnessed several amazing advancements in our understanding of the regulation of HSV gene expression. Taken together, these ®ndings reveal intricacies of gene regulation that were unimaginable when the general scheme of viral gene expression was ®rst described over 20 years ago. As several of the examples cited in this brief review illustrate, the virus has evolved various mechanisms to regulate the orderly and ef®cient expression of its genetic program. These mechanisms, which are directly mediated by virus-encoded regulatory proteins, include selective transcriptional activation of virus genes as well as post-transcriptional control of viral mRNA. Nevertheless, in spite of the progress of the last few years, there are still some very basic gaps in our understanding of the regulation of HSV gene expression. Furthermore, as noted in multiple instances, the infected cell cannot be considered as a passive bystander in which the virus expresses its genetic program by simply substituting its own mRNA for that of the cell, using cellular machinery for transcription, mRNA processing, and translation. Rather, the virus fundamentally alters the cell to divert key cellular constituents from their normal uses and to redirect these components to the viral genome for its own needs. It has been pointed out that HSV is a scavenger, selectively picking out cellular targets during the course of its evolution for subversion to its own goals (see discussion in Van Sant et al., 1999; Advani et al., 2000a). While this is likely true to some extent for most viruses, the ways in which a virus coopts the cell undoubtedly vary enormously, depending upon the complexity of the virus, its life cycle, and the idiosyncrasies of the cells it infects. Understanding the interactions of HSV and the cell during infection is absolutely essential to a complete understanding of how viral gene expression is regulated. In turn, elucidation of these virus±cell interactions often reveals unexpected ®ndings about the interworkings of the cell itself. Thus, a quarter of a century after its early use as a model system for eukaryotic gene regulation, HSV remains an important as well as a legitimate tool for investigating fundamental eukaryotic cellular processes.
5. Note added in proof The newest edition of Fields Virology, which contain updated reviews of herpes simplex virus and other herpes viruses, is now in press (Fields Virology, 4th Edition. Knipe, D.M., Howley, P.M. (Eds.), Lippincott Williams & Wilkins, Philadelphia, PA).
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