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Arvin A, Campadelli-Fiume G, Mocarski E, et al., editors. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press; 2007.

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Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis.

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Chapter 33Molecular basis of HSV latency and reactivation

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Primary infection with HSV-1 or HSV-2 results in productive replication of the virus at the site of infection, following the pattern of gene expression described elsewhere in this volume. During this initial phase, virus enters sensory neurons via their termini and retrograde transport takes the genome to the neuronal nuclei in the sensory ganglia that innervate the infected dermatome. At early times after infection, virus replication occurs in ganglionic neurons but within a few days no virus can be detected. The genome, however, persists in neurons in a latent state from which it reactivates periodically to resume replication and produce infectious virus. This reactivation event may be “spontaneous” but is generally thought to be provoked by stress stimuli that act on the neuron, or at a peripheral site innervated by the infected ganglion, or systemically. Three phases of latency are recognized. Establishment occurs during the period following primary infection, and although virus replication can be detected in a proportion of neurons during this phase, the initiation and normal progression of productive infection and cell death is arrested in those neurons destined to become latently infected. Unravelling the way in which the seemingly inexorable progression of the gene expression program is blocked constitutes a major challenge for the molecular virologist. The maintenance phase of latency is characterized by the lifelong retention of the HSV genome in a silent state, characterized by repression of all viral lytic genes. One region, encoding the latency-associated transcripts (LATs), remains active during latency. Questions relating to the maintenance of the latent state focus on the structure of the genome, the mechanisms that silence it, and the specific properties of the LAT transcription unit that enable it to remain active. During the reactivation phase the silent genome responds to cellular signals that provoke the resumption of viral gene expression. The molecular basis for this dramatic functional reversal is poorly understood and is the subject of considerable research effort. In view of the specific association of LAT with the latent state, questions concerning the role of this transcript pervade all aspects of latency.

Model systems to study latency

Animal systems provide the most relevant means of studying HSV latency. In mice, latency can be established efficiently and relatively reproducibly after inoculation with HSV-1 in the cornea, ear, footpad, or at other peripheral sites. The virus replicates at the site of inoculation, and as a consequence the exact dose applied to nerve termini is undefined and changing over the first few days. These factors complicate quantitative aspects of latency studies. Reactivation is difficult to achieve in mice and most investigations have relied on explantation of ganglia, with subsequent culture in the laboratory, to recover virus (Stevens and Cook, 1971). An in vivo protocol has been developed in which reactivation is achieved by exposure of mice to transient hyperthermia (Sawtell and Thompson, 1992b). While this method is inefficient in terms of the number of reactivation events per animal, it represents the most relevant mouse model currently available for paralleling latency in humans. In the rabbit, inoculation of the cornea results in latency that, for certain strains of HSV-1 such as McKrea, is characterized by long-term periodic virus shedding, described as spontaneous reactivation. Production of virus can be enhanced by procedures such as iontophoresis of epinephrine into the eye. For HSV-2, inoculation into the guinea pig vagina results in latency in which virus is periodically shed to form lesions that can be scored. Therefore, the rabbit and guinea pig provide reasonable models for HSV-1 and HSV-2, respectively, in humans but they are more difficult and expensive to use than the mouse. All animal systems entail inoculation with relatively high virus doses, often just short of fatal, and it is by no means clear how this relates to natural infection. A further problem is the considerable variation in the efficiency of establishment of latency and reactivation between strains of HSV, a factor that frequently causes confusion when comparisons are made between the results from different laboratories.

As a more tractable system, infection of cultured neurons has been investigated (Wilcox and Johnson, 1988; Arthur et al., 2001). These cells are susceptible to lytic infection with HSV, but if measures are taken to prevent virus replication, long-term retention of the viral genome can be achieved. LAT can be detected in a proportion of neurons, but all other viral genes are repressed. Production of virus can be induced by removal of nerve growth factor (NGF), inhibition of histone deacetylases, or various treatments that activate signal transduction pathways (Smith et al., 1992). Alternatively, cultures may be made from ganglia dissected from mice harboring latent HSV; in this case, heat shock or treatment with dexamethasone are the most effective reactivation stimuli (Halford et al., 1996).

The final type of model involves the infection of standard tissue culture cells, usually human fibroblasts, with HSV-1 mutants that are impaired for immediate early (IE) gene expression and thus do not kill cells (Preston and Nicholl, 1997; Samaniego et al., 1998). The viral genome is retained in a quiescent state in which all gene expression, including that of LAT, is repressed. The only known way of reactivating quiescent virus is to provide the HSV IE protein ICP0 by superinfection of cultures. Fibroblast systems may mimic some, but certainly not all, aspects of latency.

The latent genome

Latent HSV DNA does not contain detectable termini and almost certainly exists as a circular episome, in contrast to the linear state in the virus particle (Rock and Fraser, 1983; Efstathiou et al., 1986). Quiescent genomes stably retained in fibroblasts are also circular (Jamieson et al., 1995; Jackson and DeLuca, 2003). Various methods of quantifying viral DNA load revealed that latently infected neurons must contain, on average, many more than one HSV genome copy per infected cell. This conclusion has been verified by the use of “contextual analysis” (CXA), in which individual neurons or small groups of cells are separated and analyzed by polymerase chain reaction (PCR) (Sawtell, 1997). The latent viral genome copy number varied generally between 1 and 100, but a small proportion of neurons contained more than 1000 viral DNA molecules per cell. Likely, the retention of such high copy numbers has an influence on neuronal physiology, and recent studies have shown that latently infected ganglia contain increased levels of certain cellular gene products (Kramer et al., 2003). Furthermore, analysis of latent DNA at a gross level is skewed towards the few neurons containing thousands of viral genomes. Surprisingly, for reasons that are not understood, latent viral DNA cannot be detected by in situ hybridization (ISH), therefore in situ PCR has been applied to investigate the number of neurons that harbor HSV genomes. This approach shows that many more cells contain DNA than are detected by ISH for LAT; thus, LAT is not an unambiguous marker for latent HSV. Laser capture microdissection, in which individual neurons are excised and analyzed by PCR, confirmed that viral genomes can be isolated from LAT-negative (LAT-) neurons and essentially agreed with the quantification from CXA (Chen et al., 2002). The conclusion that there is a population of latently infected neurons that does not express LAT may depend on the sensitivity of ISH, since a study using in situ RT-PCR suggested that LAT was present in all HSV DNA-containing neurons, albeit at low concentration in many (Ramakrishnan et al., 1996).

In cells, silencing of large gene blocks occurs at the level of chromatin structure, and it is therefore suspected that an organization of this type applies to the latent viral genome. One study has addressed this issue and found that all regions of HSV DNA examined, including the LAT region, exhibit a regular nucleosomal pattern in mouse brain stem (Deshmane and Fraser, 1989). Interpretation of this result is complicated by the fact that reactivation from brain stem is inefficient and, unfortunately, it was not possible to obtain sufficient material from trigeminal ganglia for similar analyses. More recently, the application of chromatin immunoprecipitation (ChiP) assays has demonstrated the importance of histone modifications in the maintenance of latency. It is well established that post-translational modification of the amino terminal tails of histones is involved in the regulation of transcription. Thus hyperacetylation of histones is generally associated with an “open chromatin” conformation and transcriptional activity, whilst histone hypoacetylation is associated with condensed chromatin and gene silencing. Recent work on HSV-1 suggests that chromatinization of the viral genome and certain accompanying histone modifications offer a means to regulate virus gene expression during lytic infection (Herrera and Triezenberg, 2004; Kent et al., 2004). In the context of latency it is of particular significance that ChiP assays have shown the LAT promoter to be enriched with acetylated histone H3 whilst representative lytic cycle promoters exhibit a decreased association with acetylated histones (Kubat et al., 2004a,b). The demonstration that enrichment of acetylated histones on the ICP0 promoter following the application of a reactivation stimulus by ganglionic explantation strongly supports the view that genome de-repression is linked to the acetylation status of histones positioned on lytic cycle promoters (Amelio et al., 2006). Furthermore, it has been shown that a LAT-mutant exhibits enrichment of histone modifications associated with transcriptional activation during latency, suggesting that LAT-encoded functions facilitate maintenance of a repressed chromatinized genome (Wang et al., 2005). Since HSV replication in ganglia precedes latency, it has long been suspected that some viral genomes are derived from residual replication intermediates rather than from virions delivered from the periphery. During infection with TK-mutants, which replicate at the site of inoculation but not in neurons, high copy number retention of TK-virus genomes is possible and therefore some neurons can receive hundreds of viruses from the periphery (Thompson and Sawtell, 2000). In general, however, TK-mutants deposit less latent DNA than wild type virus. However, depending on the site of inoculation, TK-mutants replicate less efficiently peripherally. Thus, apparently normal latency can be established without viral replication in neurons.

The latency-associated transcripts

The only transcripts detectable during latency are the LATs, which map to the viral repeats flanking UL (Fig. 33.1). These have been detected in latently infected neuronal tissues from experimentally infected animals and following natural infection in humans (Stevens et al., 1987). Similar transcripts are synthesized during latent infection by HSV-2 and other alphaherpesviruses such as bovine herpesvirus-1 (BHV-1) and pseudorabies virus.

Fig. 33.1. Location and organization of HSV-1 LATs.

Fig. 33.1

Location and organization of HSV-1 LATs. The IRL/IRS region of the genome is expanded in part (a). In addition to the LATs, the positions of lytic cycle genes and a set of transcripts known as L/STs, which specify ORFs O and P, are shown. The functions (more...)

Structure of LATs

In HSV-1, the LATs comprise a series of colinear predominantly nuclear transcripts. They consist of a highly abundant non-polyadenylated major species of 2.0 kb that is derived by splicing from a less abundant precursor RNA termed minor (m) LAT. The mLAT is transcribed antisense to the ICP0 gene and extends to a polyadenylation signal in the short repeat region. Based on the sequence analysis of HSV-1 strain 17, in LAT spans nucleotides 118 801 to 127 143 and consists of a primary transcript of 8.3 kb. Current evidence supports the view that the 2.0 kb major LAT is an unusually stable intron which is present to at least 40 000 copies per cell. The stability of this RNA is a consequence of inefficient debranching of the intron, due to the presence of a unique non-consensus guanosine branchpoint resulting in persistence of major LAT as a lariat. Further splicing of the 2.0 kb major LAT RNA occurs within neurons to produce an additional stable RNA species of 1.5 kb, which is also considered to accumulate as a stable lariat (Zabolotny et al., 1997). A less complex pattern of transcription is observed during lytic infection of cells in culture. In this setting, synthesis of the 2.0 kb LAT can be detected late in infection but there is a notable absence of the 1.5 kb major LAT species. Furthermore, a fully processed transcript composed of the spliced exons of the primary transcript has not been detected during productive or latent infection, presumably reflecting the rapid degradation of this RNA species. ISH studies of latently infected sensory neurons have shown that major LATs have a diffuse nuclear localization pattern whereas mLATs are localized within discrete nuclear foci that may represent sites of accumulation or synthesis (Arthur et al., 1993). In contrast, during productive infection of cells in culture the 2 kb LAT intron is also found in the cytoplasm and associates with both ribosomal and splicing complexes in infected cells (Ahmed and Fraser, 2001). More recently it has been shown that herpesviruses, including HSV-1, encode micro (mi) RNAs (Pfeffer et al., 2005; Cui et al., 2006). Interestingly, a single miRNA generated from the exon 1 region of LATs has been shown to exert an anti-apoptotic effect by targeting transforming growth factor (TGF) beta and SMAD3 expression (Gupta et al., 2006).

The LAT promoter

Analyses of the HSV-1 DNA sequence upstream from the minor LAT transcription start site identified a TATA box (nt 118 647), a CAAT box (nt 118 647), two CREB binding sites, and SP1 binding sites, making this a candidate LAT promoter element (LAP1). To define the role played by LAP1 in LAT synthesis, a small fragment including the TATA box was deleted (Dobson et al., 1989). Although such a virus could establish latency, no LATs were produced. In addition when the rabbit beta-globin gene was inserted downstream of the TATA box, beta-globin specific RNA, but no major LATs, were transcribed in latently infected neurons. These data are consistent with latent phase transcription initiating from LAP1 to produce the large mLAT species, which is subsequently processed to generate the stable major LAT species. Considerable effort has gone into studying the activity of LAP1 in transient assays. These studies have revealed that this promoter has a high basal activity in a variety of non-neuronal cell types and shows enhanced activity in cells of neuronal origin, an observation consistent with the identification of neuron specific transcription factors which bind to upstream regions of the promoter. Since LATs are expressed to high levels in only a small proportion of latently infected neurons it is likely that their expression is tightly regulated during the various stages of latency. In support of this view, transient promoter assays in PC12 cells have identified cis-acting sequences which are required for activation by NGF and sodium butyrate, which mediate their affects via the Ras and Raf signalling pathways (Frazier et al., 1996). The in vivo significance of these observations is unclear, although it has been suggested that the upregulation of LATs via expression of neurotrophins could function to block reactivation.

LAP1 contains two cAMP response elements (CREs), located at −38 bp (CRE1) and −77 bp (CRE2) relative to the LAT transcription start site, which appear to play an important role in virus reactivation. The CRE1 element has been shown to facilitate both epinephrine-induced reactivation in rabbits and reactivation in mice induced by hyperthermic stress or explantation of ganglia (Bloom et al., 1996, 1997; Marquart et al., 2001). Although these studies suggested that cAMP mediated up-regulation of LATs may be associated with reactivation, it is of interest to note that an in vitro neuronal latency model has linked expression of inducible cAMP repressors (ICERs) with downregulation of LATs and subsequent virus reactivation (Colgin et al., 2001). Located adjacent to the LAP1 transcription start site is a binding site for ICP4 which functions to repress expression of LATs during lytic cycle replication. The mechanism by which the LAT promoter remains active during latency and escapes the otherwise global repression which is so efficiently imposed on the latent genome has been subject to much investigation. The observation that LAP1 deleted viruses are able to express 2 kb major LAT during lytic infection in culture but not during latency in vivo implied the existence of a second promoter between LAP1 and the start of the major LAT intron (Nicosia et al., 1993). A 600 bp fragment within this region was subsequently shown to exhibit promoter activity and was designated LAP2. This second latency associated promoter drives low level reporter gene expression when inserted at an ectopic site in the virus genome. However, despite its designation it would appear that LAP2 functions principally during lytic infection and appears to make a minimal contribution to LATs expression during latency (Chen et al., 1995).

LAP1 is insufficient to mediate long-term latent phase expression, because insertion of reporter genes downstream of LAP1 results in only transient latent phase gene expression. Such studies indicate that although LAP1 contains elements necessary for neuronal expression, additional regulatory sequences are necessary for long-term promoter activity. A long-term expression element (LTE) has been shown to reside downstream of the LAP transcription start site and corresponds to a 1.5 kb fragment which contains an enhancer element and LAP2. Despite extensive efforts it has proven difficult to genetically dissect the downstream LTE sequence. This raises the possibility that the LTE cooperates with LAP1 to direct latent phase transcription (Lachmann and Efstathiou, 1997; Berthomme et al., 2001).

Major LAT ORFs

Major LAT contains two prominent ORFs with the potential to encode proteins of 30 and 15 kd (Fig. 33.1). The lack of conservation of these ORFs between HSV-1 and HSV-2 and the lack of any detectable in vivo latency phenotypes of mutants in which these ORFs are disrupted has suggested that they are unlikely to be of functional significance. Nonetheless, cell lines expressing the 30 kd ORF2 can support the replication of virus mutants defective for IE gene expression and therefore that the ORF can overcome the repression characteristic of quiescent HSV (Thomas et al., 2002). This raises the possibility that this ORF could play a role in reactivation. The significance of these observations remains unclear since there is currently no evidence for the expression of this ORF during infection or following the induction of reactivation from latency; further research in this area is clearly warranted.

Establishment of latency

Specific features of the neuron must be crucial for the interruption of the normal gene expression program during the establishment of latency. The point of arrest in neurons is not known at present, but a number of possibilities, not necessarily mutually exclusive, have been proposed. A description that includes all of the current data and hypotheses in a consistent manner cannot be presented, thus the concepts that are currently favored will be considered separately.

A block to HSV IE transcription

The hypothesis that viral IE transcription fails in neurons has its origins in the observation from tissue culture studies that synthesis of IE proteins is essential for virus replication. HSV mutants with mutations that prevent IE gene expression are not cytotoxic but instead are retained in a quiescent state; therefore, artificial measures to block IE protein production lead to a latency-like interaction in fibroblasts. In mouse ganglia, neurons express either viral antigens or LAT but rarely both during the first few days after infection, suggesting that early events determine the outcome of infection and that latency is incompatible with productive replication (Margolis et al., 1992). Virus mutants that are unable to replicate in neurons still can establish latency. In particular, long term latent promoter activity was observed after inoculation of mice with high doses of HSV-1 mutants that lack the three major transcription activators VP16, ICP0 and ICP4 (Marshall et al., 2000). These mutants enter neurons directly without replicating at a peripheral site. This approach represents the closest available to direct infection of the target cell. The results show that latency can be established in the absence of IE proteins; thus, a natural block to IE transcription in neurons is compatible with the latent state. Ideas on the mechanism by which gene expression in neurons may fail at this stage focus on the requirement for the formation of a TAATGARAT-binding multiprotein complex between VP16, Oct-1 and HCF to initiate IE transcription.

The infecting virion must travel long distances to reach the ganglion, and the structure of the subviral particle that is ultimately delivered to the neuronal nucleus is not known. One idea is that VP16, a tegument protein, fails to be transported to the ganglion with the viral genome, due either to physical loss during retrograde transport or to different uncoating mechanisms in the neuron (Kristie and Roizman, 1988). Alternatively, correct phosphorylation of VP16, especially at serine residue 375 within the Oct-1/HCF recognition domain, is required for its transcriptional activity, and this modification may be affected in neurons (O’Reilly et al., 1997). Absence of functional VP16, even if Oct-1 and HCF are present, would be expected to reduce IE transcription and might, by analogy with observations in cell cultures, predispose the genome to latency.

Oct-1 is a ubiquitous cellular protein initially defined by its ability to bind to the ‘octamer’ element ATGCAAAT. The protein participates in a variety of important cellular processes including transcription, and is utilized by many viruses for gene expression or replication. Oct-1 contains a ‘POU’ domain, which contains the DNA-binding elements and the sites for interaction with many different proteins including VP16. Sensory neurons contain Oct-1 in a form that is functional in vitro; thus it is unlikely that absence of this factor underlies a failure of IE transcription (Hagmann et al., 1995). It is possible, however, that neuron-specific members of the POU-containing family interfere with the binding of Oct-1 to viral target sequences. Many POU-containing proteins bind to TAATGARAT elements in IE promoters but do not interact with VP16, and it would be expected that such proteins could compete with Oct-1 and thereby block gene activation (Latchman, 1999). Rodent Oct-1 varies from the human protein specifically at a few residues that are important for binding of human Oct-1 to VP16 and HCF (Cleary et al., 1993). Thus, VP16 forms the multiprotein complex less efficiently with murine Oct-1, raising concerns about the relevance of the mouse models of latency. Possibly, latency is relatively favored over lytic replication in mice compared with humans. Murine Oct-1 must function to some extent in vivo, however, because HSV-1 VP16 mutants are severely attenuated for replication in mice; if Oct-1 were inactive, the absence of VP16 function would probably be inconsequential.

HCF is a large cellular protein of 2035 amino acids that undergoes internal proteolytic cleavage but nonetheless can participate in activation of transcription with only a heterodimer of the critical N- and C- terminal fragments. The N-terminal portion contains six repeats with homology to the Drosophila protein Kelch, that are predicted to form a propeller-like structure which binds VP16 (Wilson et al., 1993). One major function of HCF appears to be stabilization of the Oct-1/VP16/HCF complex, but more recent studies suggest that HCF itself contains activating regions that may contribute to stimulation of gene expression (Lociano and Wilson, 2002). In proliferating cells HCF is associated with chromatin and is important for cell division, since a cell line harboring a temperature sensitive mutation in HCF arrests predominantly at G0/G1 upon shift to the non-permissive temperature. In sensory neurons in vivo, HCF appears to be cytoplasmic, possibly reflecting the non-dividing state of the cells (Kristie et al., 1999). This localization, if maintained after infection, would prevent activation of IE transcription through the VP16-mediated pathway. Cellular proteins have been identified that, like VP16, contain the short motif D/E HXY which interacts with the Kelch domain of HCF. One of these, named LZIP or Luman, is cytoplasmic in tissue culture cells and, when over-expressed, redistributes HCF from the nucleus to the endoplasmic reticulum (ER) (Freiman and Herr, 1997; Lu and Misra, 2000). Transfected tissue culture cells expressing Luman are impaired for productive HSV-1 replication, presumably because HCF is sequestered at the ER. Another HCF-binding protein, Zhangfei, is selectively expressed in human neurons and also blocks HSV-1 replication when ectopically expressed in tissue culture cells, possibly by counteracting VP16 (Akhova et al., 2005). Clearly, if Luman, Zhangfei or other HCF-binding proteins are present in neurons, activation of IE transcription may be impaired, due to relocation of HCF to the ER, to competition for VP16-binding sites, or to interaction with VP16.

Role of LAT in the establishment of latency

Expression of LAT is not essential for any phase of latency, but there is considerable evidence that it plays a modulatory role. When LAT+ and LAT− viruses are compared virus production at the periphery and in the ganglion is generally equivalent, although early studies suggested that LAT− mutants produce greater quantities of lytic transcripts and proteins in neurons (Garber et al., 1997). In general, however, LAT− mutants reactivate inefficiently and much experimentation has centered on whether this reflects a defect in reactivation per se or is a consequence of reduced ability to establish latency. Analysis of ganglionic viral DNA contents by direct PCR yields equivocal results, with some investigators reporting a deficit of around three-fold and others detecting no significant difference. Errors in these estimations are inherently large, thus relevant differences might not score as statistically significant. The application of CXA revealed that corneal infection with LAT− mutants results in approximately threefold fewer latently infected neurons in trigeminal ganglia, although the HSV-1 genome content distribution within cells was indistinguishable from that of mice infected with a LAT+ virus (Thompson and Sawtell, 1997). These results suggest that LAT affects the number of neurons that ultimately harbor the latent genome rather than copy number within individual cells, and this conclusion is supported by investigation of the effect of LAT on neuronal survival (Perng et al., 2000). Infected rabbit ganglia exhibited greater neuronal apoptosis after infection with a LAT− virus than with a LAT+ counterpart. This effect was maximal at 7 days post-infection, and, surprisingly, few apoptotic neurons were detected at 3 days post-infection, when virus replication was at its peak. It is proposed that LAT has an anti-apoptotic activity that could result in a greater number of neurons surviving in animals infected with the LAT+ virus, thereby increasing establishment of latency. In mice, the basic observation that LAT improves neuronal survival also holds, although there is currently debate concerning whether death is through apoptosis or an alternative route (Thompson and Sawtell, 2001; Ahmed et al., 2002). In tissue culture cells, expression of LAT from transfected plasmids or viruses inhibits apoptosis induced by toxic agents or by virus infection itself, supporting the idea of an anti-apoptotic role (Inman et al., 2001). Furthermore, recent data showing that a miRNA encoded by the HSV-1 LAT gene regulates apoptosis induction by modulating TGF-beta signalling adds considerable support to the view that an important biological function of LATs is to prevent neuronal apoptosis during latency establishment and/or reactivation (Gupta et al., 2006).

LAT has been proposed to block IE gene expression, possibly by antisense inhibition of ICP0 synthesis, an hypothesis that could explain the greater toxicity of LAT-mutants for neurons. Cultured neuroblastoma cells transformed stably to express the 2 kb LAT exhibited reduced permissiveness to HSV-1 infection and a reduction in the levels of all IE-specific mRNAs, suggesting an inhibitory effect of LAT on IE RNA production through a trans-acting mechanism (Mador et al., 1998). However, no reduction in ICP0-specific transcript or protein levels was found in human 293T cells engineered to express 2 kb LAT (Burton et al., 2003).

Alternative models for establishment of latency

In studies with cultured neurons ICP0 was not detected in the nucleus, even though ICP0-specific RNA was expressed (Chen et al., 2000). The reasons for the failure to detect the protein are not clear, although post-transcriptional mechanisms are implicated. The absence of ICP0 might predispose the virus to latency.

All models for the establishment of latency are complicated by the fact that neurons are not inherently resistant to HSV infection because a proportion is able to support productive replication during the first few days after inoculation of animals. There is some evidence that specific neuronal subtypes may differ in susceptibility, but an absolute distinction between permissive and non-permissive cells has not been made to date. Most of the viral DNA produced during the acute phase is eliminated by a rapidly evolving immune response; however, there remains the possibility that some of the latent genomes are derived from replicated molecules rather than transport from peripheral sites. Studies with TK-mutants argue against this hypothesis for snout and corneal inoculation of mice, but in a flank inoculation model evidence was obtained for retention of replicated DNA in neurons that directly innervate the site of infection (Simmons et al., 1992).

An all-encompassing model does not exist to describe the molecular basis for the establishment of latency. If the idea of an early decision between lytic infection and latency, with a primary block at the level of IE gene expression, is accepted, then there would be no apoptotic stimulus (in the form of de novo synthesized viral proteins) to the neuron. This is difficult to reconcile with the hypothesis that LAT antagonizes a response to the presence of viral proteins, which presupposes that the gene expression program proceeds past the IE stage. Possibly, there is heterogeneity in the responses of individual infected neurons, such that some escape an IE block but are arrested at a later stage by LAT. Understanding the cause of neuronal death in infected ganglia is critical to a resolution of these issues. The effect of LAT on the establishment of latency is anatomical site-specific, since LAT− mutants apparently show no difference from LAT+ HSV-1 when latency in dorsal root ganglia is examined after inoculation of the footpad (Sawtell and Thompson, 1992a).

Maintenance of latency

The stability of the latent state, together with the failure to detect viral gene expression apart from that of LAT, supports the concept that the majority of the genome is in a silent state that can be reversed only by specific triggers. Studies in the mouse using sensitive RT-PCR, however, demonstrated that transcripts from the ICP4 and TK regions of the genome could be detected in ganglia during latency (Kramer et al., 1998). This observation is supported by experiments in which sections from many mouse ganglia were analyzed by ISH (Feldman et al., 2002). Approximately one neuron per 10 sections was found to be positive for transcripts representing the lytic genes ICP4, TK and glycoprotein C. In addition, antigen positive neurons were detected at approximately the same frequency, and these cells were surrounded by an immune infiltrate. The most reasonable explanation for the results is that a few neurons support viral gene expression in the mouse, a view that is supported by the finding that interferon gamma and CD8+ T cells are present in murine ganglia at latent times, suggesting that active immune surveillance may operate to maintain latency (Cantin et al., 1995; Khanna et al., 2003). Further discussion of these results is given elsewhere in this volume. Therefore, although the majority of latent genomes are retained in an untranscribed state, the possibility exists that some neurons express HSV-specific proteins and are prevented from producing virus by host immune responses.


Since viral gene products characteristic of the lytic cycle cannot, in general, be detected in latently infected neurons, cellular mechanisms must be important for reactivation. The crucial cellular events are not understood at the molecular level and are still vaguely described as applying ‘stress’ to the neuron. Furthermore, the models for reactivation may rely on very different cellular stimuli and hence the mechanisms involved may vary between both animals and systems. For instance, explantation is probably a more severe stress than in vivo treatments. A further serious complication arises from the fact that reactivation is an inefficient process with only a small proportion of the neurons that harbor viral genomes responding by production of virus. This means that the genomes detected at a gross level during latency may not represent those able to reactivate, with the latter possibly forming a small subset of the total. In addition, in a comparison between HSV-1 strains that differ in their abilities to respond to hyperthermia in vivo, the efficiency of reactivation correlated with the genome copy number distribution but not the number of neurons harboring latent virus (Sawtell, 1998). Therefore, the neurons containing large amounts of HSV DNA may be more susceptible to reactivation stimuli in vivo.

Models for reactivation depend critically on understanding the mechanism of establishment of latency. Thus, if the view is taken that a block in IE transcription leads to establishment, the route to reactivation can be subdivided into two basic concepts, depending on the consequences of the IE block. If, as in fibroblast models, failure of IE gene expression results in conversion of the genome into a quiescent state that is disrupted by ICP0 but is unresponsive to changes in cell physiology such as activation of signal transduction pathways, it follows that reactivation must be provoked either by the action of cellular proteins that mimic the activity of ICP0 or by induction of ICP0 synthesis. An alternative, more popular, view is that viral promoters are not repressed thoroughly, as in fibroblasts, but are inactive and potentially responsive to cellular signals provided by reactivation stimuli. The two models overlap if the genome is generally repressed but the ICP0 promoter specifically escapes repression. In this case, reactivation stimuli would initially be targeted to the ICP0 promoter, with the subsequent reversal of genome repression by the ICP0 protein. A role for LAT in reactivation is suggested by a number of experimental observations, although the interpretation of the data again depends on the events leading to establishment.

The role of ICP0 in reactivation

ICP0 was first characterized as a transcription activator that is not sequence-specific, but recent studies have shown that its primary mode of action is as an ubiquitin E3 ligase that mediates the targeted proteolysis of cellular proteins, particularly those of the nuclear structures known as ND10 (Everett, 2000; Van Sant et al., 2001; Boutell et al., 2002). Indeed, ICP0 rapidly and effectively mediates the disruption of all ND10 in the cell, with accompanying degradation of many of the component proteins. Since transcriptionally active input HSV genomes initially associate with ND10, it is thought that ICP0 creates an environment that is conducive to transcription, probably by directing the destruction of cellular repressors. Histone deacetylases (HDACs) promote the formation of inactive chromatin, thus it is interesting that ICP0 interacts with HDACs 4, 5 and 7 (Lomonte et al., 2004). ICP0 also dissociates HDAC 1 and 2 from CoREST/REST, a protein complex that represses transcription, thereby possibly relieving repression (Gu et al., 2005). These data suggest an important role for ICP0 in antagonizing histone-mediated gene silencing. The dramatic reversal of the quiescent state by ICP0 in cell culture suggests that this protein may be important for reactivation of latent HSV. Early in vivo studies showed that ICP0-deficient mutants were impaired for latency, as measured by reactivation efficiency after explantation, but it was not possible to distinguish between a true effect on reactivation and inefficient establishment due to the known reduction in replication at the periphery and in the ganglion. Immunosuppression of mice enables ICP0 null mutants to establish latency as efficiently as wild-type virus as judged by latent genome copy number, and ICP0 null mutants exhibit reduced reactivation efficiency in the explant model even when viral DNA loads in the ganglia are equivalent (Halford and Schaffer, 2001). ICP0 is therefore important for explant reactivation, but the exact stage at which it functions is unclear. Explantation might specifically induce the synthesis of ICP0, but an alternative interpretation is that ICP0 merely improves the replication, and hence detection, of HSV-1 once the reactivation stimulus has acted. The former hypothesis predicts that the promoter, or other important sequences controlling ICP0, contains elements that respond to reactivation stimuli.

The ICP0 promoter as a possible target for reactivation signals

The ICP0 promoter has many motifs, in addition to the TAATGARATs, that bind transcription factors, and these sites might be targets for reactivation stimuli (Fig. 33.2). Nucleotides –79 to –97 bind Olf-1, a neuron-specific factor that activates transcription (Devireddy and Jones, 2000), and sequences between –74 and –89 are recognized by two proteins, NF-Y and one of unknown identity (named F1), in the human neuroblastoma line IMR-32 (O’Rourke and O’Hare, 1993). Gene array analysis demonstrated that explantation of ganglia induces the synthesis of Bcl-3 (as well as other gene products) in neurons (Tsavachidou et al., 2001). This is interesting because Bcl-3 associates with a dimer of the p50 subunit of NF-κB, and the ICP0 promoter contains NF-κB consensus binding sites at –51 and –273. Phosphorylation influences the binding of Bcl-3 to p50, thus explantation may activate kinases that promote the interaction of these proteins. Bcl-3 also interacts with Tip60, a histone acetylase, and therefore may mediate its effects by recruiting this protein and modifying chromatin structure at the ICP0 promoter. In ganglia, HCF is found in the cytoplasm of neurons but is transported to the nucleus within 20 minutes of explantation (Kristie et al., 1999). If the HSV genome, or strategic regions such as the ICP0 promoter, is available for transcription then HCF, by virtue of its intrinsic activation domain, could trigger the viral gene expression program. This hypothesis requires that a mechanism exists for localizing HCF to the promoter, and binding to Oct-1 is the obvious candidate. However, HCF interacts with the ETS family member GABP, which binds to motifs of consensus CGGAAR (Vogel and Kristie, 2000). There are GABP recognition sites in the ICP0 promoter, and these might direct activation of transcription through a GABP/HCF complex. Another HCF-binding protein, Luman, is cytoplasmic in tissue culture cells but is, in essence, a basic leucine zipper transcription factor of the ATF/CREB family that can bind to CREs and activate transcription in an HCF-dependent manner (Lu and Misra, 2000). In tissue culture cells, Luman is released from the ER by the action of the site 1 protease, an enzyme that catalyzes the regulated intramembrane proteolysis (RIP) of membrane-bound transcription factors, releasing them for transport to the nucleus and activation of transcription (Raggo et al., 2002). Various stress stimuli trigger RIP, thus reactivation signals may result in the release of Luman from the ER of neurons, thereby relocating a complex of this protein plus HCF to the nucleus. The proposed site of action is a CRE at position –67 in the ICP0 promoter.

Fig. 33.2. Binding sites for transcription factors in the ICP0 promoter.

Fig. 33.2

Binding sites for transcription factors in the ICP0 promoter. The region to nucleotide −400 is shown, with the −100 to −30 sequences expanded. The major TAATGARAT element (TG), and two TAATGARAT homologies that have not been demonstrated (more...)

Hypotheses on the significance of transcription factor binding to the ICP0 promoter must include the possibility that such binding may be relevant to replication in neurons at the early stages of infection rather than to reactivation. In addition, all of the above ideas must take account of studies on an HSV-1 mutant deleted for nucleotides –70 to –420 in the ICP0 promoter, thus lacking most of the factor-binding sequences mentioned above (Davido and Leib, 1996). This mutant established latency and displayed normal reactivation efficiency in the explantation system even though replication in cell culture was impaired, suggesting that the region between –70 and –420 in the ICP0 promoter, which includes most of the important known elements controlling expression in cell culture systems, does not contain critical target sequences for explant reactivation of latent virus. Notably, the NF-κB binding site at –51 and the CRE at –67 lie outwith the dispensable region.

Cellular reactivation signals

Among the many changes that occur in neurons following explantation of ganglia, the cyclin-dependent kinases cdk2, cdk4 and cdk7 exhibit alterations in abundance and location (Davido et al., 2002). Increases in the level of cdk2 were observed, and this enzyme was found predominantly in the nucleus. In the case of cdk4, the protein was found mainly in the cytoplasm immediately after plating of explanted ganglia, but became nuclear during culture. A dramatic drop in cdk7 levels occurred within the first day of explant. HSV-1-specific antigens were found exclusively in those neurons containing nuclear cdk2 and cdk4, suggesting that changes in the kinases might be required for reactivation. Roscovitine, an inhibitor of cdk2 that blocks HSV-1 replication in tissue culture, also prevented virus reactivation. Furthermore, no HSV-1 antigen reactivity could be detected in the presence of roscovitine, suggesting that the inhibitor blocks reactivation at an early stage rather than during spread of virus in the explanted ganglion. In tissue culture systems, roscovitine affects many aspects of HSV-1 replication, but it is noteworthy that the compound blocks the function of ICP0 due to alteration of post-translational modification. Therefore, cdk2 and cdk4 may be important for reactivation due to their roles in ensuring the activity of ICP0. In cultured primary rat neurons, withdrawal of NGF results in the rapid resumption of virus replication, to an extent mimicking one of the effects of explantation, in which the in vivo supply of NGF is disrupted (Wilcox and Johnson, 1988). Inhibition of deacetylases also reactivates latent virus in cultured neurons, suggesting a requirement for modification of chromatin structure (Arthur et al., 2001). Treatment of cells with agents that activate signal transduction pathways through cAMP-mediated mechanisms is effective, and recent studies have indicated an involvement of inducible cAMP early repressors (ICER) in this process (Colgin et al., 2001). ICER can heterodimerize with CREB/ATF transcription family members that mediate the transcriptional changes induced by cAMP, but since ICER lacks an activation domain the complexes act as repressors when bound to CREs. Expression of ICER itself is activated by cAMP and, intriguingly, by heat stress of neurons. Crucially, reactivation of latent HSV-1 was induced by infection of neuronal cultures with an adenovirus recombinant expressing ICER. In concert with reactivation, the levels of LAT decreased, leading to the suggestion that the known CREs in the LAT promoter mediate repression by ICER. This observation is difficult to reconcile with suggestions of a positive role for LAT in reactivation, as discussed below. However, in a model that uses cultured cells from dissociated ganglia of latently infected mice, transient heat shock or addition of dexamethasone induced reactivation but elevation of cAMP levels did not (Halford et al., 1996). The nature of the reactivating stimuli therefore differs in the various cell culture systems currently available.

The role of LAT in reactivation

Early work ascertained that, in most cases, LAT− virus mutants reactivate less efficiently than their LAT+ counterparts. This observation was made in mice, for both explant and in vivo reactivation, in rabbits, and in guinea pigs infected with HSV-2. The conclusion that LAT has a role in reactivation is therefore widely accepted. This assumption is complicated by the findings, discussed above, that LAT− mutants establish latency less efficiently in some systems; clearly if fewer neurons harbor HSV genomes, a lower reactivation potential would be expected. Studies in mice, analyzing neuronal DNA contents by CXA, concluded that the impaired reactivation of LAT− mutants in trigeminal ganglia can be entirely accounted for by reduced establishment of latency (Thompson and Sawtell, 1997). Tellingly, it was possible to increase the efficiency of establishment by LAT− mutants to that of wild-type HSV-1 if hyperthermic treatment was applied during the first three days after infection. An equivalent rise in in vivo reactivation frequency to wild-type levels accompanied the increased establishment, strongly suggesting that the primary role of LAT in the mouse trigeminal ganglion is at the level of establishment of latency. In the rabbit eye model, LAT− mutants exhibit reduced efficiency of both spontaneous and induced reactivation. Analysis of latent DNA levels is difficult in this model, however, and although most studies conclude that LAT− and LAT+ mutants establish latency with equivalent efficiencies, variation in the data could obscure a three-fold difference. The question of whether LAT affects establishment or reactivation, or both, in the rabbit remains open. Intriguingly, replacement of the LAT region of HSV-2 with the equivalent region from HSV-1 revealed a role of LAT in anatomical-site specificity of reactivation (Yoshikawa et al., 1996). The recombinant acquired HSV-1-like characteristics, displaying an increased response to iontophoresis of epinephrine in the rabbit but a reduced reactivation frequency in the guinea pig. Only the first 1.5 kb of the mLAT transcript, representing the LAP2 region and part of the stable LAT, is required for efficient spontaneous reactivation in the rabbit (Perng et al., 1996). Re-introduction of sequences encoding this region, plus 1.8 kbp of the LAT promoter, between the UL37 and UL38 coding sequences of a LAT− mutant restored the defect and resulted in a virus exhibiting normal reactivation phenotype. Comparison between strains suggested that none of the ORFs that can be detected in the 1.5 kbp fragment is functionally important for reactivation (Drolet et al., 1998). In addition, inhibition of apoptosis by HSV-1 LAT in tissue culture cells also maps to the 1.5 kbp region (Inman et al., 2001). Thus, in the rabbit, the 1.5 kbp region is thought to mediate increased spontaneous reactivation by virtue of its anti-apoptotic activity. This view is strengthened by the finding that the reduced reactivation efficiency of LAT-viruses can be reversed by insertion of sequences that encode a baculovirus anti-apoptotic protein (Jin et al., 2005). Therefore, the anti-apoptotic function of the LAT region may be important for prolonging survival of the reactivating cells and increasing the production of infectious virus. Deletion of a subfragment of the 1.5 kbp region, consisting of a part of LAP2, dramatically reduced the efficiency of epinephrine-induced reactivation in the rabbit (Bloom et al., 1996). This deleted region lies within the mLAT region and does not affect the accumulation of major LAT; thus, presumably either the expression of a transcript or a cis effect accounts for the activity of the LAP2-derived element and probably the entire 1.5 kbp region.

All of the ideas described above on the mechanism of reactivation assume that latency is essentially “static,” with a switch required to reverse the silencing of the genome. The alternative “dynamic” model, in which continual low level production of virus occurs with lesions only occurring sporadically, does not readily fit with known ideas of viral gene expression but may be consistent with the detection of CD8+ T cells in ganglia (Khanna et al., 2003). The use of PCR has demonstrated that aymptomatic shedding of HSV-1 and HSV-2 occurs with surprisingly high frequency in humans (Wald et al., 1997), suggesting that the dynamic model, an interaction which could be envisaged as a slow persistent infection, deserves consideration.

Concluding remarks

Latency is clearly very complex at the molecular level, and the difficulties inherent in the model systems ensure that it will not be unraveled easily. The non-uniformity of latency, in terms of viral genome copy number, LAT expression and nature of reactivation stimulus, may be of fundamental benefit to the virus. If latency was uniform, a single stimulus might induce reactivation in the entire latent reservoir and result in clearance of the virus from the host. Perhaps the different virus/cell interactions respond to different host signals, explaining why it has been so difficult to arrive at a simple model for the molecular basis of latency.


We thank Valerie Preston for constructive comments on the manuscript, and Robin Lachmann for help with Figure 33.1.


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