Intermittent therapy with helicase-primase inhibitor IM-250 efficiently controls recurrent herpes disease and reduces reactivation of latent HSV.

Herpes is a contagious life-long infection with persistently high incidence and prevalence, causing significant disease worldwide. Current therapies have efficacy against active HSV infections but no impact on the latent viral reservoir in neurons. Thus, despite treatment, disease recurs from latency and the infectious potential remains unaffected within patients. Here, efficacy of the helicase-primase inhibitor (HPI) IM-250 against chronic neuronal HSV infections utilizing two classic herpes in vivo latency/reactivation animal models (intravaginal guinea pig HSV-2 infection model and ocular mouse HSV-1 infection model) is presented. Intermittent therapy of infected animals with 4-7 cycles of IM-250 during latency silences subsequent recurrences analyzed up to 6 months. In contrast to common experience, our studies show that the latent reservoir is indeed accessible to antiviral therapy altering the latent viral reservoir such that reactivation frequency can be reduced significantly by prior IM-250 treatment. We provide evidence that antiviral treatment during HSV latency can reduce future reactivation from the latent reservoir, supporting a conceptual shift in the antiviral field, and reframing what is achievable with respect to therapy of latent neuronal HSV infections.

Alphaherpesvirus Latency and Reactivation with a Focus on Herpes Simplex Virus.

We are at an interesting time in the understanding of alpha herpesvirus latency and reactivation and their implications to human disease. Conceptual advances have come from both animal and neuronal culture models. This review focuses on the concept that the tegument protein and viral transactivator VP16 plays a major role in the transition from latency to the lytic cycle. During acute infection, regulation of VP16 transactivation balances spread in the nervous system, establishment of latent infections and virulence. Reactivation is dependent on this transactivator to drive entry into the lytic cycle. In vivo de novo expression of VP16 protein is mediated by sequences conferring pre-immediate early transcription embedded in the normally leaky late promoter. In vitro, alternate mechanisms regulating VP16 expression in the context of latency have come from the SCG neuron culture model and include the concepts that (i) generalized transcriptional derepression of the viral genome and sequestration of VP16 in the cytoplasm for ~48 hours (Phase I) precedes and is required for VP16-dependent reactivation (Phase II); and (ii) a histone methyl/phospho switch during Phase I is required for Phase II reactivation. The challenge to the field is reconciling these data into a unified model of virus reactivation. The task of compiling this review was uncomfortably humbling, as if cataloging the stars in the universe. While not completely dark, our night sky is missing a multitude of studies which are among the many points of light contributing to our field. This article is a focused review in which we discuss from the vantage point of our expertise, just a handful of concepts that have or are emerging. A lookback at some of the pioneering work that grounds our field is also included.

Resolution of herpes simplex virus reactivation in vivo results in neuronal destruction.

A fundamental question in herpes simplex virus (HSV) pathogenesis is the consequence of viral reactivation to the neuron. Evidence supporting both post-reactivation survival and demise is published. The exceedingly rare nature of this event at the neuronal level in the sensory ganglion has limited direct examination of this important question. In this study, an in-depth in vivo analysis of the resolution of reactivation was undertaken. Latently infected C57BL/6 mice were induced to reactivate in vivo by hyperthermic stress. Infectious virus was detected in a high percentage (60-80%) of the trigeminal ganglia from these mice at 20 hours post-reactivation stimulus, but declined by 48 hours post-stimulus (0-13%). With increasing time post-reactivation stimulus, the percentage of reactivating neurons surrounded by a cellular cuff increased, which correlated with a decrease in detectable infectious virus and number of viral protein positive neurons. Importantly, in addition to intact viral protein positive neurons, fragmented viral protein positive neurons morphologically consistent with apoptotic bodies and containing cleaved caspase-3 were detected. The frequency of this phenotype increased through time post-reactivation. These fragmented neurons were surrounded by Iba1+ cells, consistent with phagocytic removal of dead neurons. Evidence of neuronal destruction post-reactivation prompted re-examination of the previously reported non-cytolytic role of T cells in controlling reactivation. Latently infected mice were treated with anti-CD4/CD8 antibodies prior to induced reactivation. Neither infectious virus titers nor neuronal fragmentation were altered. In contrast, when viral DNA replication was blocked during reactivation, fragmentation was not observed even though viral proteins were expressed. Our data demonstrate that at least a portion of reactivating neurons are destroyed. Although no evidence for direct T cell mediated antigen recognition in this process was apparent, inhibition of viral DNA replication blocked neuronal fragmentation. These unexpected findings raise new questions about the resolution of HSV reactivation in the host nervous system.

HSV Mutant Generation and Dual Detection Methods for Gaining Insight into Latent/Lytic Cycles In Vivo.

Two important components of a useful strategy to examine viral gene function, regulation, and pathogenesis in vivo are (1) a highly efficient protocol to generate viral mutants that limits undesired mutation and retains full replication competency in vivo, and (2) an efficient system to detect and quantify viral promoter activity and gene expression in rare cells in vivo and to gain insight into the surrounding tissue environment. Our strategy and protocols for generating, characterizing, and employing HSV viral promoter/reporter mutants in vivo are provided in this two-part chapter.

Targeted Promoter Replacement Reveals That Herpes Simplex Virus Type-1 and 2 Specific VP16 Promoters Direct Distinct Rates of Entry Into the Lytic Program in Sensory Neurons in vivo.

Infection and life-long residence in the human nervous system is central to herpes simplex virus (HSV) pathogenesis. Access is gained through innervating axonal projections of sensory neurons. This distinct mode of entry separates the viral genome from tegument proteins, including the potent transactivator of viral IE genes, VP16. This, in turn, promotes a balance between lytic and latent infection which underlies the ability of the virus to invade, disseminate, and set up a large reservoir of latent infections. In the mouse ocular model, TG neurons marked as either "latent" or "lytic" at 48 h postinfection indicated that these programs were selected early and were considered distinct and mutually exclusive. More recently, a temporal analysis of viral program selection revealed a default latent-like state that begins at ~18 h postinfection and in individual neurons, precedes entry into the viral lytic cycle. Studies using refined viral mutants demonstrated that transition out of this latent program depended upon the transactivation function of VP16. Pursuit of the apparent incongruity between the established leaky-late kinetics of VP16 expression with a "preimmediate-early" function led to the discovery of an unrecognized regulatory feature of the HSV-1 VP16 promoter near/downstream of its TATA box. Among three potential sites identified was a putative Egr-1/Sp1 site. Here, we report that a refined mutation of this site, while having no impact on replication in cultured cells or cornea, resulted in ~100-fold reduction in lytic infection in TG in vivo. Notably, the HSV-2 VP16 promoter has 13 direct tandem-repeats upstream of its TATA box forming multiple potential overlapping Egr-1/Sp1 sites. Thus, despite different structures, these promoters might share function in directing the preimmediate-early VP16 protein expression. To test this, the HSV-1 VP16 promoter/5'UTR was replaced by the HSV-2 VP16 promoter/5'UTR in the HSV-1 backbone. Compared to the genomically repaired isolate, the HSV-2 VP16 promoter/5'UTR (1) accelerated the transition into the lytic cycle, and enhanced (2) virulence, and (3) entry into the lytic cycle following a reactivation stressor. These gain-of-function phenotypes support the hypothesis that the VP16 promoter regulates the latent/lytic boundary in neurons and that the HSV-1 and HSV-2 promoter/5'UTRs encode distinct thresholds for this transition.

Infectious Herpes Simplex Virus in the Brain Stem Is Correlated with Reactivation in the Trigeminal Ganglia.

Herpes simplex virus (HSV) establishes latency in neurons of the peripheral and central nervous systems (CNS). Evidence is mounting that HSV latency and reactivation in the nervous system has the potential to promote neurodegenerative processes. Understanding how this occurs is an important human health goal. In the mouse model, in vivo viral reactivation in the peripheral nervous system, triggered by hyperthermic stress, has been well characterized with respect to frequency and cell type. However, characterization of in vivo reactivation in the CNS is extremely limited. Further, it remains unclear whether virus reactivated in the peripheral nervous system is transported to the CNS in an infectious form, how often this occurs, and what parameters underlie the efficiency and outcomes of this process. In this study, reactivation was quantified in the trigeminal ganglia (TG) and the brain stem from the same latently infected animal using direct assays of equivalent sensitivity. Reactivation was detected more frequently in the TG than in the brain stem and, in all but one case, the amount of virus recovered was greater in the TG than that detected in the brain stem. Viral protein positive neurons were observed in the TG, but a cellular source for reactivation in the brain stem was not identified, despite serially sectioning and examining the entire tissue (0/6 brain stems). These findings suggest that infectious virus detected in the brain stem is primarily the result of transport of reactivated virus from the TG into the brain stem.IMPORTANCE Latent herpes simplex virus (HSV) DNA has been detected in the central nervous systems (CNS) of humans postmortem, and infection with HSV has been correlated with the development of neurodegenerative diseases. However, whether HSV can directly reactivate in the CNS and/or infectious virus can be transported to the CNS following reactivation in peripheral ganglia has been unclear. In this study, infectious virus was recovered from both the trigeminal ganglia and the brain stem of latently infected mice following a reactivation stimulus, but a higher frequency of reactivation and increased titers of infectious virus were recovered from the trigeminal ganglia. Viral proteins were detected in neurons of the trigeminal ganglia, but a cellular source of infectious virus could not be identified in the brain stem. These results suggest that infectious virus is transported from the ganglia to the CNS following reactivation but do not exclude the potential for direct reactivation in the CNS.

Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis.

Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact contribution is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML NBs, leading to the formation of viral DNA-containing PML NBs (vDCP NBs), and the complete silencing of HSV-1. Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP and Re-ChIP show that vDCP NBs-associated latent/quiescent viral genomes are chromatinized almost exclusively with H3.3 modified on its lysine (K) 9 by trimethylation, consistent with an interaction of the H3.3 chaperones with multiple viral loci and with the transcriptional silencing of HSV-1. Only simultaneous inactivation of both H3.3 chaperone complexes has a significant impact on the deposition of H3.3 on viral genomes, suggesting a compensation mechanism. In contrast, the sole depletion of PML significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. vDCP NBs-associated HSV-1 genomes are not definitively silenced since the destabilization of vDCP NBs by ICP0, which is essential for HSV-1 reactivation in vivo, allows the recovery of a transcriptional lytic program and the replication of viral genomes. Consequently, the present study demonstrates a specific chromatin regulation of vDCP NBs-associated latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML NB/histone H3.3/H3.3 chaperone axis.

Inferred father-to-son transmission of herpes simplex virus results in near-perfect preservation of viral genome identity and in vivo phenotypes.

High throughout sequencing has provided an unprecedented view of the circulating diversity of all classes of human herpesviruses. For herpes simplex virus 1 (HSV-1), we and others have previously published data demonstrating sequence diversity between hosts. However the extent of variation during transmission events, or in one host over years of chronic infection, remain unknown. Here we present an initial example of full characterization of viruses isolated from a father to son transmission event. The likely occasion of transmission occurred 17 years before the strains were isolated, enabling a first view of the degree of virus conservation after decades of recurrences, including transmission and adaptation to a new host. We have characterized the pathogenicity of these strains in a mouse ocular model of infection, and sequenced the full viral genomes. Surprisingly, we find that these two viruses have preserved their phenotype and genotype nearly perfectly during inferred transmission from father to son, and during nearly two decades of episodes of recurrent disease in each human host. Given the close genetic relationship of these two hosts, it remains to be seen whether or not this conservation of sequence will occur during non-familial transmission events.

Analysis of Herpes Simplex Virus Reactivation in Explant Reveals a Method-Dependent Difference in Measured Timing of Reactivation.

Herpes simplex virus (HSV) infection is widespread in the human population. Following orofacial infection, HSV establishes latency in innervating sensory neurons, primarily located in the trigeminal ganglia. A central feature of HSV pathogenesis is the ability to periodically reactivate in those neurons and be transported back to the body surface. Both transmission and disease, such as keratitis, encephalitis, and neurodegeneration, have been linked to reactivation. Despite invaluable insights obtained from model systems, interactions between viral and host functions that regulate reactivation are still incompletely understood. Various assays are used for measuring reactivation in animal models, but there have been limited comparisons between methods and the accuracy of detecting the timing of reactivation and the corresponding amount of infectious virus produced in the ganglia per reactivation event. Here, we directly compare two approaches for measuring reactivation in latently infected explanted ganglia by sampling media from the explanted cultures or by homogenization of the ganglia and compare the results to viral protein expression in the whole ganglia. We show that infectious virus detection by direct homogenization of explanted ganglia correlates with viral protein expression, but detection of infectious virus in medium samples from explanted cultures does not occur until extensive spread of virus is observed in the ganglia. The medium-sampling method is therefore not reflective of the initial timing of reactivation, and the additional variables influencing spread of virus in the ganglia should be considered when interpreting results obtained using this method.IMPORTANCE The development of treatments to prevent and/or treat HSV infection rely upon understanding viral and host factors that influence reactivation. Progress is dependent on experimental methods that accurately measure the frequency and timing of reactivation in latently infected neurons. In this study, two methods for detecting reactivation using the explant model are compared. We show through direct tissue homogenization that reactivation occurs much earlier than can be detected by the indirect method of sampling media from explanted cultures. Thus, the sampling method does not detect the initial timing of reactivation, and results obtained using this method are subject to additional variables with the potential to obscure reactivation outcomes.

De Novo Herpes Simplex Virus VP16 Expression Gates a Dynamic Programmatic Transition and Sets the Latent/Lytic Balance during Acute Infection in Trigeminal Ganglia.

The life long relationship between herpes simplex virus and its host hinges on the ability of the virus to aggressively replicate in epithelial cells at the site of infection and transport into the nervous system through axons innervating the infection site. Interaction between the virus and the sensory neuron represents a pivot point where largely unknown mechanisms lead to a latent or a lytic infection in the neuron. Regulation at this pivot point is critical for balancing two objectives, efficient widespread seeding of the nervous system and host survival. By combining genetic and in vivo in approaches, our studies reveal that the balance between latent and lytic programs is a process occurring early in the trigeminal ganglion. Unexpectedly, activation of the latent program precedes entry into the lytic program by 12 -14hrs. Importantly, at the individual neuronal level, the lytic program begins as a transition out of this acute stage latent program and this escape from the default latent program is regulated by de novo VP16 expression. Our findings support a model in which regulated de novo VP16 expression in the neuron mediates entry into the lytic cycle during the earliest stages of virus infection in vivo. These findings support the hypothesis that the loose association of VP16 with the viral tegument combined with sensory axon length and transport mechanisms serve to limit arrival of virion associated VP16 into neuronal nuclei favoring latency. Further, our findings point to specialized features of the VP16 promoter that control the de novo expression of VP16 in neurons and this regulation is a key component in setting the balance between lytic and latent infections in the nervous system.

Herpes simplex virus and the lexicon of latency and reactivation: a call for defining terms and building an integrated collective framework.

The field of herpes simplex virus (HSV) latency and reactivation has been marked by controversy, which is not unexpected considering the complexities of the biology involved. While controversy is an important tool for digging to the bottom of difficult issues, we propose that unproductive conflict in the field arises in part from poorly defined terminology and the need for a collective framework. The uses of advanced global molecular and next-generation sequencing approaches and an increasing array of in vitro model systems have provided new molecular-level insights into HSV latency and reactivation, with the promise of expanding our concepts of these processes. However, our current framework and language are inadequate to effectively integrate new data streams into the established theories. In this brief perspective, we look back into the past to examine when and how the lexicon of HSV latency and reactivation arose in the literature and its evolution. We propose to open a dialogue among investigators for the purpose of updating and clearly defining terms used to describe these processes and to build a collective integrated framework to move our field forward.

A forward phenotypically driven unbiased genetic analysis of host genes that moderate herpes simplex virus virulence and stromal keratitis in mice.

Both viral and host genetics affect the outcome of herpes simplex virus type 1 (HSV-1) infection in humans and experimental models. Little is known about specific host gene variants and molecular networks that influence herpetic disease progression, severity, and episodic reactivation. To identify such host gene variants we have initiated a forward genetic analysis using the expanded family of BXD strains, all derived from crosses between C57BL/6J and DBA/2J strains of mice. One parent is highly resistant and one highly susceptible to HSV-1. Both strains have also been fully sequenced, greatly facilitating the search for genetic modifiers that contribute to differences in HSV-1 infection. We monitored diverse disease phenotypes following infection with HSV-1 strain 17syn+ including percent mortality (herpes simplex encephalitis, HSE), body weight loss, severity of herpetic stromal keratitis (HSK), spleen weight, serum neutralizing antibody titers, and viral titers in tear films in BXD strains. A significant quantitative trait locus (QTL) on chromosome (Chr) 16 was found to associate with both percent mortality and HSK severity. Importantly, this QTL maps close to a human QTL and the gene proposed to be associated with the frequency of recurrent herpetic labialis (cold sores). This suggests that a single host locus may influence these seemingly diverse HSV-1 pathogenic phenotypes by as yet unknown mechanisms. Additional suggestive QTLs for percent mortality were identified--one on Chr X that is epistatically associated with that on Chr 16. As would be anticipated the Chr 16 QTL also modulated weight loss, reaching significance in females. A second significant QTL for maximum weight loss in male and female mice was mapped to Chr 12. To our knowledge this is the first report of a host genetic locus that modulates the severity of both herpetic disease in the nervous system and herpetic stromal keratitis.

Herpes simplex virus mutant generation and dual-detection methods for gaining insight into latent/lytic cycles in vivo.

Two important components to a useful strategy to examine viral gene regulation in vivo are (1) a highly efficient protocol to generate viral mutants that limits undesired mutation and retains full replication competency in vivo and (2) an efficient system to detect and quantify viral promoter activity in rare cells in vivo. Our strategy and protocols for generating, characterizing, and employing HSV viral promoter/reporter mutants in vivo are provided in this two-part chapter.

HSV-1 genome subnuclear positioning and associations with host-cell PML-NBs and centromeres regulate LAT locus transcription during latency in neurons.

Major human pathologies are caused by nuclear replicative viruses establishing life-long latent infection in their host. During latency the genomes of these viruses are intimately interacting with the cell nucleus environment. A hallmark of herpes simplex virus type 1 (HSV-1) latency establishment is the shutdown of lytic genes expression and the concomitant induction of the latency associated (LAT) transcripts. Although the setting up and the maintenance of the latent genetic program is most likely dependent on a subtle interplay between viral and nuclear factors, this remains uninvestigated. Combining the use of in situ fluorescent-based approaches and high-resolution microscopic analysis, we show that HSV-1 genomes adopt specific nuclear patterns in sensory neurons of latently infected mice (28 days post-inoculation, d.p.i.). Latent HSV-1 genomes display two major patterns, called "Single" and "Multiple", which associate with centromeres, and with promyelocytic leukemia nuclear bodies (PML-NBs) as viral DNA-containing PML-NBs (DCP-NBs). 3D-image reconstruction of DCP-NBs shows that PML forms a shell around viral genomes and associated Daxx and ATRX, two PML partners within PML-NBs. During latency establishment (6 d.p.i.), infected mouse TGs display, at the level of the whole TG and in individual cells, a substantial increase of PML amount consistent with the interferon-mediated antiviral role of PML. "Single" and "Multiple" patterns are reminiscent of low and high-viral genome copy-containing neurons. We show that LAT expression is significantly favored within the "Multiple" pattern, which underlines a heterogeneity of LAT expression dependent on the viral genome copy number, pattern acquisition, and association with nuclear domains. Infection of PML-knockout mice demonstrates that PML/PML-NBs are involved in virus nuclear pattern acquisition, and negatively regulate the expression of the LAT. This study demonstrates that nuclear domains including PML-NBs and centromeres are functionally involved in the control of HSV-1 latency, and represent a key level of host/virus interaction.

Opposing biological functions of tryptophan catabolizing enzymes during intracellular infection.

Recent studies have underscored physiological and pathophysiological roles for the tryptophan-degrading enzyme indolamine 2,3-dioxygenase (IDO) in immune counterregulation. However, IDO was first recognized as an antimicrobial effector, restricting tryptophan availability to Toxoplasma gondii and other pathogens in vitro. The biological relevance of these findings came under question when infectious phenotypes were not forthcoming in IDO-deficient mice. The recent discovery of an IDO homolog, IDO-2, suggested that the issue deserved reexamination. IDO inhibition during murine toxoplasmosis led to 100% mortality, with increased parasite burdens and no evident effects on the immune response. Similar studies revealed a counterregulatory role for IDO during leishmaniasis (restraining effector immune responses and parasite clearance), and no evident role for IDO in herpes simplex virus type 1 (HSV-1) infection. Thus, IDO plays biologically important roles in the host response to diverse intracellular infections, but the dominant nature of this role--antimicrobial or immunoregulatory--is pathogen-specific.

VP16 serine 375 is a critical determinant of herpes simplex virus exit from latency in vivo.

Development of novel prevention and treatment strategies for herpes simplex virus (HSV) mediated diseases is dependent upon an accurate understanding of the central molecular events underlying the regulation of latency and reactivation. We have recently shown that the transactivation function of the virion protein VP16 is a critical determinant in the exit from latency in vivo. HSV-1 strain SJO2 carries a single serine to alanine substitution at position 375 in VP16 which disrupts its interaction with its essential co-activator Oct-1. Here we report that SJO2 is severely impaired in its ability to exit latency in vivo. This result reinforces our prior observations with VP16 transactivation mutant, in1814, in which VP16 interaction with Oct-1 is also disrupted and solidifies the importance of the VP16-Oct-1 interaction in the early steps in HSV-1 reactivation.

The herpes simplex virus type 1 latency associated transcript locus is required for the maintenance of reactivation competent latent infections.

Herpes simplex virus (HSV) establishes latent infections in sensory neurons from which it can periodically reactivate and cause recurrent disease and transmission to new hosts. Little is known about the virally encoded mechanisms that influence the maintenance of HSV latent infectious and modulate the frequency of virus reactivation from the latent state. Here, we report that the latency associated transcript locus of HSV-1 is required for long-term maintenance of reactivation competent latent infections.

Recurrent vaginal shedding of herpes simplex type 2 virus in the mouse and effects of antiviral therapy.

A mouse model of recurrent herpes simplex type 2 (HSV-2) would improve our understanding of the immunobiology of recurrent disease and provide a useful model for evaluating antiviral treatments. We developed a model to evaluate recurrent vaginal HSV-2 shedding using high-dose acyclovir (ACV) therapy beginning at 3 days post infection (dpi). Treatment with 150mg/kg of ACV for 10 days increased survival to 80% following vaginal challenge with HSV-2 strain 186 and to 100% after challenge with strain MS. We then evaluated recurrent vaginal HSV-2 shedding in surviving mice. Although infectious virus was not detected in vaginal samples after 21dpi, viral DNA was detectable by PCR in 80% of mice (47/59) on at least 1 day, while no animal was positive for virus on every day. ACV therapy administered from day 21 to 31 significantly reduced recurrent virus shedding during this period from 7.3% (8/109 swabs) to 0.8% (1/126 swabs) (p=0.013). Lastly, ACV-rescued HSV-2-infected mice treated with cyclophosphamide at 35 and 38dpi rapidly succumbed, indicating that this model can be used to study immune control of the persistent infection. Thus, this model provides an inexpensive model for evaluating therapeutic strategies and immune control of persistent HSV.

Therapeutic implications of new insights into the critical role of VP16 in initiating the earliest stages of HSV reactivation from latency.

Reactivation of herpes simplex virus (HSV) is a leading cause of fatal encephalitis in the USA and recurrent herpetic keratitis is a major infectious cause of blindness. There is no effective vaccine and no cure for HSV latency. While current antiviral drugs reduce viral replication, none prevent the initiation of reactivation in the nervous system and, thus, chronic inflammatory damage proceeds. The discovery that HSV VP16 is necessary for the exit from latency represents the first potential target for preventing the chronic inflammatory insult associated with HSV reactivation. Blocking VP16 transactivation would reduce the spread of the virus in the population and, importantly, presumably reduce or prevent the pathological long term chronic inflammation in the nervous system.

De novo synthesis of VP16 coordinates the exit from HSV latency in vivo.

The mechanism controlling the exit from herpes simplex virus latency (HSV) is of central importance to recurrent disease and transmission of infection, yet interactions between host and viral functions that govern this process remain unclear. The cascade of HSV gene transcription is initiated by the multifunctional virion protein VP16, which is expressed late in the viral replication cycle. Currently, it is widely accepted that VP16 transactivating function is not involved in the exit from latency. Utilizing the mouse ocular model of HSV pathogenesis together with genetically engineered viral mutants and assays to quantify latency and the exit from latency at the single neuron level, we show that in vivo (i) the VP16 promoter confers distinct regulation critical for viral replication in the trigeminal ganglion (TG) during the acute phase of infection and (ii) the transactivation function of VP16 (VP16TF) is uniquely required for the exit from latency. TG neurons latently infected with the VP16TF mutant in1814 do not express detectable viral proteins following stress, whereas viruses with mutations in the other major viral transcription regulators ICP0 and ICP4 do exit the latent state. Analysis of a VP16 promoter/reporter mutant in the background of in1814 demonstrates that the VP16 promoter is activated in latently infected neurons following stress in the absence of other viral proteins. These findings support the novel hypothesis that de novo expression of VP16 regulates entry into the lytic program in neurons at all phases of the viral life cycle. HSV reactivation from latency conforms to a model in which stochastic derepression of the VP16 promoter and expression of VP16 initiates entry into the lytic cycle.