Herpes simplex virus 1 (HSV-1) undergoes primary acute lytic replication in mucosal epithelial cells and then spreads to establish latent infection in peripheral neurons including sensory and autonomic neurons. Latent HSV-1 reactivates to cause recurrent cold sores but can also cause more severe disease including keratitis and encephalitis. Animal models have been used extensively to study HSV-1 latent infection, while human sensory neuronal systems have been difficult to establish or make scalable. To address neuron-intrinsic mechanisms of HSV-1 latent infection, we developed a protocol to rapidly differentiate human-inducible pluripotent stem cells (hiPSCs) into sensory neurons. The differentiated neurons are excitable and express functional ion channels. We established conditions for latent infection with HSV-1 in these cells that show i) no infectious virus, ii) reduced lytic gene expression, iii) efficient latency-associated transcript expression, and iv) viral heterochromatin. Latent HSV-1 can be reactivated by previously known stimuli including forskolin and PI3Ki. Therefore, this scalable human iPSC-derived sensory neuron system is a promising model to explore mechanisms of HSV-1 latent infection in human neurons.IMPORTANCEHerpes simplex viruses are prevalent human pathogens that cause significant morbidity and mortality in neonates and adults. They undergo acute infection in the mucosae and spread to peripheral neurons including sensory and autonomic neurons, where they establish lifelong latent infection. Reactivation from latency leads to recurrent infection and disease, including central nervous system infection that can be life-threatening. Experimental studies of HSV latency have mostly been conducted in animals, which may differ from the human situation. In this study, we establish a system for differentiation of human-inducible pluripotent stem cells into sensory neurons and for latent infection and reactivation by herpes simplex virus 1. This system will enable studies of the mechanism of HSV latent infection in human sensory neurons and therapeutic approaches to curtail it.

MicroRNA miR-138, which is highly expressed in neurons, represses herpes simplex virus 1 (HSV-1) lytic cycle genes by targeting viral ICP0 messenger RNA, thereby promoting viral latency in mice. We found that overexpressed miR-138 also represses lytic processes independently of ICP0 in murine and human neuronal cells; therefore, we investigated whether miR-138 has targets besides ICP0. Using genome-wide RNA sequencing/photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation followed by short interfering RNA knockdown of candidate targets, we identified the host Oct-1 and Foxc1 messenger mRNAs as miR-138’s targets, whose gene products are transcription factors important for HSV-1 replication in neuronal cells. OCT-1 has a known role in the initiation of HSV transcription. Overexpression of FOXC1, which was not known to affect HSV-1, promoted HSV-1 replication in murine neurons and ganglia. CRISPR–Cas9 knockout of FOXC1 reduced viral replication, lytic gene expression and miR-138 repression in murine neuronal cells. FOXC1 also collaborated with ICP0 to decrease heterochromatin on viral genes and compensated for the defect of an ICP0-null virus. In summary, miR-138 targets ICP0, Oct-1 and Foxc1 to repress HSV-1 lytic cycle genes and promote epigenetic gene silencing, which together enable favourable conditions for latent infection. Genome-wide RNA sequencing/PAR-CLIP and short interfering RNA knockdown are combined to show that neuronal microRNA miR-138 targets ICP0, Oct-1 and Foxc1 to repress HSV-1 lytic cycle genes and promote epigenetic gene silencing, thereby favouring viral latency.

ABSTRACT Herpes simplex virus 1 (HSV-1) establishes latent infection in neurons via a variety of epigenetic mechanisms that silence its genome. The cellular CCCTC-binding factor (CTCF) functions as a mediator of transcriptional control and chromatin organization and has binding sites in the HSV-1 genome. We constructed an HSV-1 deletion mutant that lacked a pair of CTCF-binding sites ( CTRL2 ) within the latency-associated transcript ( LAT ) coding sequences and found that loss of these CTCF-binding sites did not alter lytic replication or levels of establishment of latent infection, but their deletion reduced the ability of the virus to reactivate from latent infection. We also observed increased heterochromatin modifications on viral chromatin over the LAT promoter and intron. We therefore propose that CTCF binding at the CTRL2 sites acts as a chromatin insulator to keep viral chromatin in a form that is poised for reactivation, a state which we call poised latency. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a human pathogen that persists for the lifetime of the host as a result of its ability to establish latent infection within sensory neurons. The mechanism by which HSV-1 transitions from the lytic to latent infection program is largely unknown; however, HSV-1 is able to coopt cellular silencing mechanisms to facilitate the suppression of lytic gene expression. Here, we demonstrate that the cellular CCCTC-binding factor (CTCF)-binding site within the latency associated transcript ( LAT ) region is critical for the maintenance of a specific local chromatin structure. Additionally, loss of CTCF binding has detrimental effects on the ability to reactivate from latent infection. These results argue that CTCF plays a critical role in epigenetic regulation of viral gene expression to establish and/or maintain a form of latent infection that can reactivate efficiently.

Many viruses undergo an acute infection in the host organism and then are cleared by the ensuing host immune response, but other viruses establish a persistent infection involving a latent infection or a chronic infection. Latent infection by the herpesviruses or human immunodeficiency virus involves epigenetic silencing of the DNA genome or proviral genome, respectively. Latent infection was previously thought to be a default pathway resulting from infection of a nonpermissive cell, but recent studies have shown that viral gene products can promote epigenetic silencing and latent infection. This review will summarize the viral gene products that have been shown to promote epigenetic silencing of the genomes and their potential for therapeutics to target these viral gene products and disrupt or lock in latent infection.

A collection of genomic DNA sequences of herpes simplex virus (HSV) strains has been defined and analyzed, and some information is available about genomic stability upon limited passage of viruses in culture. The nature of genomic change upon extensive laboratory passage remains to be determined. In this report we review the history of the HSV-1 KOS laboratory strain and the related KOS1.1 laboratory sub-strain, also called KOS (M), and determine the complete genomic sequence of an early passage stock of the KOS laboratory sub-strain and a laboratory stock of the KOS1.1 sub-strain. The genomes of the two sub-strains are highly similar with only five coding changes, 20 non-coding changes, and about twenty non-ORF sequence changes. The coding changes could potentially explain the KOS1.1 phenotypic properties of increased replication at high temperature and reduced neuroinvasiveness. The study also provides sequence markers to define the provenance of specific laboratory KOS virus stocks.

ABSTRACT Herpesviruses must contend with host cell epigenetic silencing responses acting on their genomes upon entry into the host cell nucleus. In this study, we confirmed that unchromatinized herpes simplex virus 1 (HSV-1) genomes enter primary human foreskin fibroblasts and are rapidly subjected to assembly of nucleosomes and association with repressive heterochromatin modifications such as histone 3 (H3) lysine 9-trimethylation (H3K9me3) and lysine 27-trimethylation (H3K27me3) during the first 1 to 2 h postinfection. Kinetic analysis of the modulation of nucleosomes and heterochromatin modifications over the course of lytic infection demonstrates a progressive removal that coincided with initiation of viral gene expression. We obtained evidence for three phases of heterochromatin removal from an early gene promoter: an initial removal of histones and heterochromatin not dependent on ICP0, a second ICP0-dependent round of removal of H3K9me3 that is independent of viral DNA synthesis, and a third phase of H3K27me3 removal that is dependent on ICP0 and viral DNA synthesis. The presence of ICP0 in transfected cells is also sufficient to promote removal of histones and H3K9me3 modifications of cotransfected genes. Overall, these results show that ICP0 promotes histone removal, a reduction of H3K9me3 modifications, and a later indirect reduction of H3K27me3 modifications following viral early gene expression and DNA synthesis. Therefore, HSV ICP0 promotes the reversal of host epigenetic silencing mechanisms by several mechanisms. IMPORTANCE The human pathogen herpes simplex virus (HSV) has evolved multiple strategies to counteract host-mediated epigenetic silencing during productive infection. However, the mechanisms by which viral and cellular effectors contribute to these processes are not well defined. The results from this study demonstrate that HSV counteracts host epigenetic repression in a dynamic stepwise process to remove histone 3 (H3) and subsequently target specific heterochromatin modifications in two distinct waves. This provides the first evidence of a stepwise reversal of host epigenetic silencing by viral proteins. This work also suggests that targets capable of disrupting the kinetics of epigenetic regulation could serve as potential antiviral therapeutic agents.

ABSTRACT Latent infections by viruses usually involve minimizing viral protein expression so that the host immune system cannot recognize the infected cell through the viral peptides presented on its cell surface. Herpes simplex virus (HSV), for example, is thought to express noncoding RNAs such as latency-associated transcripts (LATs) and microRNAs (miRNAs) as the only abundant viral gene products during latent infection. Here we describe analysis of HSV-1 mutant viruses, providing strong genetic evidence that HSV-infected cell protein 0 (ICP0) is expressed during establishment and/or maintenance of latent infection in murine sensory neurons in vivo. Studies of an ICP0 nonsense mutant virus showed that ICP0 promotes heterochromatin and latent and lytic transcription, arguing that ICP0 is expressed and functional. We propose that ICP0 promotes transcription of LATs during establishment or maintenance of HSV latent infection, much as it promotes lytic gene transcription. This report introduces the new concept that a lytic viral protein can be expressed during latent infection and can serve dual roles to regulate viral chromatin to optimize latent infection in addition to its role in epigenetic regulation during lytic infection. An additional implication of the results is that ICP0 might serve as a target for an antiviral therapeutic acting on lytic and latent infections. IMPORTANCE Latent infection by viruses usually involves minimizing viral protein synthesis so that the host immune system cannot recognize the infected cells and eliminate them. Herpes simplex virus has been thought to express only noncoding RNAs as abundant gene products during latency. In this study, we found genetic evidence that an HSV lytic protein is functional during latent infection, and this protein may provide a new target for antivirals that target both lytic and latent infections.

The herpesviruses are characterized by their ability to undergo a productive infection upon infection of the host organism and then spread to establish a latent infection where they persist in the host. Later, under conditions of stress or other environmental stimuli, they reactivate to undergo a productive infection and spread to new host organisms. Herpes simplex virus (HSV), in particular, undergoes a productive infection in the mucosal epithelium and spreads to sensory neurons where it undergoes a latent infection. Reactivation leads to virus shedding but can also lead to recurrent disease. Much of the herpetic disease occurs following reactivation from latent infection, and all of the available antivirals target lytic infection. Thus, an understanding of the mechanisms of establishment and reactivation of HSV from latent infection is important for devising approaches for controlling HSV establishment, maintenance, and reactivation from latent infection. In epithelial cells, the HSV genome is initially silenced by host epigenetic mechanisms, but the virion protein 16 (VP16) and immediate early (IE) infected cell protein 0 reverse the heterochromatin modifications and promote euchromatin modifications on viral chromatin to promote lytic gene transcription, allowing lytic infection to proceed (1). In sensory neurons, VP16 cannot localize to the nucleus and/or assemble into nuclear complexes to activate IE gene expression, so the lytic cascade of gene expression is not initiated. Instead, a neuron-specific promoter in the viral genome drives the expression of the latency-associated transcript (LAT), which is processed into a stable lariat intron and microRNAs (miRNAs) (2). LAT is reported to enhance neuronal survival in several ways: (i) down-regulating lytic gene expression (3) by LAT sequences acting as a long noncoding RNA and promoting epigenetic silencing of lytic genes (4, 5); (ii) serving as a precursor for viral miRNAs that inhibit lytic gene expression (6); (iii) inhibiting apoptosis (7); and (iv) promoting of survival of infected neurons by nonapoptotic mechanisms (8). Thus, understanding of the mechanisms regulating the expression of LAT is critical for antiviral strategies targeting latent infection. In PNAS, Shu et al. (9) identify Activating Transcription Factor 3 (ATF3) as a host factor that induces expression of LAT in infected cells in culture and plays a role in maintaining latent infection in murine trigeminal ganglion sensory neurons.

ABSTRACT Arabidopsis encodes five double-stranded RNA binding (DRB) proteins. DRB1 and DRB2 are involved in microRNA (miRNA) biogenesis, while DRB4 functions in cytoplasmic posttranscriptional small interfering RNA (siRNA) pathways. DRB3 and DRB5 are not involved in double-stranded RNA (dsRNA) processing but assist in silencing transcripts targeted by DRB2-associated miRNAs. The goal of this study was to determine which, if any, of the DRB proteins might also participate in a nuclear siRNA pathway that leads to geminivirus genome methylation. Here, we demonstrate that DRB3 functions with Dicer-like 3 (DCL3) and Argonaute 4 (AGO4) in methylation-mediated antiviral defense. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses, using an RNA-directed DNA methylation (RdDM) pathway similar to that used to suppress endogenous invasive DNAs such as transposons. Chromatin methylation inhibits virus replication and transcription, and methylation-deficient host plants are hypersusceptible to geminivirus infection. Using a panel of drb mutants, we found that drb3 plants uniquely exhibit a similar hypersensitivity and that viral genome methylation is substantially reduced in drb3 compared to wild-type plants. In addition, like dcl3 and ago4 mutants, drb3 plants fail to recover from infection and cannot accomplish the viral genome hypermethylation that is invariably observed in asymptomatic, recovered tissues. Small RNA analysis, bimolecular fluorescence complementation, and coimmunoprecipitation experiments show that DRB3 acts downstream of siRNA biogenesis and suggest that it associates with DCL3 and AGO4 in distinct subnuclear compartments. These studies reveal that in addition to its previously established role in the miRNA pathway, DRB3 also functions in antiviral RdDM. IMPORTANCE Plants use RNA-directed DNA methylation (RdDM) as an epigenetic defense against geminiviruses. RNA silencing pathways in Arabidopsis include five double-stranded RNA binding proteins (DRBs) related to Drosophila R2D2 and mammalian TRBP and PACT. While DRB proteins have defined roles in miRNA and cytoplasmic siRNA pathways, a role in nuclear RdDM was elusive. Here, we used the geminivirus system to show that DRB3 is involved in methylation-mediated antiviral defense. Beginning with a panel of Arabidopsis drb mutants, we demonstrated that drb3 plants uniquely show enhanced susceptibility to geminiviruses. Further, like dcl3 and ago4 mutants, drb3 plants fail to hypermethylate the viral genome, a requirement for host recovery. We also show that DRB3 physically interacts with the RdDM pathway components DCL3 and AGO4 in the nucleus. This work highlights the utility of geminiviruses as models for de novo RdDM and places DRB3 protein in this fundamental epigenetic pathway.

Herpesviruses are highly prevalent and maintain lifelong latent reservoirs, thus posing challenges to the control of herpetic disease despite the availability of antiviral pharmaceuticals that target viral DNA replication. The initiation of herpes simplex virus infection and reactivation from latency is dependent on a transcriptional coactivator complex that contains two required histone demethylases, LSD1 (lysine-specific demethylase 1) and a member of the JMJD2 family (Jumonji C domain-containing protein 2). Inhibition of either of these enzymes results in heterochromatic suppression of the viral genome and blocks infection and reactivation in vitro. We demonstrate that viral infection can be epigenetically suppressed in three animal models of herpes simplex virus infection and disease. Treating animals with the monoamine oxidase inhibitor tranylcypromine to inhibit LSD1 suppressed viral lytic infection, subclinical shedding, and reactivation from latency in vivo. This phenotypic suppression was correlated with enhanced epigenetic suppression of the viral genome and suggests that, even during latency, the chromatin state of the virus is dynamic. Therefore, epi-pharmaceuticals may represent a promising approach to treat herpetic diseases.

DNA methylation is a fundamental epigenetic modification that regulates gene expression and represses endogenous transposons and invading DNA viruses. As a counter-defense, the geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a betasatellite called DNA β. This study presents evidence that suppression of methylation-mediated TGS by the sole betasatellite-encoded protein, βC1, is crucial to the association of Tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB). We show that TYLCCNB complements Beet curly top virus (BCTV) L2- mutants deficient for methylation inhibition and TGS suppression, and that cytosine methylation levels in BCTV and TYLCCNV genomes, as well as the host genome, are substantially reduced by TYLCCNB or βC1 expression. We also demonstrate that while TYLCCNB or βC1 expression can reverse TGS, TYLCCNV by itself is ineffective. Thus its AC2/AL2 protein, known to have suppression activity in other geminiviruses, is likely a natural mutant in this respect. A yeast two-hybrid screen of candidate proteins, followed by bimolecular fluorescence complementation analysis, revealed that βC1 interacts with S-adenosyl homocysteine hydrolase (SAHH), a methyl cycle enzyme required for TGS. We further demonstrate that βC1 protein inhibits SAHH activity in vitro. That βC1 and other geminivirus proteins target the methyl cycle suggests that limiting its product, S-adenosyl methionine, may be a common viral strategy for methylation interference. We propose that inhibition of methylation and TGS by βC1 stabilizes geminivirus/betasatellite complexes.

It is well-established that plants use cytoplasmic, post-transcriptional gene silencing (PTGS) as a defense against RNA viruses and DNA virus transcripts. More recently, it has become clear that small RNA-directed methylation leading to transcriptional gene silencing (TGS) is also used as a defense against DNA virus chromatin. Here we use the DNA-containing geminiviruses as models to discuss what is currently known about both types of antiviral silencing, and viral suppression of PTGS and TGS as a counterdefense.

ABSTRACT Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2− mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.