As with all Herpesviruses, Herpes simplex virus (HSV) has both a lytic replication phase and a latency‐reactivation cycle. During lytic replication, there is an ordered cascade of viral gene expression that leads to the synthesis of infectious viral progeny. In contrast, latency is characterized by the lack of significant lytic gene expression and the absence of infectious virus. Reactivation from latency is characterized by the re‐entry of the virus into the lytic replication cycle and the production of recurrent disease. This unit describes the establishment of the mouse sensory neuron model of HSV‐1 latency‐reactivation as a useful in vivo system for the analysis of mechanisms involved in latency and reactivation. Assays including the determination of viral yields, immunohistochemical/immunofluorescent detection of viral antigens, and mRNA quantitation are used in experiments designed to investigate the network of cellular and viral proteins regulating HSV‐1 lytic infection, latency, and reactivation. © 2014 by John Wiley & Sons, Inc.

To globally profile circRNAs, we employ RNA-Sequencing paired with chimeric junction analysis for alpha-, beta-, and gamma-herpesvirus infection. We find circRNAs are, as a population, resistant to host shutoff. We validate this observation using ectopic expression assays of human and murine herpesvirus endoribonucleases. During lytic infection, four circRNAs are commonly induced across all subfamilies of human herpesviruses, suggesting a shared mechanism of regulation. We test one such mechanism, namely how interferon-stimulation influences circRNA expression. 67 circRNAs are upregulated by either interferon-β or -γ treatment, with half of these also upregulated during lytic infection. Using gain and loss of function studies we find an interferon-stimulated circRNA, circRELL1, inhibits lytic Herpes Simplex Virus-1 infection. We previously reported circRELL1 inhibits lytic Kaposi sarcoma-associated herpesvirus infection, suggesting a pan-herpesvirus antiviral activity. We propose a two-pronged model in which interferon-stimulated genes may encode both mRNA and circRNA with antiviral activity. This is critical in cases of host shutoff, such as alpha- and gamma-herpesvirus infection, where the mRNA products are degraded but circRNAs escape.

Herpesviruses require the host transcriptional machinery, inducing significant changes in gene expression to prioritize viral transcripts. We examined alpha- and gamma-herpesvirus alterations to a type of alternative splicing, namely circular RNA (circRNA) synthesis. We developed “Circrnas in Host And viRuses anaLysis pIpEline” (CHARLIE) to facilitate viral profiling. This method identified thousands of back-splicing variants, including circRNA common to lytic and latent phases of infection. Ours is the first report of Herpes Simplex Virus-1 circRNAs, including species derived from ICP0 and the latency-associated transcript. We characterized back-splicing cis - and trans -elements, and found viral circRNAs resistant to spliceosome perturbation and lacking canonical splice donor-acceptors. Subsequent loss-of-function studies of host RNA ligases (RTCB, RLIG1) revealed instances of decreased viral back splicing. Using eCLIP and 4sU-Sequencing, we determined that the KSHV RNA-binding protein, ORF57, enhanced synthesis for a subset of viral and host circRNAs. Our work explores unique splicing mechanisms driven by lytic infection, and identifies a class of transcripts with the potential to function in replication, persistence, or tumorigenesis.

Herpes simplex virus is responsible for a substantial worldwide disease burden. An initial infection leads to the establishment of a lifelong persistent infection in sensory neurons.

Epigenetic modulation of herpes simplex virus (HSV) immediate early (IE) genes is a critical parameter governing lytic infection, latency, and viral reactivation. Multiple factors, including epigenetic complexes associated with the cellular transcriptional coactivator HCF-1, modulate the state of HSV-1 chromatin. Although some aspects of HSV-1 chromatin biology have been elucidated, many epigenetic factors controlling HSV-1 chromatin accessibility and transcriptional regulation remain unknown. To identify novel epigenetic regulators of the initial stage of HSV-1 infection, an epigenetic chemical probe library was screened for molecules that impacted viral IE gene expression. This screen identified several epigenetic inhibitors that modulated IE expression. Notably, UNC0379, an inhibitor of the histone H4K20 mono-methyltransferase SETD8, potently suppressed HSV-1 IE gene transcription during lytic infection. UNC0379 treatment also repressed HSV-1 reactivation in a mouse ganglia explant model, and topical application suppressed primary ocular infection in vivo . Importantly, SETD8 inhibition resulted in enhanced heterochromatin suppression and reduced viral genome accessibility, as evident by increased repressive H3K9me3 marks and decreased ATAC-seq signal across the HSV-1 genome. Reduced IE transcription was concomitant with disruption of H4K20me1 deposition and impaired recruitment of HCF-1 and RNA polymerase II (RNAPII). These findings are consistent with the established roles of SETD8 in promoting chromatin accessibility through H4K20me1 deposition and in regulating transcriptional elongation, suggesting that SETD8 may facilitate multiple steps in HSV-1 IE gene expression. Overall, these results identified SETD8 as an essential epigenetic modulator of HSV-1 chromatin accessibility and represent a promising novel antiviral therapeutic target. IMPORTANCE: Herpes simplex virus is a highly prevalent human pathogen, with persistence of the viral genome in sensory neurons serving as a reservoir for recurrent disease and viral shedding. Epigenetic pharmaceuticals have proven to be valuable tools in elucidating the chromatin landscape of the viral genome and its impact on viral gene expression. With the use of a chemical library screen, the histone methyltransferase SETD8 was identified as a key factor required for promoting accessibility of the herpesvirus genome to transcriptional regulators. Importantly, pharmacological inhibition of SETD8 suppressed herpes simplex virus lytic infection, reduced viral reactivation in sensory neurons, and when applied topically, inhibited primary ocular infection of mice. Collectively, these findings establish SETD8 as a critical regulator of viral gene expression during lytic infection and the initiation of reactivation from latency. These results highlight SETD8 as a potential novel target for antiviral therapy.