Interferon induced circRNAs escape herpesvirus host shutoff and suppress lytic infection.

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-ß 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.

Interferon induced circRNAs escape herpesvirus host shutoff and suppress lytic infection.

A first line of defense during infection is expression of interferon (IFN)-stimulated gene products which suppress viral lytic infection. To combat this, herpesviruses express endoribonucleases to deplete host RNAs. Here we demonstrate that IFN-induced circular RNAs (circRNAs) can escape viral-mediated degradation. We performed comparative circRNA expression profiling for representative alpha- (Herpes simplex virus-1, HSV-1), beta- (human cytomegalovirus, HCMV), and gamma-herpesviruses (Kaposi sarcoma herpesvirus, KSHV; murine gamma-herpesvirus 68, MHV68). Strikingly, we found that circRNAs are, as a population, resistant to host shutoff. This observation was confirmed by ectopic expression assays of human and murine herpesvirus endoribonucleases. During primary lytic infection, ten circRNAs were commonly regulated across all subfamilies of human herpesviruses, suggesting a common mechanism of regulation. We tested one such mechanism, namely how interferon-stimulation influences circRNA expression. 67 circRNAs were upregulated by either IFN-ß or -γ treatment, with half of these also upregulated during lytic infection. Using gain and loss of function studies we found an interferon-stimulated circRNA, circRELL1, inhibited lytic HSV-1 infection. We have previously reported circRELL1 inhibits lytic KSHV 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.

Deletion of the Transcriptional Coactivator HCF-1 In Vivo Impairs the Removal of Repressive Heterochromatin from Latent HSV Genomes and Suppresses the Initiation of Viral Reactivation.

Transcription of herpes simplex virus 1 (HSV-1) immediate early (IE) genes is controlled at multiple levels by the cellular transcriptional coactivator, HCF-1. HCF-1 is complexed with epigenetic factors that prevent silencing of the viral genome upon infection, transcription factors that drive initiation of IE gene expression, and transcription elongation factors required to circumvent RNAPII pausing at IE genes and promote productive IE mRNA synthesis. Significantly, the coactivator is also implicated in the control of viral reactivation from latency in sensory neurons based on studies that demonstrate that HCF-1-associated epigenetic and transcriptional elongation complexes are critical to initiate IE expression and viral reactivation. Here, an HCF-1 conditional knockout mouse model (HCF-1cKO) was derived to probe the role and significance of HCF-1 in the regulation of HSV-1 latency/reactivation in vivo. Upon deletion of HCF-1 in sensory neurons, there is a striking reduction in the number of latently infected neurons that initiate viral reactivation. Importantly, this correlated with a defect in the removal of repressive chromatin associated with latent viral genomes. These data demonstrate that HCF-1 is a critical regulatory factor that governs the initiation of HSV reactivation, in part, by promoting the transition of latent viral genomes from a repressed heterochromatic state. IMPORTANCE 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. Periodic reactivation can result in recurrent oral and genital lesions to more significant ocular disease. Despite the significance of this pathogen, many of the regulatory factors and molecular mechanisms that govern the viral latency-reactivation cycles have yet to be elucidated. Initiation of both lytic infection and reactivation are dependent on the expression of the viral immediate early genes. In vivo deletion of a central component of the IE regulatory paradigm, the cellular transcriptional coactivator HCF-1, reduces the epigenetic transition of latent viral genomes, thus suppressing HSV reactivation. These observations define HCF-1 as a critical regulator that controls the initiation of HSV reactivation from latency in vivo and contribute to understanding of the molecular mechanisms that govern viral reactivation.

A TRUSTED targeted mass spectrometry assay for pan-herpesvirus protein detection.

The presence and abundance of viral proteins within host cells are part of the essential signatures of the cellular stages of viral infections. However, methods that can comprehensively detect and quantify these proteins are still limited, particularly for viruses with large protein coding capacity. Here, we design and experimentally validate a mass spectrometry-based Targeted herpesviRUS proTEin Detection (TRUSTED) assay for monitoring human viruses representing the three Herpesviridae subfamilies-herpes simplex virus type 1, human cytomegalovirus (HCMV), and Kaposi sarcoma-associated herpesvirus. We demonstrate assay applicability for (1) capturing the temporal cascades of viral replication, (2) detecting proteins throughout a range of virus concentrations and in in vivo models of infection, (3) assessing the effects of clinical therapeutic agents and sirtuin-modulating compounds, (4) studies using different laboratory and clinical viral strains, and (5) discovering a role for carbamoyl phosphate synthetase 1 in supporting HCMV replication.

Genome-wide Screens Identify Lineage- and Tumor-Specific Genes Modulating MHC-I- and MHC-II-Restricted Immunosurveillance of Human Lymphomas.

Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.

Inhibition of the Super Elongation Complex Suppresses Herpes Simplex Virus Immediate Early Gene Expression, Lytic Infection, and Reactivation from Latency.

Induction of herpes simplex virus (HSV) immediate early (IE) gene transcription promotes the initiation of lytic infection and reactivation from latency in sensory neurons. IE genes are transcribed by the cellular RNA polymerase II (RNAPII) and regulated by multiple transcription factors and coactivators. The HCF-1 cellular coactivator plays a central role in driving IE expression at multiple stages through interactions with transcription factors, chromatin modulation complexes, and transcription elongation components, including the active super elongation complex/P-TEFb (SEC-P-TEFb). Here, we demonstrate that the SEC occupies the promoters of HSV IE genes during the initiation of lytic infection and during reactivation from latency. Specific inhibitors of the SEC suppress viral IE expression and block the spread of HSV infection. Significantly, these inhibitors also block the initiation of viral reactivation from latency in sensory ganglia. The potent suppression of IE gene expression by SEC inhibitors indicates that transcriptional elongation represents a determining rate-limiting stage in HSV IE gene transcription and that the SEC plays a critical role in driving productive elongation during both phases of the viral life cycle. Most importantly, this supports the model that signal-mediated induction of SEC-P-TEFb levels can promote reactivation of a population of poised latent genomes.IMPORTANCE HSV infections can cause pathologies ranging from recurrent lesions to significant ocular disease. Initiation of lytic infection and reactivation from latency in sensory neurons are dependent on the induced expression of the viral immediate early genes. Transcription of these genes is controlled at multiple levels, including modulation of the chromatin state of the viral genome and appropriate recruitment of transcription factors and coactivators. Following initiation of transcription, IE genes are subject to a key regulatory stage in which transcriptional elongation rates are controlled by the activity of the super elongation complex. Inhibition of the SEC blocks both lytic infection and reactivation from latency in sensory neurons. In addition to providing insights into the mechanisms controlling viral infection and reactivation, inhibitors of critical components such as the SEC may represent novel antivirals.

Non-classical Immunity Controls Microbiota Impact on Skin Immunity and Tissue Repair.

Mammalian barrier surfaces are constitutively colonized by numerous microorganisms. We explored how the microbiota was sensed by the immune system and the defining properties of such responses. Here, we show that a skin commensal can induce T cell responses in a manner that is restricted to non-classical MHC class I molecules. These responses are uncoupled from inflammation and highly distinct from pathogen-induced cells. Commensal-specific T cells express a defined gene signature that is characterized by expression of effector genes together with immunoregulatory and tissue-repair signatures. As such, non-classical MHCI-restricted commensal-specific immune responses not only promoted protection to pathogens, but also accelerated skin wound closure. Thus, the microbiota can induce a highly physiological and pleiotropic form of adaptive immunity that couples antimicrobial function with tissue repair. Our work also reveals that non-classical MHC class I molecules, an evolutionarily ancient arm of the immune system, can promote homeostatic immunity to the microbiota.

Inhibitors of the Histone Methyltransferases EZH2/1 Induce a Potent Antiviral State and Suppress Infection by Diverse Viral Pathogens.

Epigenetic regulation is based on a network of complexes that modulate the chromatin character and structure of the genome to impact gene expression, cell fate, and development. Thus, epigenetic modulators represent novel therapeutic targets used to treat a range of diseases, including malignancies. Infectious pathogens such as herpesviruses are also regulated by cellular epigenetic machinery, and epigenetic therapeutics represent a novel approach used to control infection, persistence, and the resulting recurrent disease. The histone H3K27 methyltransferases EZH2 and EZH1 (EZH2/1) are epigenetic repressors that suppress gene transcription via propagation of repressive H3K27me3-enriched chromatin domains. However, while EZH2/1 are implicated in the repression of herpesviral gene expression, inhibitors of these enzymes suppressed primary herpes simplex virus (HSV) infection in vitro and in vivo Furthermore, these compounds blocked lytic viral replication following induction of HSV reactivation in latently infected sensory ganglia. Suppression correlated with the induction of multiple inflammatory, stress, and antipathogen pathways, as well as enhanced recruitment of immune cells to in vivo infection sites. Importantly, EZH2/1 inhibitors induced a cellular antiviral state that also suppressed infection with DNA (human cytomegalovirus, adenovirus) and RNA (Zika virus) viruses. Thus, EZH2/1 inhibitors have considerable potential as general antivirals through the activation of cellular antiviral and immune responses.IMPORTANCE A significant proportion of the world's population is infected with herpes simplex virus. Primary infection and subsequent recurrent reactivation can result in diseases ranging from mild lesions to severe ocular or neurological damage. Herpesviruses are subject to epigenetic regulation that modulates viral gene expression, lytic replication, and latency-reactivation cycles. Thus, epigenetic pharmaceuticals have the potential to alter the course of infection and disease. Here, while the histone methyltransferases EZH2/1 are implicated in the suppression of herpesviruses, inhibitors of these repressors unexpectedly suppress viral infection in vitro and in vivo by induction of key components of cellular innate defense pathways. These inhibitors suppress infection by multiple viral pathogens, indicating their potential as broad-spectrum antivirals.

Transcriptional Elongation of HSV Immediate Early Genes by the Super Elongation Complex Drives Lytic Infection and Reactivation from Latency.

The cellular transcriptional coactivator HCF-1 is required for initiation of herpes simplex virus (HSV) lytic infection and for reactivation from latency in sensory neurons. HCF-1 stabilizes the viral Immediate Early (IE) gene enhancer complex and mediates chromatin transitions to promote IE transcription initiation. In infected cells, HCF-1 was also found to be associated with a network of transcription elongation components including the super elongation complex (SEC). IE genes exhibit characteristics of genes controlled by transcriptional elongation, and the SEC-P-TEFb complex is specifically required to drive the levels of productive IE mRNAs. Significantly, compounds that enhance the levels of SEC-P-TEFb also potently stimulated HSV reactivation from latency both in a sensory ganglia model system and in vivo. Thus, transcriptional elongation of HSV IE genes is a key limiting parameter governing both the initiation of HSV infection and reactivation of latent genomes.

Neuronal Stress Pathway Mediating a Histone Methyl/Phospho Switch Is Required for Herpes Simplex Virus Reactivation.

Herpes simplex virus (HSV) reactivation from latent neuronal infection requires stimulation of lytic gene expression from promoters associated with repressive heterochromatin. Various neuronal stresses trigger reactivation, but how these stimuli activate silenced promoters remains unknown. We show that a neuronal pathway involving activation of c-Jun N-terminal kinase (JNK), common to many stress responses, is essential for initial HSV gene expression during reactivation. This JNK activation in neurons is mediated by dual leucine zipper kinase (DLK) and JNK-interacting protein 3 (JIP3), which direct JNK toward stress responses instead of other cellular functions. Surprisingly, JNK-mediated viral gene induction occurs independently of histone demethylases that remove repressive lysine modifications. Rather, JNK signaling results in a histone methyl/phospho switch on HSV lytic promoters, a mechanism permitting gene expression in the presence of repressive lysine methylation. JNK is present on viral promoters during reactivation, thereby linking a neuronal-specific stress pathway and HSV reactivation from latency.

Quantitative Analysis of HSV Gene Expression during Lytic Infection.

Herpes Simplex Virus (HSV) is a human pathogen that establishes latency and undergoes periodic reactivation, resulting in chronic recurrent lytic infection. HSV lytic infection is characterized by an organized cascade of three gene classes; however, successful transcription and expression of the first, the immediate early class, is critical to the overall success of viral infection. This initial event of lytic infection is also highly dependent on host cell factors. This unit uses RNA interference and small molecule inhibitors to examine the role of host and viral proteins in HSV lytic infection. Methods detailing isolation of viral and host RNA and genomic DNA followed by quantitative real-time PCR allow characterization of impacts on viral transcription and replication, respectively. Western blots can be used to confirm quantitative PCR results. This combination of protocols represents a starting point for researchers interested in virus-host interactions during HSV lytic infection.

Analysis of HSV Viral Reactivation in Explants of Sensory Neurons.

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.

Epigenetic repression of herpes simplex virus infection by the nucleosome remodeler CHD3.

UNLABELLED: Upon infection, the genome of herpes simplex virus is rapidly incorporated into nucleosomes displaying histone modifications characteristic of heterochromatic structures. The initiation of infection requires complex viral-cellular interactions that ultimately circumvent this repression by utilizing host cell enzymes to remove repressive histone marks and install those that promote viral gene expression. The reversion of repression and activation of viral gene expression is mediated by the cellular coactivator HCF-1 in association with histone demethylases and methyltransferases. However, the mechanisms and the components that are involved in the initial repression remain unclear. In this study, the chromatin remodeler chromodomain helicase DNA binding (CHD3) protein is identified as an important component of the initial repression of the herpesvirus genome. CHD3 localizes to early viral foci and suppresses viral gene expression. Depletion of CHD3 results in enhanced viral immediate early gene expression and an increase in the number of transcriptionally active viral genomes in the cell. Importantly, CHD3 can recognize the repressive histone marks that have been detected in the chromatin associated with the viral genome and this remodeler is important for ultimately reducing the levels of accessible viral genomes. A model is presented in which CHD3 represses viral infection in opposition to the actions of the HCF-1 coactivator complex. This dynamic, at least in part, determines the initiation of viral infection. IMPORTANCE: Chromatin modulation of herpesvirus infection is a dynamic process involving regulatory components that mediate suppression and those that promote viral gene expression and the progression of infection. The mechanisms by which the host cell employs the assembly and modulation of chromatin as an antiviral defense strategy against an invading herpesvirus remain unclear. This study defines a critical cellular component that mediates the initial repression of infecting HSV genomes and contributes to understanding the dynamics of this complex interplay between host cell and viral pathogen.

Persistent human herpesvirus-6 infection in patients with an inherited form of the virus.

Human herpesvirus-6 (HHV-6)A and 6B are ubiquitous betaherpesviruses viruses with lymphotropic and neurotropic potential. As reported earlier, these viruses establish latency by integration into the telomeres of host chromosomes. Chromosomally integrated HHV-6 (CIHHV-6) can be transmitted vertically from parent to child. Some CIHHV-6 patients are suffering from neurological symptoms, while others remain asymptomatic. Four patients with CIHHV-6 and CNS dysfunction were treated with valganciclovir or foscarnet. HHV-6 replication was detected by reverse transcriptase polymerase chain reaction amplification of a late envelope glycoprotein. In this study we also compared the inherited and persistent HHV-6 viruses by DNA sequencing. The prevalence of CIHHV-6 in this cohort of adult patients from the USA suffering from a wide range of neurological symptoms including long-term fatigue were found significantly greater than the reported 0.8% in the general population. Long-term antiviral therapy inhibited HHV-6 replication as documented by loss of viral mRNA production. Sequence comparison of the mRNA and the inherited viral genome revealed that the transcript is produced by an exogenous virus. In conclusion, the data presented here document that some individuals with CIHHV-6 are infected persistently with exogenous HHV-6 strains that lead to a wide range of neurological symptoms; the proposed name for this condition is inherited herpesvirus 6 syndrome or IHS.

Mapping the telomere integrated genome of human herpesvirus 6A and 6B.

Human herpesvirus 6B (HHV-6B) is the causative agent of roseola infantum. HHV-6A and 6B can reactivate in immunosuppressed individuals and are linked with severe inflammatory response, organ rejection and central nervous system diseases. About 0.85% of the US and UK population carries an integrated HHV-6 genome in all nucleated cells through germline transmission. We have previously reported that the HHV-6A genome integrated in telomeres of patients suffering from neurological dysfunction and also in telomeres of tissue culture cells. We now report that HHV-6B also integrates in telomeres during latency. Detailed mapping of the integrated viral genomes demonstrates that a single HHV-6 genome integrates and telomere repeats join the left end of the integrated viral genome. When HEK-293 cells carrying integrated HHV-6A were exposed to the histone deacetylase inhibitor Trichostatin A, circularization and/or formation of concatamers were detected and this assay could be used to distinguish between lytic replication and latency.

Targeting the JMJD2 histone demethylases to epigenetically control herpesvirus infection and reactivation from latency.

Chromatin and the chromatin modulation machinery not only provide a regulatory matrix for enabling cellular functions such as DNA replication and transcription but also regulate the infectious cycles of many DNA viruses. Elucidation of the components and mechanisms involved in this regulation is providing targets for the development of new antiviral therapies. Initiation of infection by herpes simplex virus (HSV) requires the activity of several cellular chromatin modification enzymes including the histone demethylases LSD1 and the family of JMJD2 proteins that promote transcriptional activation of the initial set of viral genes. Depletion of the JMJD2 members or inhibition of their activity with a new drug results in repression of expression of viral immediate early genes and abrogation of infection. This inhibitor also represses the reactivation of HSV from the latent state in sensory neurons. Like HSV, the ß-herpesvirus human cytomegalovirus also requires the activity of LSD1 and the JMJD2s to initiate infection, thus demonstrating the potential of this chromatin-based inhibitor to be useful against a variety of different viruses.

The molecular biology of human herpesvirus-6 latency and telomere integration.

The genomes of herpesviruses establish latency as a circular episome. However, Human herpesvirus-6 (HHV-6) has been shown to specifically integrate into the telomeres of chromosomes during latency and vertically transmit through the germ-line. This review will focus on the telomere integration of HHV-6, the potential viral and cellular genes that mediate integration, and the clinical impact on the host.

The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro.

Previous research has suggested that human herpesvirus-6 (HHV-6) may integrate into host cell chromosomes and be vertically transmitted in the germ line, but the evidence--primarily fluorescence in situ hybridization (FISH)--is indirect. We sought, first, to definitively test these two hypotheses. Peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per milliliter of blood. FISH confirmed that HHV-6 DNA colocalized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, and that the integration site was identical among members of the same family. Integration of the HHV-6 genome into TTAGGG telomere repeats was confirmed by additional methods and sequencing of the integration site. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome. We next asked whether HHV-6A infection of naïve cell lines could lead to integration. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6, the virus integrated into telomeres. Reactivation of integrated HHV-6A virus from individuals' PBMCs as well as cell lines was successfully accomplished by compounds known to induce latent herpesvirus replication. Finally, no circular episomal forms were detected even by PCR. Taken together, the data suggest that HHV-6 is unique among human herpesviruses: it specifically and efficiently integrates into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.