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Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin.

Varicella zoster virus (VZV), a human alphaherpesvirus, causes varicella during primary infection. VZV reactivation from neuronal latency may cause herpes zoster, post herpetic neuralgia (PHN) and other neurologic syndromes. To investigate VZV neuropathogenesis, we developed a model using human dorsal root ganglia (DRG) xenografts in immunodeficient (SCID) mice. The SCID DRG model provides an opportunity to examine characteristics of VZV infection that occur in the context of the specialized architecture of DRG, in which nerve cell bodies are ensheathed by satellite glial cells (SGC) which support neuronal homeostasis. We hypothesized that VZV exhibits neuron-subtype specific tropism and that VZV tropism for SGC contributes to VZV-related ganglionopathy. Based on quantitative analyses of viral and cell protein expression in DRG tissue sections, we demonstrated that, whereas DRG neurons had an immature neuronal phenotype prior to implantation, subtype heterogeneity was observed within 20 weeks and SGC retained the capacity to maintain neuronal homeostasis longterm. Profiling VZV protein expression in DRG neurons showed that VZV enters peripherin+ nociceptive and RT97+ mechanoreceptive neurons by both axonal transport and contiguous spread from SGC, but replication in RT97+ neurons is blocked. Restriction occurs even when the SGC surrounding the neuronal cell body were infected and after entry and ORF61 expression, but before IE62 or IE63 protein expression. Notably, although contiguous VZV spread with loss of SGC support would be predicted to affect survival of both nociceptive and mechanoreceptive neurons, RT97+ neurons showed selective loss relative to peripherin+ neurons at later times in DRG infection. Profiling cell factors that were upregulated in VZV-infected DRG indicated that VZV infection induced marked pro-inflammatory responses, as well as proteins of the interferon pathway and neuroprotective responses. These neuropathologic changes observed in sensory ganglia infected with VZV may help to explain the neurologic sequelae often associated with zoster and PHN.

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins.

Varicella zoster virus (VZV), a human alphaherpesvirus, causes varicella during primary infection. VZV reactivation from neuronal latency may cause herpes zoster, post herpetic neuralgia (PHN) and other neurologic syndromes. To investigate VZV neuropathogenesis, we developed a model using human dorsal root ganglia (DRG) xenografts in immunodeficient (SCID) mice. The SCID DRG model provides an opportunity to examine characteristics of VZV infection that occur in the context of the specialized architecture of DRG, in which nerve cell bodies are ensheathed by satellite glial cells (SGC) which support neuronal homeostasis. We hypothesized that VZV exhibits neuron-subtype specific tropism and that VZV tropism for SGC contributes to VZV-related ganglionopathy. Based on quantitative analyses of viral and cell protein expression in DRG tissue sections, we demonstrated that, whereas DRG neurons had an immature neuronal phenotype prior to implantation, subtype heterogeneity was observed within 20 weeks and SGC retained the capacity to maintain neuronal homeostasis longterm. Profiling VZV protein expression in DRG neurons showed that VZV enters peripherin+ nociceptive and RT97+ mechanoreceptive neurons by both axonal transport and contiguous spread from SGC, but replication in RT97+ neurons is blocked. Restriction occurs even when the SGC surrounding the neuronal cell body were infected and after entry and ORF61 expression, but before IE62 or IE63 protein expression. Notably, although contiguous VZV spread with loss of SGC support would be predicted to affect survival of both nociceptive and mechanoreceptive neurons, RT97+ neurons showed selective loss relative to peripherin+ neurons at later times in DRG infection. Profiling cell factors that were upregulated in VZV-infected DRG indicated that VZV infection induced marked pro-inflammatory responses, as well as proteins of the interferon pathway and neuroprotective responses. These neuropathologic changes observed in sensory ganglia infected with VZV may help to explain the neurologic sequelae often associated with zoster and PHN. [ABSTRACT FROM AUTHOR]

As the Coronavirus Disease 2019 (COVID-19) pandemic continues, there is a growing concern regarding the relationship between viral infections and neuropathic pain. Chronic neuropathic pain resulting from virus-induced neural dysfunction has emerged as a significant issue currently faced. However, the molecular mechanisms underlying this phenomenon remain unclear, and clinical treatment outcomes are often suboptimal. Therefore, delving into the relationship between viral infections and neuropathic pain, exploring the pathophysiological characteristics and molecular mechanisms of different viral pain models, can contribute to the discovery of potential therapeutic targets and methods, thereby enhancing pain relief and improving the quality of life for patients. This review focuses on HIV-related neuropathic pain (HNP), postherpetic neuralgia (PHN), and neuropathic pain caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections, examining rodent models and relevant cellular molecular pathways. Through elucidating the connection between viral infections and neuropathic pain, it aims to delineate the current limitations and challenges faced by treatments, thereby providing insights and directions for future clinical practice and research. [ABSTRACT FROM AUTHOR]

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that respond to riboflavin biosynthesis and cytokines through TCR-dependent and -independent pathways, respectively. MAIT cell activation plays an immunoprotective role against several pathogens, however the functional capacity of MAIT cells following direct infection or exposure to infectious agents remains poorly defined. We investigated the impact of Varicella Zoster Virus (VZV) on blood-derived MAIT cells and report virus-mediated impairment of activation, cytokine production, and altered transcription factor expression by VZV infected (antigen+) and VZV exposed (antigen-) MAIT cells in response to TCR-dependent and -independent stimulation. Furthermore, we reveal that suppression of VZV exposed (antigen-) MAIT cells is not mediated by a soluble factor from neighbouring VZV infected (antigen+) MAIT cells. Finally, we demonstrate that VZV impairs the cytolytic potential of MAIT cells in response to riboflavin synthesising bacteria. In summary, we report a virus-mediated immune-evasion strategy that disarms MAIT cell responses. Author summary: Mucosal-associated invariant T (MAIT) cells are a uniquely specialised and substantial innate-T cell population that can rapidly respond to diverse bacterial and fungal pathogens through T cell receptor dependent recognition of riboflavin synthesis derived metabolite antigens. Additionally, MAIT cells can be triggered by local pro-inflammatory cues such as cytokines; therefore extending their functionality to non-riboflavin pathogens such as viral infections. Despite the capacity of MAIT cells to play a protective role against several classes of pathogens, there remains a dearth of studies investigating direct pathogenic suppression of MAIT cell functionality. Here, we investigate a previously uncharacterised interplay between MAIT cells and the causative agent of varicella (chickenpox) and shingles (zoster): Varicella Zoster Virus (VZV). VZV successfully infects and establishes lifelong latency within the host; in part to their ability to effectively manipulate several innate and adaptive axes of the host immune response. In this study, we report that VZV profoundly impairs MAIT cell activation in response to both riboflavin synthesis and cytokine stimulation, therefore resulting in a downstream paralysis of several effector functions such as cytokine production and cytotoxic potential. This work highlights a previously uncharacterised strategy of viral pathogens to effectively target and restrict the MAIT cell effector response. [ABSTRACT FROM AUTHOR]

Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs) that target cellular IRFs and/or other innate-immune and stress signaling regulators and suppress the cellular response to viral infection and replication. For vIRF-1, cellular protein targets include IRFs, p53, p53-activating ATM kinase, BH3-only proteins, and antiviral signaling effectors MAVS and STING; vIRF-1 inhibits each, with demonstrated or likely promotion of HHV-8 de novo infection and productive replication. Here, we identify direct interactions of vIRF-1 with STAT3 and STAT-activating Janus kinase TYK2 (the latter reported previously by us to be inhibited by vIRF-1) and suppression by vIRF-1 of cytokine-induced STAT3 activation. Suppression of active, phosphorylated STAT3 (pSTAT3) by vIRF-1 was evident in transfected cells and vIRF-1 ablation in lytically-reactivated recombinant-HHV-8-infected cells led to increased levels of pSTAT3. Using a panel of vIRF-1 deletion variants, regions of vIRF-1 required for interactions with STAT3 and TYK2 were identified, which enabled correlation of STAT3 signaling inhibition by vIRF-1 with TYK2 binding, independently of STAT3 interaction. A viral mutant expressing vIRF-1 deletion-variant Δ198–222 refractory for TYK2 interaction and pSTAT3 suppression was severely compromised for productive replication. Conversely, expression of phosphatase-resistant, protractedly-active STAT3 led to impaired HHV-8 replication. Cells infected with HHV-8 mutants expressing STAT3-refractory vIRF-1 deletion variants or depleted of STAT3 displayed reduced vIRF-1 expression, while custom-peptide-promoted STAT3 interaction could effect increased vIRF-1 expression and enhanced virus replication. Taken together, our data identify vIRF-1 targeting and inhibition of TYK2 as a mechanism of STAT3-signaling suppression and critical for HHV-8 productive replication, the importance of specific pSTAT3 levels for replication, positive roles of STAT3 and vIRF-1-STAT3 interaction in vIRF-1 expression, and significant contributions to lytic replication of STAT3 targeting by vIRF-1. Author summary: HHV-8 is involved etiologically in Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease, which occur predominantly in the context of HIV co-infection. Effective treatments for these diseases, in which both latent and lytic modes of HHV-8 infection are implicated, are lacking. Understanding the molecular biology of HHV-8 and the virus-cellular interactions critical for virus productive and latent infection is important for the development of novel antiviral and therapeutic strategies. HHV-8 vIRF-1 is of demonstrated importance to both latency and lytic replication, contributing to latently-infected PEL cell viability and being required for efficient virus production. This study identifies direct interactions of vIRF-1 with signaling protein STAT3 and STAT-activating TYK2 kinase and correlates the respective interactions with vIRF-1 expression and suppression of STAT3 activation in infected cells along with promotion of productive replication. These data provide a foundation for the development of novel and specific antiviral agents targeting these newly-identified direct interactions. [ABSTRACT FROM AUTHOR]

Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek's disease is a devastating herpesviral disease of chickens caused by Marek's disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5' ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems. Author summary: In the natural host, the transcriptome and proteome of many herpesviruses are poorly defined. Here, we evaluated the viral transcriptome and proteome in feather follicle epithelial skin cells of chickens infected with Marek's disease virus (MDV), an important poultry pathogen as well as an excellent model for skin-tropic human alphaherpesvirus replication in skin cells. Using fluorescently tagged virus, we significantly enriched the number of infected cells sampled from live chickens, greatly enhancing the detection of viral transcripts and proteins within a host background. We carefully defined the in vivo transcriptome using both short-read RNA-Seq and long-read full-length transcript sequencing, and deep MS/MS-based proteomics from the enriched virus-infected feather follicle samples confirmed the translation of most transcripts and identified novel expressed peptides supportive of an increasingly complex translational and regulatory viral landscape. The demonstrated deep peptide sequencing capability can serve as a template for future in vivo work in herpesviral proteomics. [ABSTRACT FROM AUTHOR]

G-quadruplex (G4) formed by repetitive guanosine-rich sequences plays important roles in diverse cellular processes; however, its roles in viral infection are not fully understood. In this study, we investigated the genome-wide distribution of G4-forming sequences (G4 motifs) in Varicella-Zoster virus (VZV) and found that G4 motifs are enriched in the internal repeat short and the terminal repeat short regions flanking the unique short region and also in some reiteration (R) sequence regions. A high density of G4 motifs in the R2 region was found on the template strand of ORF14, which encodes glycoprotein C (gC), a virulent factor for viral growth in skin. Analyses such as circular dichroism spectroscopy, thermal difference spectra, and native polyacrylamide gel electrophoresis with oligodeoxynucleotides demonstrated that several G4 motifs in ORF14 form stable G4 structures. In transfection assays, gC expression from the G4-disrupted ORF14 gene was increased at the transcriptional level and became more resistant to suppression by G4-ligand treatment. The recombinant virus containing the G4-disrupted ORF14 gene expressed a higher level of gC mRNA, while it showed a slightly reduced growth. This G4-disrupted ORF14 virus produced smaller plaques than the wild-type virus. Our results demonstrate that G4 formation via reiteration sequences suppresses gC expression during VZV infection and regulates viral cell-to-cell spread. Author summary: G-quadruplexes (G4s) are found in herpesvirus genomes. However, whether G4 formation regulates the growth and pathogenesis of Varicella-Zoster virus (VZV), which causes chickenpox in children and shingles in older adults, is unclear. Here, we identified ~150 putative G4-forming sequences (G4 motifs) in the VZV genome and found that these sequences are enriched in the repeat regions in the genome. In particular, the G4 motifs found in the R2 reiteration region are located on the template strand of ORF14 that encodes glycoprotein C (gC). We demonstrate that several G4 motifs in R2 form anti-parallel G4 structures and that the formation of these G4s suppresses gC expression at the transcription level during virus infection. We also show that the G4-mediated attenuation of gC expression promotes viral spread to neighboring cells. Therefore, VZV has evolved to control gC expression through G4 formation by reiteration sequences for regulation of viral cell-to-cell spread. [ABSTRACT FROM AUTHOR]

Primary infection with varicella-zoster virus (VZV) causes varicella and the establishment of lifelong latency in sensory ganglion neurons. In one-third of infected individuals VZV reactivates from latency to cause herpes zoster, often complicated by difficult-to-treat chronic pain. Experimental infection of non-human primates with simian varicella virus (SVV) recapitulates most features of human VZV disease, thereby providing the opportunity to study the pathogenesis of varicella and herpes zoster in vivo. However, compared to VZV, the transcriptome and the full coding potential of SVV remains incompletely understood. Here, we performed direct long-read RNA sequencing to annotate the SVV transcriptome in lytically SVV-infected African green monkey (AGM) and rhesus macaque (RM) kidney epithelial cells. We refined structures of canonical SVV transcripts and uncovered numerous RNA isoforms, splicing events, fusion transcripts and non-coding RNAs, mostly unique to SVV. We verified the expression of canonical and newly identified SVV transcripts in vivo, using lung samples from acutely SVV-infected cynomolgus macaques. Expression of selected transcript isoforms, including those located in the unique left-end of the SVV genome, was confirmed by reverse transcription PCR. Finally, we performed detailed characterization of the SVV homologue of the VZV latency-associated transcript (VLT), located antisense to ORF61. Analogous to VZV VLT, SVV VLT is multiply spliced and numerous isoforms are generated using alternative transcription start sites and extensive splicing. Conversely, low level expression of a single spliced SVV VLT isoform defines in vivo latency. Notably, the genomic location of VLT core exons is highly conserved between SVV and VZV. This work thus highlights the complexity of lytic SVV gene expression and provides new insights into the molecular biology underlying lytic and latent SVV infection. The identification of the SVV VLT homolog further underlines the value of the SVV non-human primate model to develop new strategies for prevention of herpes zoster. Author summary: Varicella-zoster virus (VZV)–a ubiquitous human pathogen–infects most individuals during childhood, leading to chickenpox, after which the virus persists in the host for decades. Later in life, VZV reactivates to cause shingles, frequently associated with difficult-to-treat chronic pain. Our limited understanding of the viral life-cycle hampers the development of more effective treatment options. Simian varicella virus (SVV) is the non-human primate homologue of VZV and causes a natural disease in Old World monkeys with clinical, pathological, and immunological features resembling human VZV infection. However, it is unclear how similar both viruses are at the molecular level. Here, we have revisited the genome-wide transcriptional activity of SVV during lytic infection of kidney epithelial cells derived from two non-human primate species and validated expression of newly identified viral transcripts in lung tissue from SVV-infected animals. Together, this has led to the identification of numerous alternative RNA isoforms, mostly unique to SVV, and some of which may have functional implications for the virus. Notably, we defined the SVV latency-associated transcript, which is highly similar to its VZV counterpart. In conclusion, our study shows the value of understanding the molecular biology of a given animal model and identifies potentially conserved mechanism of latency. [ABSTRACT FROM AUTHOR]

STING is a nodal point for cellular innate immune response to microbial infections, autoimmunity and cancer; it triggers the synthesis of the antiviral proteins, type I interferons. Many DNA viruses, including Herpes Simplex Virus 1 (HSV1), trigger STING signaling causing inhibition of virus replication. Here, we report that HSV1 evades this antiviral immune response by inducing a cellular microRNA, miR-24, which binds to the 3' untranslated region of STING mRNA and inhibits its translation. Expression of the gene encoding miR-24 is induced by the transcription factor AP1 and activated by MAP kinases in HSV1-infected cells. Introduction of exogenous miR-24 or prior activation of MAPKs, causes further enhancement of HSV1 replication in STING-expressing cells. Conversely, transfection of antimiR-24 inhibits virus replication in those cells. HSV1 infection of mice causes neuropathy and death; using two routes of infection, we demonstrated that intracranial injection of antimiR-24 alleviates both morbidity and mortality of the infected mice. Our studies reveal a new immune evasion strategy adopted by HSV1 through the regulation of STING and demonstrates that it can be exploited to enhance STING's antiviral action. Author summary: The type I interferon system is the first line of cellular antiviral innate immune response. Virus infection is recognized by various pattern recognition receptors in the infected cell and it activates the interferon system to inhibit virus replication. However, viruses have evolved various mechanisms to evade the cellular immune response and enhance viral replication. Our study uncovers an immune evasion strategy used by the Herpes Simplex virus to circumvent the cGAS/STING signaling pathway which is the pivotal innate immune response to combat DNA virus replication. miR-24 induction by HSV1 targets STING and hence, dampens Type I Immune response against the virus. The induction of miR-24 is regulated by virus induced MAPK activation, which are also required during early lytic cycles of HSV1 replication and is indispensable for HSV1 reactivation from latency in neurons; depicting a new direct co-relation between MAPK activation and HSV1 replication orchestrated through cellular miR-24. Silencing of miR-24 in mice brain curtails viral replication and disease severity. Overall, these results indicate possible therapeutic use of stable antimiR-24 against HSV1 and other diseases that are alleviated by STING. [ABSTRACT FROM AUTHOR]

Herpes zoster, the result of varicella-zoster virus (VZV) reactivation, is frequently complicated by difficult-to-treat chronic pain states termed postherpetic neuralgia (PHN). While there are no animal models of VZV-induced pain following viral reactivation, subcutaneous VZV inoculation of the rat causes long-term nocifensive behaviors indicative of mechanical and thermal hypersensitivity. Previous studies using UV-inactivated VZV in the rat model suggest viral gene expression is required for the development of pain behaviors. However, it remains unclear if complete infection processes are needed for VZV to induce hypersensitivity in this host. To further assess how gene expression and replication contribute, we developed and characterized three replication-conditional VZV using a protein degron system to achieve drug-dependent stability of essential viral proteins. Each virus was then assessed for induction of hypersensitivity in rats under replication permissive and nonpermissive conditions. VZV with a degron fused to ORF9p, a late structural protein that is required for virion assembly, induced nocifensive behaviors under both replication permissive and nonpermissive conditions, indicating that complete VZV replication is dispensable for the induction of hypersensitivity. This conclusion was confirmed by showing that a genetic deletion recombinant VZV lacking DNA packaging protein ORF54p still induced prolonged hypersensitivities in the rat. In contrast, VZV with a degron fused to the essential IE4 or IE63 proteins, which are involved in early gene regulation of expression, induced nocifensive behaviors only under replication permissive conditions, indicating importance of early gene expression events for induction of hypersensitivity. These data establish that while early viral gene expression is required for the development of nocifensive behaviors in the rat, complete replication is dispensable. We postulate this model reflects events leading to clinical PHN, in which a population of ganglionic neurons become abortively infected with VZV during reactivation and survive, but host signaling becomes altered in order to transmit ongoing pain. Author summary: Acute and chronic pain are common complications of herpes zoster, but the mechanisms underlying the transition to chronic pain states remain poorly understood. Varicella-zoster virus (VZV)-inoculated rats develop persistent behaviors that indicate the development of prolonged hypersensitivity that may model postherpetic neuralgia (PHN) seen in the clinic. To address the requirements of viral gene expression and replication for the induction of pain, we developed mutant VZV and applied them to rat models of PHN. Our results reveal the production of essential VZV regulatory proteins is required to induce nocifensive behaviors, but that full productive virus replication is dispensable. These data suggest a mechanism for pain induction that involves the early expression of VZV regulatory proteins in abortively infected neurons after herpes zoster that may cause aberrant host pain signaling and PHN. [ABSTRACT FROM AUTHOR]

Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae, including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109KSQD112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109AAAA112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae. Author summary: Herpesviruses are ubiquitous infectious agents of medical and economic importance, including varicella-zoster virus (VZV), which causes chicken pox and shingles. A unifying theme of herpesviruses is their mechanism of entry into host cells, membrane fusion, via a core complex of virally expressed envelope glycoproteins gB, gH and gL. Of these, the primary fusogen, gB, is activated by the heterodimer gH-gL through an unknown mechanism and enables the virus envelope to merge with cell membranes to release the DNA containing capsid into the cytoplasm to initiate infection. By using a human antibody that neutralizes VZV we have recently demonstrated that the initiation of membrane fusion is associated with the crown region of gB. Here, we use cryogenic electron microscopy to compare the structure of this human neutralizing antibody, 93k, to a non-neutralizing antibody SG2. Surprisingly, both antibodies bind to the crown of gB with considerable overlap of their footprints on gB with one important exception, SG2 does not bind to a flexible region in the gB N-terminus. Mutations incorporated into this flexible region disrupts gB mediated membrane fusion and significantly impairs VZV propagation, identifying an Achilles heel in viral replication. [ABSTRACT FROM AUTHOR]

Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin pathogenesis based on studies with gB and gH hyperfusogenic VZV mutants. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Two structurally related non-antibiotic macrolides, tacrolimus and pimecrolimus, both significantly increased VZV gB/gH-gL mediated cell fusion. These compounds form a drug-protein complex with FKBP1A, which binds to calcineurin and specifically inhibits calcineurin phosphatase activity. Inhibition of calcineurin phosphatase activity also enhanced both herpes simplex virus-1 fusion complex and syncytin-1 mediated cell fusion, indicating a broad role of calcineurin in modulating this process. To characterize the role of calcineurin phosphatase activity in VZV gB/gH-gL mediated fusion, a series of biochemical, biological and infectivity assays was performed. Pimecrolimus-induced, enhanced cell fusion was significantly reduced by shRNA knockdown of FKBP1A, further supporting the role of calcineurin phosphatase activity in fusion regulation. Importantly, inhibition of calcineurin phosphatase activity during VZV infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with the previously reported phenotypes of gB and gH hyperfusogenic VZV mutants. Seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited were identified as potential downstream factors involved in fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis. Author summary: VZV is a medically important human virus that causes varicella (chicken pox) and reactivates as zoster (shingles). Postherpetic neuralgia (PHN) is a common sequela of zoster, a persistent nerve pain that can last from months to years after zoster. Refractory to antiviral drugs and pain treatment, PHN significantly impacts quality of life. Despite the success of VZV live-attenuated vaccines in the healthy population, immunocompromised individuals are still vulnerable to significant morbidity. The recently approved subunit zoster vaccine is effective but whether it works against varicella is not known. Therefore, a better understanding of the molecular basis of VZV pathogenesis would provide significant advances towards alternative treatments. A hallmark of VZV pathogenesis is syncytia formation observed in infected skin and nerve ganglia. VZV-induced cell fusion is mediated by the viral fusion complex comprised of glycoproteins gB, gH, and gL. Dysregulation of this fusion process leads to impaired VZV infection in skin. Here, we report that inhibition of a cellular phosphatase, calcineurin, significantly increased gB/gH-gL mediated cell fusion, and intensified syncytia formation during VZV replication, but suppressed virus spread. Our study demonstrated that calcineurin phosphatase activity regulates VZV-induced cell fusion, providing a new perspective for potential antiviral strategies that target host factors. [ABSTRACT FROM AUTHOR]

Herpesviruses are known to encode a number of inhibitors of host cell death, including Rip Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein, open reading frame (ORF) 20, that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1-expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies. Author summary: Rip homotypic interaction motifs (RHIMs) are found in host proteins that can signal for programmed cell death and in viral proteins that can prevent it. Complexes stabilized by intermolecular interactions involving RHIMs have a fibrillar amyloid structure. We have identified a novel RHIM within the ORF20 protein expressed by Varicella zoster virus (VZV) that forms amyloid-based complexes with human cellular RHIMs. Whereas other herpesvirus RHIMs inhibit necroptosis, this new VZV RHIM targets the host RHIM-containing protein ZBP1 to inhibit apoptosis during infection. This is the first study to demonstrate the importance of the ZBP1 pathway in VZV infection and to identify the role of a viral RHIM in apoptosis inhibition. It broadens our understanding of host defense pathways and demonstrates how a decoy amyloid strategy is employed by pathogens to circumvent the host response. [ABSTRACT FROM AUTHOR]

Cytomegaloviruses (order Herpesvirales) display remarkable species-specificity as a result of long-term co-evolution with their mammalian hosts. Human cytomegalovirus (HCMV) is exquisitely adapted to our species and displays high genetic diversity. We leveraged information on inter-species divergence of primate-infecting cytomegaloviruses and intra-species diversity of clinical isolates to provide a genome-wide picture of HCMV adaptation across different time-frames. During adaptation to the human host, core viral genes were commonly targeted by positive selection. Functional characterization of adaptive mutations in the primase gene (UL70) indicated that selection favored amino acid replacements that decrease viral replication in human fibroblasts, suggesting evolution towards viral temperance. HCMV intra-species diversity was largely governed by immune system-driven selective pressure, with several adaptive variants located in antigenic domains. A significant excess of positively selected sites was also detected in the signal peptides (SPs) of viral proteins, indicating that, although they are removed from mature proteins, SPs can contribute to viral adaptation. Functional characterization of one of these SPs indicated that adaptive variants modulate the timing of cleavage by the signal peptidase and the dynamics of glycoprotein intracellular trafficking. We thus used evolutionary information to generate experimentally-testable hypotheses on the functional effect of HCMV genetic diversity and we define modulators of viral phenotypes. Author summary: Human cytomegalovirus (HCMV), which represents the most common infectious cause of birth defects, is perfectly adapted to infect humans. We performed a two-tier analysis of HCMV evolution, by describing selective events that occurred during HCMV adaptation to our species and by identifying more recently emerged adaptive variants in clinical isolates. We show that distinct viral genes were targeted by natural selection over different time frames and we generate a catalog of adaptive variants that represent candidate determinants of viral phenotypic variation. As a proof of concept, we show that adaptive changes in the viral primase modulate viral growth in vitro and that selected variants in the UL144 signal peptide affect glycoprotein intracellular trafficking. [ABSTRACT FROM AUTHOR]

Varicella zoster virus (VZV) is a lymphotropic alpha-herpesvirinae subfamily member that produces varicella on primary infection and causes zoster, vascular disease and vision loss upon reactivation from latency. VZV-infected peripheral blood mononuclear cells (PBMCs) disseminate virus to distal organs to produce clinical disease. To assess immune evasion strategies elicited by VZV that may contribute to dissemination of infection, human PBMCs and VZV-specific CD8+ T cells (V-CD8+) were mock- or VZV-infected and analyzed for immunoinhibitory protein PD-1, PD-L1, PD-L2, CTLA-4, LAG-3 and TIM-3 expression using flow cytometry. All VZV-infected PBMCs (monocytes, NK, NKT, B cells, CD4+ and CD8+ T cells) and V-CD8+ showed significant elevations in PD-L1 expression compared to uninfected cells. VZV induced PD-L2 expression in B cells and V-CD8+. Only VZV-infected CD8+ T cells, NKT cells and V-CD8+ upregulated PD-1 expression, the immunoinhibitory receptor for PD-L1/PD-L2. VZV induced CTLA-4 expression only in V-CD8+ and no significant changes in LAG-3 or TIM-3 expression were observed in V-CD8+ or PBMC T cells. To test whether PD-L1, PD-L2 or CTLA-4 regulates V-CD8+ effector function, autologous PBMCs were VZV-infected and co-cultured with V-CD8+ cells in the presence of blocking antibodies against PD-L1, PD-L2 or CTLA-4; ELISAs revealed significant elevations in IFNγ only upon blocking of PD-L1. Together, these results identified additional immune cells that are permissive to VZV infection (monocytes, B cells and NKT cells); along with a novel mechanism for inhibiting CD8+ T cell effector function through induction of PD-L1 expression. [ABSTRACT FROM AUTHOR]

Varicella zoster virus (VZV) is a ubiquitous human alphaherpesvirus, responsible for varicella upon primary infection and herpes zoster following reactivation from latency. To establish lifelong infection, VZV employs strategies to evade and manipulate the immune system to its advantage in disseminating virus. As innate lymphocytes, natural killer (NK) cells are part of the early immune response to infection, and have been implicated in controlling VZV infection in patients. Understanding of how VZV directly interacts with NK cells, however, has not been investigated in detail. In this study, we provide the first evidence that VZV is capable of infecting human NK cells from peripheral blood in vitro. VZV infection of NK cells is productive, supporting the full kinetic cascade of viral gene expression and producing new infectious virus which was transmitted to epithelial cells in culture. We determined by flow cytometry that NK cell infection with VZV was not only preferential for the mature CD56dim NK cell subset, but also drove acquisition of the terminally-differentiated maturity marker CD57. Interpretation of high dimensional flow cytometry data with tSNE analysis revealed that culture of NK cells with VZV also induced a potent loss of expression of the low-affinity IgG Fc receptor CD16 on the cell surface. Notably, VZV infection of NK cells upregulated surface expression of chemokine receptors associated with trafficking to the skin –a crucial site in VZV disease where highly infectious lesions develop. We demonstrate that VZV actively manipulates the NK cell phenotype through productive infection, and propose a potential role for NK cells in VZV pathogenesis. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 3 (STAT3) is a key regulator of numerous physiological functions, including the immune response. As pathogens elicit an acute phase response with concerted activation of STAT3, they are confronted with two evolutionary options: either curtail it or employ it. This has important consequences for the host, since abnormal STAT3 function is associated with cancer development and other diseases. This review provides a comprehensive outline of how human viruses cope with STAT3-mediated inflammation and how this affects the host. Finally, we discuss STAT3 as a potential target for antiviral therapy. [ABSTRACT FROM AUTHOR]

A close relative of poliovirus, enterovirus 71 (EV71) is regarded as an important neurotropic virus of serious public health concern. EV71 causes Hand, Foot and Mouth Disease and has been associated with neurological complications in young children. Our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. Here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during EV71 infection of motor-neuron NSC-34 cells, which are found at the neuromuscular junctions where EV71 is believed to enter the central nervous system. Meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in NSC-34 cells. Among the candidate proteins, we focused our attention on prohibitin (PHB), a protein that is involved in multiple cellular functions and the target of anti-cancer drug Rocaglamide (Roc-A). We demonstrated that cell surface-expressed PHB is involved in EV71 entry into neuronal cells specifically, while membrane-bound mitochondrial PHB associates with the virus replication complex and facilitates viral replication. Furthermore, Roc-A treatment of EV71-infected neuronal cells reduced significantly virus yields. However, the inhibitory effect of Roc-A on PHB in NSC-34 cells was not through blocking the CRAF/MEK/ERK pathway as previously reported. Instead, Roc-A treated NSC-34 cells had lower mitochondria-associated PHB and lower ATP levels that correlated with impaired mitochondria integrity. In vivo, EV71-infected mice treated with Roc-A survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. Together, this study uncovers PHB as the first host factor that is specifically involved in EV71 neuropathogenesis and a potential drug target to limit neurological complications. [ABSTRACT FROM AUTHOR]

The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear lamina in response to herpesviral or inherent cellular stimuli. In essence, Pin1 represents a regulatory effector of lamina disassembly that promotes the nuclear pore-independent egress of herpesviral capsids. [ABSTRACT FROM AUTHOR]

Varicella zoster virus (VZV) latency in sensory and autonomic neurons has remained enigmatic and difficult to study, and experimental reactivation has not yet been achieved. We have previously shown that human embryonic stem cell (hESC)-derived neurons are permissive to a productive and spreading VZV infection. We now demonstrate that hESC-derived neurons can also host a persistent non-productive infection lasting for weeks which can subsequently be reactivated by multiple experimental stimuli. Quiescent infections were established by exposing neurons to low titer cell-free VZV either by using acyclovir or by infection of axons in compartmented microfluidic chambers without acyclovir. VZV DNA and low levels of viral transcription were detectable by qPCR for up to seven weeks. Quiescently-infected human neuronal cultures were induced to undergo renewed viral gene and protein expression by growth factor removal or by inhibition of PI3-Kinase activity. Strikingly, incubation of cultures induced to reactivate at a lower temperature (34°C) resulted in enhanced VZV reactivation, resulting in spreading, productive infections. Comparison of VZV genome transcription in quiescently-infected to productively-infected neurons using RNASeq revealed preferential transcription from specific genome regions, especially the duplicated regions. These experiments establish a powerful new system for modeling the VZV latent state, and reveal a potential role for temperature in VZV reactivation and disease. [ABSTRACT FROM AUTHOR]

Varicella zoster virus (VZV) causes chickenpox in humans and, subsequently, establishes latency in the sensory ganglia from where it reactivates to cause herpes zoster. Infection of rhesus macaques with simian varicella virus (SVV) recapitulates VZV pathogenesis in humans thus representing a suitable animal model for VZV infection. While the type I interferon (IFN) response has been shown to affect VZV replication, the virus employs counter mechanisms to prevent the induction of anti-viral IFN stimulated genes (ISG). Here, we demonstrate that SVV inhibits type I IFN-activated signal transduction via the JAK-STAT pathway. SVV-infected rhesus fibroblasts were refractory to IFN stimulation displaying reduced protein levels of IRF9 and lacking STAT2 phosphorylation. Since previous work implicated involvement of the VZV immediate early gene product ORF63 in preventing ISG-induction we studied the role of SVV ORF63 in generating resistance to IFN treatment. Interestingly, SVV ORF63 did not affect STAT2 phosphorylation but caused IRF9 degradation in a proteasome-dependent manner, suggesting that SVV employs multiple mechanisms to counteract the effect of IFN. Control of SVV ORF63 protein levels via fusion to a dihydrofolate reductase (DHFR)-degradation domain additionally confirmed its requirement for viral replication. Our results also show a prominent reduction of IRF9 and inhibition of STAT2 phosphorylation in VZV-infected cells. In addition, cells expressing VZV ORF63 blocked IFN-stimulation and displayed reduced levels of the IRF9 protein. Taken together, our data suggest that varicella ORF63 prevents ISG-induction both directly via IRF9 degradation and indirectly via transcriptional control of viral proteins that interfere with STAT2 phosphorylation. SVV and VZV thus encode multiple viral gene products that tightly control IFN-induced anti-viral responses. [ABSTRACT FROM AUTHOR]

The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1) infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR) resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins) to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dose (HD), but not low dose (LD), HSV1 inoculation exhibited spontaneous reactivation. Following hyperthermia stress (HS), the majority of HD inoculated mice developed HSV1 encephalitis (HSE) rapidly and synchronously, whereas for LD inoculated mice reactivated HSV1 persisted only transiently in trigeminal ganglia (Tg). T cells, but not B cells, were required to suppress spontaneous reactivation in HD inoculated latently infected mice. Transfer of HSV1 memory but not OVA specific or naïve T cells prior to HS blocked IVR, revealing the utility of this powerful Rag latency model for studying immune mechanisms involved in control of reactivation. Crossing Rag mice to various knockout strains and infecting them with wild type or mutant HSV1 strains is expected to provide novel insights into the role of specific cellular and viral genes in reactivation, thereby facilitating identification of new targets with the potential to block reactivation. [ABSTRACT FROM AUTHOR]

Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress. [ABSTRACT FROM AUTHOR]

The conserved herpesvirus fusion complex consists of glycoproteins gB, gH, and gL which is critical for virion envelope fusion with the cell membrane during entry. For Varicella Zoster Virus (VZV), the complex is necessary for cell-cell fusion and presumed to mediate entry. VZV causes syncytia formation via cell-cell fusion in skin and in sensory ganglia during VZV reactivation, leading to neuronal damage, a potential contributory factor for the debilitating condition of postherpetic neuralgia. The gH cytoplasmic domain (gHcyt) is linked to the regulation of gB/gH-gL-mediated cell fusion as demonstrated by increased cell fusion in vitro by an eight amino acid (aa834-841) truncation of the gHcyt. The gHcyt regulation was identified to be dependent on the physical presence of the domain, and not of specific motifs or biochemical properties as substitution of aa834-841 with V5, cMyc, and hydrophobic or hydrophilic sequences did not affect fusion. The importance of the gHcyt length was corroborated by stepwise deletions of aa834-841 causing incremental increases in cell fusion, independent of gH surface expression and endocytosis. Consistent with the fusion assay, truncating the gHcyt in the viral genome caused exaggerated syncytia formation and significant reduction in viral titers. Importantly, infection of human skin xenografts in SCID mice was severely impaired by the truncation while maintaining the gHcyt length with the V5 substitution preserved typical replication in vitro and in skin. A role for the gHcyt in modulating the functions of the gB cytoplasmic domain (gBcyt) is proposed as the gHcyt truncation substantially enhanced cell fusion in the presence of the gB[Y881F] mutation. The significant reduction in skin infection caused by hyperfusogenic mutations in either the gHcyt or gBcyt demonstrates that both domains are critical for regulating syncytia formation and failure to control cell fusion, rather than enhancing viral spread, is severely detrimental to VZV pathogenesis. [ABSTRACT FROM AUTHOR]

Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome. [ABSTRACT FROM AUTHOR]

Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells. [ABSTRACT FROM AUTHOR]

Varicella-zoster virus (VZV) causes varicella, establishes a life-long latent infection of ganglia and reactivates to cause herpes zoster. The cell types that transport VZV from the respiratory tract to skin and ganglia during primary infection are unknown. Clinical, pathological, virological and immunological features of simian varicella virus (SVV) infection of non-human primates parallel those of primary VZV infection in humans. To identify the host cell types involved in virus dissemination and pathology, we infected African green monkeys intratracheally with recombinant SVV expressing enhanced green fluorescent protein (SVV-EGFP) and with wild-type SVV (SVV-wt) as a control. The SVV-infected cell types and virus kinetics were determined by flow cytometry and immunohistochemistry, and virus culture and SVV-specific real-time PCR, respectively. All monkeys developed fever and skin rash. Except for pneumonitis, pathology produced by SVV-EGFP was less compared to SVV-wt. In lungs, SVV infected alveolar myeloid cells and T-cells. During viremia the virus preferentially infected memory T-cells, initially central memory T-cells and subsequently effector memory T-cells. In early non-vesicular stages of varicella, SVV was seen mainly in perivascular skin infiltrates composed of macrophages, dendritic cells, dendrocytes and memory T-cells, implicating hematogenous spread. In ganglia, SVV was found primarily in neurons and occasionally in memory T-cells adjacent to neurons. In conclusion, the data suggest the role of memory T-cells in disseminating SVV to its target organs during primary infection of its natural and immunocompetent host. [ABSTRACT FROM AUTHOR]

The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC−/−), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system. [ABSTRACT FROM AUTHOR]

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level. [ABSTRACT FROM AUTHOR]

Primary infection with varicella zoster virus (VZV) results in varicella (more commonly known as chickenpox) after which VZV establishes latency in sensory ganglia. VZV can reactivate to cause herpes zoster (shingles), a debilitating disease that affects one million individuals in the US alone annually. Current vaccines against varicella (Varivax) and herpes zoster (Zostavax) are not 100% efficacious. Specifically, studies have shown that 1 dose of varivax can lead to breakthrough varicella, albeit rarely, in children and a 2-dose regimen is now recommended. Similarly, although Zostavax results in a 50% reduction in HZ cases, a significant number of recipients remain at risk. To design more efficacious vaccines, we need a better understanding of the immune response to VZV. Clinical observations suggest that T cell immunity plays a more critical role in the protection against VZV primary infection and reactivation. However, no studies to date have directly tested this hypothesis due to the scarcity of animal models that recapitulate the immune response to VZV. We have recently shown that SVV infection of rhesus macaques models the hallmarks of primary VZV infection in children. In this study, we used this model to experimentally determine the role of CD4, CD8 and B cell responses in the resolution of primary SVV infection in unvaccinated animals. Data presented in this manuscript show that while CD20 depletion leads to a significant delay and decrease in the antibody response to SVV, loss of B cells does not alter the severity of varicella or the kinetics/magnitude of the T cell response. Loss of CD8 T cells resulted in slightly higher viral loads and prolonged viremia. In contrast, CD4 depletion led to higher viral loads, prolonged viremia and disseminated varicella. CD4 depleted animals also had delayed and reduced antibody and CD8 T cell responses. These results are similar to clinical observations that children with agammaglobulinemia have uncomplicated varicella whereas children with T cell deficiencies are at increased risk of progressive varicella with significant complications. Moreover, our studies indicate that CD4 T cell responses to SVV play a more critical role than antibody or CD8 T cell responses in the control of primary SVV infection and suggest that one potential mechanism for enhancing the efficacy of VZV vaccines is by eliciting robust CD4 T cell responses. [ABSTRACT FROM AUTHOR]

Simian varicella virus (SVV), the etiologic agent of naturally occurring varicella in primates, is genetically and antigenically closely related to human varicella zoster virus (VZV). Early attempts to develop a model of VZV pathogenesis and latency in nonhuman primates (NHP) resulted in persistent infection. More recent models successfully produced latency; however, only a minority of monkeys became viremic and seroconverted. Thus, previous NHP models were not ideally suited to analyze the immune response to SVV during acute infection and the transition to latency. Here, we show for the first time that intrabronchial inoculation of rhesus macaques with SVV closely mimics naturally occurring varicella (chickenpox) in humans. Infected monkeys developed varicella and viremia that resolved 21 days after infection. Months later, viral DNA was detected only in ganglia and not in non-ganglionic tissues. Like VZV latency in human ganglia, transcripts corresponding to SVV ORFs 21, 62, 63 and 66, but not ORF 40, were detected by RT-PCR. In addition, as described for VZV, SVV ORF 63 protein was detected in the cytoplasm of neurons in latently infected monkey ganglia by immunohistochemistry. We also present the first in depth analysis of the immune response to SVV. Infected animals produced a strong humoral and cell-mediated immune response to SVV, as assessed by immunohistology, serology and flow cytometry. Intrabronchial inoculation of rhesus macaques with SVV provides a novel model to analyze viral and immunological mechanisms of VZV latency and reactivation. [ABSTRACT FROM AUTHOR]

Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV), infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP) firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural function seen in PRV-infected animals. [ABSTRACT FROM AUTHOR]