Viral unmasking of cellular 5S rRNA pseudogene transcripts induces RIG-I-mediated immunity.

The sensor RIG-I detects double-stranded RNA derived from RNA viruses. Although RIG-I is also known to have a role in the antiviral response to DNA viruses, physiological RNA species recognized by RIG-I during infection with a DNA virus are largely unknown. Using next-generation RNA sequencing (RNAseq), we found that host-derived RNAs, most prominently 5S ribosomal RNA pseudogene 141 (RNA5SP141), bound to RIG-I during infection with herpes simplex virus 1 (HSV-1). Infection with HSV-1 induced relocalization of RNA5SP141 from the nucleus to the cytoplasm, and virus-induced shutoff of host protein synthesis downregulated the abundance of RNA5SP141-interacting proteins, which allowed RNA5SP141 to bind RIG-I and induce the expression of type I interferons. Silencing of RNA5SP141 strongly dampened the antiviral response to HSV-1 and the related virus Epstein-Barr virus (EBV), as well as influenza A virus (IAV). Our findings reveal that antiviral immunity can be triggered by host RNAs that are unshielded following depletion of their respective binding proteins by the virus.

Human TMEFF1 is a restriction factor for herpes simplex virus in the brain.

Most cases of herpes simplex virus 1 (HSV-1) encephalitis (HSE) remain unexplained1,2. Here, we report on two unrelated people who had HSE as children and are homozygous for rare deleterious variants of TMEFF1, which encodes a cell membrane protein that is preferentially expressed by brain cortical neurons. TMEFF1 interacts with the cell-surface HSV-1 receptor NECTIN-1, impairing HSV-1 glycoprotein D- and NECTIN-1-mediated fusion of the virus and the cell membrane, blocking viral entry. Genetic TMEFF1 deficiency allows HSV-1 to rapidly enter cortical neurons that are either patient specific or derived from CRISPR-Cas9-engineered human pluripotent stem cells, thereby enhancing HSV-1 translocation to the nucleus and subsequent replication. This cellular phenotype can be rescued by pretreatment with type I interferon (IFN) or the expression of exogenous wild-type TMEFF1. Moreover, ectopic expression of full-length TMEFF1 or its amino-terminal extracellular domain, but not its carboxy-terminal intracellular domain, impairs HSV-1 entry into NECTIN-1-expressing cells other than neurons, increasing their resistance to HSV-1 infection. Human TMEFF1 is therefore a host restriction factor for HSV-1 entry into cortical neurons. Its constitutively high abundance in cortical neurons protects these cells from HSV-1 infection, whereas inherited TMEFF1 deficiency renders them susceptible to this virus and can therefore underlie HSE.

TMEFF1 is a neuron-specific restriction factor for herpes simplex virus.

The brain is highly sensitive to damage caused by infection and inflammation1,2. Herpes simplex virus 1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis3. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Here we identify TMEFF1 as an HSV-1 restriction factor using genome-wide CRISPR screening. TMEFF1 is expressed specifically in neurons of the central nervous system and is not regulated by type I interferon, the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death following HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with nectin-1 and non-muscle myosin heavy chains IIA and IIB, which are core proteins in virus-cell binding and virus-cell fusion, respectively4-6. Notably, Tmeff1-/- mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the central nervous system.

Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity.

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.

Clinical trial links oncolytic immunoactivation to survival in glioblastoma.

Immunotherapy failures can result from the highly suppressive tumour microenvironment that characterizes aggressive forms of cancer such as recurrent glioblastoma (rGBM)1,2. Here we report the results of a first-in-human phase I trial in 41 patients with rGBM who were injected with CAN-3110-an oncolytic herpes virus (oHSV)3. In contrast to other clinical oHSVs, CAN-3110 retains the viral neurovirulence ICP34.5 gene transcribed by a nestin promoter; nestin is overexpressed in GBM and other invasive tumours, but not in the adult brain or healthy differentiated tissue4. These modifications confer CAN-3110 with preferential tumour replication. No dose-limiting toxicities were encountered. Positive HSV1 serology was significantly associated with both improved survival and clearance of CAN-3110 from injected tumours. Survival after treatment, particularly in individuals seropositive for HSV1, was significantly associated with (1) changes in tumour/PBMC T cell counts and clonal diversity, (2) peripheral expansion/contraction of specific T cell clonotypes; and (3) tumour transcriptomic signatures of immune activation. These results provide human validation that intralesional oHSV treatment enhances anticancer immune responses even in immunosuppressive tumour microenvironments, particularly in individuals with cognate serology to the injected virus. This provides a biological rationale for use of this oncolytic modality in cancers that are otherwise unresponsive to immunotherapy (ClinicalTrials.gov: NCT03152318 ).

S6K-STING interaction regulates cytosolic DNA-mediated activation of the transcription factor IRF3.

Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway.

The HSV-1 pUL37 protein promotes cell invasion by regulating the kinesin-1 motor.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), recruit microtubule motor proteins to invade cells. The incoming viral particle traffics to nuclei in a two-step process. First, the particle uses the dynein-dynactin motor to sustain transport to the centrosome. In neurons, this step is responsible for long-distance retrograde axonal transport and is an important component of the neuroinvasive property shared by these viruses. Second, a kinesin-dependent mechanism redirects the particle from the centrosome to the nucleus. We have reported that the kinesin motor used during the second step of invasion is assimilated into nascent virions during the previous round of infection. Here, we report that the HSV-1 pUL37 tegument protein suppresses the assimilated kinesin-1 motor during retrograde axonal transport. Region 2 (R2) of pUL37 was required for suppression and functioned independently of the autoinhibitory mechanism native to kinesin-1. Furthermore, the motor domain and proximal coiled coil of kinesin-1 were sufficient for HSV-1 assimilation, pUL37 suppression, and nuclear trafficking. pUL37 localized to the centrosome, the site of assimilated kinesin-1 activation during infection, when expressed in cells in the absence of other viral proteins; however, pUL37 did not suppress kinesin-1 in this context. These results indicate that the pUL37 tegument protein spatially and temporally regulates kinesin-1 via the amino-terminal motor region in the context of the incoming viral particle.

The importance of IFNα2A (Roferon-A) in HSV-1 latency and T cell exhaustion in ocularly infected mice.

Published studies have generated compelling results indicating that type I IFN modulates function of HSV-1 latency-associated transcript (LAT). One member of type I IFN is IFNα2A also called Roferon-A). IFNα2A has been used in monotherapy or in combination therapy with other drugs to treat viral infections and different kinds of cancer in humans. The goal of this study was to determine whether the absence of IFNα2A affects primary and latent infections in ocularly infected mice. Therefore, we generated a mouse strain lacking IFNα2A expression (IFNα2A-/-). Ocular HSV-1 replication, IFN and immune cell expressions on days 3 and 5 post infection (PI), as well as eye disease, survival, latency-reactivation, and T cell exhaustion were evaluated in ocularly infected IFNα2A-/- and wild type (WT) control mice. Absence of IFNα2A did not affect other members of the IFNα family but it affected IFNß and IFNγ expressions as well as some immune cells on day 5 PI compared to WT mice. Viral replication in the eye, eye disease, and survival amongst ocularly infected IFNα2A-/- mice were similar to that of WT infected mice. The absence of IFNα2A significantly reduced the levels of latency and T cell exhaustion but not time of reactivation compared with control mice. Our results suggest that blocking IFNα2A expression may be a useful tool in reducing latency and the subsequent side effects associated with higher levels of latency.

The HSV-1 encoded CCCTC-binding factor, CTRL2, impacts the nature of viral chromatin during HSV-1 lytic infection.

HSV-1 genomes are rapidly heterochromatinized following entry by host cells to limit viral gene expression. Efficient HSV-1 genome replication requires mechanisms that de-repress chromatin associated with the viral genome. CCCTC-binding factors, or CTCF insulators play both silencing and activating roles in cellular transcriptional regulation. Importantly, the HSV-1 genome encodes several CTCF insulators that flank IE genes, implying that individual HSV-1 encoded CTCF insulators regulate IE transcription during all stages of the HSV-1 life cycle. We previously reported that the HSV-1 encoded CTCF insulator located downstream of the LAT (CTRL2) controlled IE gene silencing during latency. To further characterize the role of this insulator during the lytic infection we leveraged a ΔCTRL2 recombinant virus to show that there was a genome replication defect that stemmed from decreased IE gene expression in fibroblasts and epithelial cells at early times following initiation of infection. Further experiments indicated that the defect in gene expression resulted from chromatin inaccessibility in the absence of the insulator. To elucidate how chromatin accessibility was altered in the absence of the CTRL2 insulator, we showed that enrichment of Alpha-thalassemia/mental retardation, X-linked chromatin remodeler (ATRX), and the histone variant H3.3, both of which are known for their roles in maintaining repressive histone markers on the HSV-1 viral genome were increased on IE regions of HSV-1. Finally, both H3K27me3 and H3K9me3 repressive histone marks remained enriched by 4 hours post infection in the absence of the CTRL2 insulator, confirming that the CTRL2 insulator is required for de-repression of IE genes of viral genomes. To our knowledge these are the first data that show that a specific CTCF insulator in the HSV-1 genome (CTRL2) regulates chromatin accessibility during the lytic infection.

The anti-apoptotic function of HSV-1 LAT in neuronal cell cultures but not its function during reactivation correlates with expression of two small non-coding RNAs, sncRNA1&2.

Multiple functions are associated with HSV-1 latency associated transcript (LAT), including establishment of latency, virus reactivation, and antiapoptotic activity. LAT encodes two sncRNAs that are not miRNAs and previously it was shown that they have antiapoptotic activity in vitro. To determine if we can separate the antiapoptotic function of LAT from its latency-reactivation function, we deleted sncRNA1 and sncRNA2 sequences in HSV-1 strain McKrae, creating ΔsncRNA1&2 recombinant virus. Deletion of the sncRNA1&2 in ΔsncRNA1&2 virus was confirmed by complete sequencing of ΔsncRNA1&2 virus and its parental virus. Replication of ΔsncRNA1&2 virus in tissue culture or in the eyes of WT infected mice was similar to that of HSV-1 strain McKrae (LAT-plus) and dLAT2903 (LAT-minus) viruses. The levels of gB DNA in trigeminal ganglia (TG) of mice latently infected with ΔsncRNA1&2 virus was intermediate to that of dLAT2903 and McKrae infected mice, while levels of LAT in TG of latently infected ΔsncRNA1&2 mice was significantly higher than in McKrae infected mice. Similarly, the levels of LAT expression in Neuro-2A cells infected with ΔsncRNA1&2 virus was significantly higher than in McKrae infected cells. Reactivation in TG of ΔsncRNA1&2 infected mice was similar to that of McKrae and time of reactivation in both groups were significantly faster than dLAT2903 infected mice. However, levels of apoptosis in Neuro-2A cells infected with ΔsncRNA1&2 virus was similar to that of dLAT2903 and significantly higher than that of McKrae infected cells. Our results suggest that the antiapoptotic function of LAT resides within the two sncRNAs, which works independently of its latency-reactivation function and it has suppressive effect on LAT expression in vivo and in vitro.

Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Herpes simplex virus spreads rapidly in human foreskin, partly driven by chemokine-induced redistribution of Nectin-1 on keratinocytes.

HSV infects keratinocytes in the epidermis of skin via nectin-1. We established a human foreskin explant infection model to investigate HSV entry and spread. HSV1 entry could only be achieved by the topical application of virus via high density microarray projections (HD-MAPs) to the epidermis, which penetrated beyond one third of its thickness, simulating in vivo microtrauma. Rapid lateral spread of HSV1 to a mean of 13 keratinocytes wide occurred after 24 hours and free virus particles were observed between keratinocytes, consistent with an intercellular route of spread. Nectin-1 staining was markedly decreased in foci of infection in the epidermis and in the human keratinocyte HaCaT cell line. Nectin-1 was redistributed, at the protein level, in adjacent uninfected cells surrounding infection, inducible by CCL3, IL-8 (or CXCL8), and possibly CXCL10 and IL-6, thus facilitating spread. These findings provide the first insights into HSV1 entry and spread in human inner foreskin in situ.

Nuclear interferon-stimulated gene product maintains heterochromatin on the herpes simplex viral genome to limit lytic infection.

Interferons (IFN) are expressed in and secreted from cells in response to virus infection, and they induce the expression of a variety of genes called interferon-stimulated genes (ISGs) in infected and surrounding cells to block viral infection and limit spread. The mechanisms of action of a number of cytoplasmic ISGs have been well defined, but little is known about the mechanism of action of nuclear ISGs. Constitutive levels of nuclear interferon-inducible protein 16 (IFI16) serve to induce innate signaling and epigenetic silencing of herpes simplex virus (HSV), but only when the HSV infected cell protein 0 (ICP0) E3 ligase, which promotes IFI16 degradation, is inactivated. In this study, we found that following IFN induction, the pool of IFI16 within the infected cell remains high and can restrict wild-type viral gene expression and replication due to both the induced levels of IFI16 and the IFI16-mediated repression of ICP0 levels. Restriction of viral gene expression is achieved by IFI16 promoting the maintenance of heterochromatin on the viral genome, which silences it epigenetically. These results indicate that a nuclear ISG can restrict gene expression and replication of a nuclear DNA virus by maintaining or preventing the removal of repressive heterochromatin associated with the viral genome.

The immunobiology of corneal HSV-1 infection and herpetic stromal keratitis.

SUMMARYHuman alphaherpesvirus 1 (HSV-1) is a highly successful neurotropic pathogen that primarily infects the epithelial cells lining the orofacial mucosa. After primary lytic replication in the oral, ocular, and nasal mucosal epithelial cells, HSV-1 establishes life-long latency in neurons within the trigeminal ganglion. Patients with compromised immune systems experience frequent reactivation of HSV-1 from latency, leading to virus entry in the sensory neurons, followed by anterograde transport and lytic replication at the innervated mucosal epithelial surface. Although recurrent infection of the corneal mucosal surface is rare, it can result in a chronic immuno-inflammatory condition called herpetic stromal keratitis (HSK). HSK leads to gradual vision loss and can cause permanent blindness in severe untreated cases. Currently, there is no cure or successful vaccine to prevent latent or recurrent HSV-1 infections, posing a significant clinical challenge to managing HSK and preventing vision loss. The conventional clinical management of HSK primarily relies on anti-virals to suppress HSV-1 replication, anti-inflammatory drugs (such as corticosteroids) to provide symptomatic relief from pain and inflammation, and surgical interventions in more severe cases to replace damaged cornea. However, each clinical treatment strategy has limitations, such as local and systemic drug toxicities and the emergence of anti-viral-resistant HSV-1 strains. In this review, we summarize the factors and immune cells involved in HSK pathogenesis and highlight alternate therapeutic strategies for successful clinical management of HSK. We also discuss the therapeutic potential of immunoregulatory cytokines and immunometabolism modulators as promising HSK therapies against emerging anti-viral-resistant HSV-1 strains.

Individual herpes simplex virus 1 (HSV-1) particles exit by exocytosis and accumulate at preferential egress sites.

The human pathogen herpes simplex virus 1 (HSV-1) produces a lifelong infection in the majority of the world's population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect are cell type dependent, not virus dependent. This preferential egress and clustering appear to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor.IMPORTANCEAlpha herpesviruses produce lifelong infections in their human and animal hosts. The majority of people in the world are infected with herpes simplex virus 1 (HSV-1), which typically causes recurrent oral or genital lesions. However, HSV-1 can also spread to the central nervous system, causing severe encephalitis, and might also contribute to the development of neurodegenerative diseases. Many of the steps of how these viruses infect and replicate inside host cells are known in depth, but the final step, exiting from the infected cell, is not fully understood. In this study, we engineered a novel variant of HSV-1 that allows us to visualize how individual virus particles exit from infected cells. With this imaging assay, we investigated preferential egress site formation in certain cell types and their contribution to the cell-cell spread of HSV-1.

c-Jun signaling during initial HSV-1 infection modulates latency to enhance later reactivation in addition to directly promoting the progression to full reactivation.

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates to permit transmission, which can result in clinical manifestations. Viral transactivators required for lytic infection are largely absent during latent infection, and therefore, HSV-1 relies on the co-option of neuronal host signaling pathways to initiate its gene expression. The activation of the neuronal c-Jun N-terminal kinase (JNK) cell stress pathway is central to initiating biphasic reactivation in response to multiple stimuli. However, how host factors work with JNK to stimulate the initial wave of gene expression (known as Phase I) or the progression to full Phase II reactivation remains unclear. Here, we found that c-Jun, the primary target downstream of neuronal JNK cell stress signaling, functions during reactivation but not during the JNK-mediated initiation of Phase I gene expression. Instead, c-Jun was required to transition from Phase I to full HSV-1 reactivation and was detected in viral replication compartments of reactivating neurons. Interestingly, we also identified a role for both c-Jun and enhanced neuronal stress during initial neuronal infection in promoting a more reactivation-competent form of HSV-1 latency. Therefore, c-Jun functions at multiple stages during the HSV latent infection of neurons to promote reactivation but not during the initial JNK-dependent Phase I. Importantly, by demonstrating that initial infection conditions can contribute to later reactivation abilities, this study highlights the potential for latently infected neurons to maintain a molecular scar of previous exposure to neuronal stressors.IMPORTANCEThe molecular mechanisms that regulate the reactivation of herpes simplex virus-1 (HSV-1) from latent infection are unknown. The host transcription and pioneer factor c-Jun is the main target of the JNK cell stress pathway that is known to be important in exit of HSV from latency. Surprisingly, we found that c-Jun does not act with JNK during exit from latency but instead promotes the transition to full reactivation. Moreover, c-Jun and enhanced neuronal stress during initial neuronal infection promoted a more reactivation-competent form of HSV-1 latency. c-Jun, therefore, functions at multiple stages during HSV-1 latent infection of neurons to promote reactivation. Importantly, this study contributes to a growing body of evidence that de novo HSV-1 infection conditions can modulate latent infection and impact future reactivation events, raising important questions on the clinical impact of stress during initial HSV-1 acquisition on future reactivation events and consequences.

Absence of CD80 reduces HSV-1 replication in the eye and delays reactivation but not latency levels.

Herpes simplex virus-1 (HSV-1) infections are among the most frequent serious viral eye infections in the U.S. and are a major cause of viral-induced blindness. HSV-1 infection is known to induce T cell activation, proliferation, and differentiation that play crucial roles in the development of virus-induced inflammatory lesions, leading to eye disease and causing chronic corneal damage. CD80 is a co-stimulatory molecule and plays a leading role in T cell differentiation. Previous efforts to limit lesion severity by controlling inflammation at the cellular level led us to ask whether mice knocked out for CD80 would show attenuated virus replication following reactivation. By evaluating the effects of CD80 activity on primary and latent infection, we found that in the absence of CD80, virus replication in the eyes and virus reactivation in latent trigeminal ganglia were both significantly reduced. However, latency in latently infected CD80-/- mice did not differ significantly from that in wild-type (WT) control mice. Reduced virus replication in the eyes of CD80-/- mice correlated with significantly expanded CD11c gene expression as compared to WT mice. Taken together, our results indicate that suppression of CD80 could offer significant beneficial therapeutic effects in the treatment of Herpes Stromal Keratitis (HSK).IMPORTANCEOf the many problems associated with recurrent ocular infection, reducing virus reactivation should be a major goal of controlling ocular herpes simplex virus-1 (HSV-1) infection. In this study, we have shown that the absence of CD80 reduces HSV-1 reactivation, which marks the establishment of a previously undescribed mechanism underlying viral immune evasion that could be exploited to better manage HSV infection.

A dual fluorescent herpes simplex virus type 1 recombinant reveals divergent outcomes of neuronal infection.

Critical stages of lytic herpes simplex virus type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 can also persist in neuronal cells via a non-replicative, transcriptionally repressed infection called latency. The regulation of lytic and latent transcriptional profiles is critical to HSV-1 pathogenesis and persistence. We sought a fluorescence-based approach to observe the outcome of neuronal HSV-1 infection at the single-cell level. To achieve this goal, we constructed and characterized a novel HSV-1 recombinant that enables discrimination between lytic and latent infection. The dual reporter HSV-1 encodes a human cytomegalovirus-immediate early (hCMV-IE) promoter-driven enhanced yellow fluorescent protein (eYFP) to visualize the establishment of infection and an endogenous mCherry-VP26 fusion to report lytic replication. We confirmed that viral gene expression, replication, and spread of infection are not altered by the incorporation of the fluorescent reporters, and fluorescent protein (FP) detection virtuously reports the progression of lytic replication. We demonstrate that the outcome of HSV-1 infection of compartmentalized primary neurons is determined by viral inoculating dose: high-dose axonal inoculation proceeds to lytic replication, whereas low-dose axonal inoculation establishes a latent HSV-1 infection. Interfering with low-dose axonal inoculation via small molecule drugs reports divergent phenotypes of eYFP and mCherry reporter detection, correlating with altered states of viral gene expression. We report that the transcriptional state of neuronal HSV-1 infection is variable in response to changes in the intracellular neuronal environment.IMPORTANCEHerpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global human population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by the regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We used a recombinant HSV-1 to evaluate viral gene expression during infection of primary neurons. Manipulation of cell signaling pathways impacts the establishment and transcriptional state of HSV-1 latency in neurons. The work here provides critical insight into the cellular and viral factors contributing to the establishment of latent HSV-1 infection.

Identification of a novel neurovirulence factor encoded by the cryptic orphan gene UL31.6 of herpes simplex virus 1.

Although the herpes simplex virus type 1 (HSV-1) genome was thought to contain approximately 80 different protein coding sequences (CDSs), recent multi-omics analyses reported HSV-1 encodes more than 200 potential CDSs. However, few of the newly identified CDSs were confirmed to be expressed at the peptide or protein level in HSV-1-infected cells. Furthermore, the impact of the proteins they encode on HSV-1 infection is largely unknown. This study focused on a newly identified CDS, UL31.6. Re-analyzation of our previous chemical proteomics data verified that UL31.6 was expressed at the peptide level in HSV-1-infected cells. Antisera raised against a viral protein encoded by UL31.6 (pUL31.6) reacted with a protein with an approximate molecular mass of 37 kDa in lysates of Vero cells infected with each of three HSV-1 strains. pUL31.6 was efficiently dissociated from virions in high-salt solution. A UL31.6-null mutation had a minimal effect on HSV-1 gene expression, replication, cell-to-cell spread, and morphogenesis in Vero cells; in contrast, it significantly reduced HSV-1 cell-to-cell spread in three neural cells but not in four non-neural cells including Vero cells. The UL31.6-null mutation also significantly reduced the mortality and viral replication in the brains of mice after intracranial infection, but had minimal effects on pathogenic manifestations in and around the eyes, and viral replication detected in the tear films of mice after ocular infection. These results indicated that pUL31.6 was a tegument protein and specifically acted as a neurovirulence factor by potentially promoting viral transmission between neuronal cells in the central nervous system.IMPORTANCERecent multi-omics analyses reported the herpes simplex virus type 1 (HSV-1) genome encodes an additional number of potential coding sequences (CDSs). However, the expressions of these CDSs at the peptide or protein levels and the biological effects of these CDSs on HSV-1 infection remain largely unknown. This study annotated a cryptic orphan CDS, termed UL31.6, an HSV-1 gene that encodes a tegument protein with an approximate molecular mass of 37 kDa, which specifically acts as a neurovirulence factor. Our study indicates that HSV-1 proteins important for viral pathogenesis remain to be identified and a comprehensive understanding of the pathogenesis of HSV-1 will require not only the identification of cryptic orphan CDSs using emerging technologies but also step-by-step and in-depth analyses of each of the cryptic orphan CDSs.

Histone H2A variant H2A.B is enriched in transcriptionally active and replicating HSV-1 lytic chromatin.

Herpes simplex virus 1 (HSV-1) transcription is restricted in latently infected neurons and the genomes are in mostly silenced chromatin, whereas all viral genes are transcribed in lytically infected cells, in which the genomes are dynamically chromatinized. Epigenetic regulation modulates HSV-1 transcription during lytic, latent, and reactivating infections but the precise mechanisms are not fully defined. Nucleosomes are dynamic: they slide, breathe, assemble, and disassemble. We and others have proposed that the most dynamic HSV-1 chromatin is transcriptionally competent, whereas the least dynamic is silenced. However, the mechanisms yielding the unusually dynamic viral chromatin remain unknown. Histone variants affect nucleosome dynamics. The dynamics of H2A, H2A.X, and macroH2A were enhanced in infected cells, whereas those of H2A.B were uniquely decreased. We constructed stably transduced cells expressing tagged histone H2A, H2A.B, macroH2A, or H2B, which assembles the H2A/H2B nucleosome dimers with all H2A variants. All H2A variants, as well as ectopic and endogenous H2B were assembled into HSV-1 chromatin evenly throughout the genome but canonical H2A was relatively depleted whereas H2A.B was enriched, particularly in the most dynamic viral chromatin. When viral transcription and DNA replication were restricted, H2A.B became as depleted from the viral chromatin through the entire genome as H2A. We propose that lytic HSV-1 nucleosomes are enriched in the dynamic variant H2A.B/H2B dimers to promote HSV-1 chromatin dynamics and transcriptional competency and conclude that the dynamics of HSV-1 chromatin are determined in part by the H2A variants. IMPORTANCE: Herpes simplex virus 1 (HSV-1) transcription is epigenetically regulated during latent and lytic infections, and epigenetic inhibitors have been proposed as potential antiviral drugs to modulate latency and reactivation. However, the detailed epigenetic mechanisms of regulation of HSV-1 transcription have not been fully characterized and may differ from those regulating cellular transcription. Whereas lytic HSV-1 chromatin is unusually dynamic, latent silenced HSV-1 chromatin is not. The mechanisms resulting in the unique dynamics of the lytic chromatin remain unknown. Here we identify the enrichment of the highly dynamic histone 2A variant H2A in the most dynamic viral chromatin, which provides a mechanistic understanding of its unique dynamics. Future work to identify the mechanisms of enrichment in H2A.B on the viral chromatin may identify novel druggable epigenetic regulators that modulate HSV-1 latency and reactivation.