Herpes simplex virus 1 (HSV-1) establishes latent infections in sensory neurons, from which HSV sporadically reactivates, often due to external stress and other stimuli. Latency and reactivation are studied using in vivo models in a variety of hosts, as well as in vitro models including primary mouse neurons, and neurons derived from human pluripotent stem cells (iPSCs). The interferon (IFN)-based neuronal innate immune response is critical in controlling HSV-1 replication and HSV-1 counters these responses, in part, through infected-cell protein 34.5 (ICP34.5). ICP34.5 also promotes neurovirulence by preventing host translational shutoff and interfering with host cell autophagy through its interaction with the autophagy regulator Beclin 1. Here we demonstrate in human iPSC-derived neurons (iNeurons) that ICP34.5 unexpectedly suppresses spontaneous reactivation and thereby is critical for maintenance of HSV-1 latency. Furthermore, our results suggest that both sustaining host translation and the interaction of ICP34.5 with Beclin 1 are important for maintaining latency in iNeurons. Experiments using primary mouse neurons show that ICP34.5 may be essential for maintaining latency but in an IRF3/7-dependent manner. In wild type mouse neurons, ICP34.5-null and ΔPP1⍺ viruses exhibited little spontaneous reactivation and had defects in induced reactivation suggesting that countering PKR-mediated responses is the key activity of ICP34.5 for enhancement of reactivation. These results highlight the value of studying HSV-1 latency and reactivation in different models and we explore explanations for how ICP34.5 may differentially impact latency in the two systems studied herein.

Herpes simplex virus 1 (HSV-1) establishes latent infections in sensory neurons, from which HSV sporadically reactivates due to external stress and other stimuli. Latency and reactivation are studied through in vivo models in a variety of hosts, as well as in vitro models using primary neurons, and neurons derived from pluripotent stem cells (iPSCs). These systems behave disparately, but the reasons remain unknown. The interferon (IFN)-based neuronal innate immune response is critical in controlling HSV-1 replication and HSV-1 counters these responses in part through infected-cell protein 34.5 (ICP34.5). ICP34.5 also promotes neurovirulence by preventing host translational shutoff and interfering with host cell autophagy. Here we demonstrate in a human iPSC neuronal model that sustaining host translation is the key activity of ICP34.5 for enhancement of reactivation. Specifically, our data shows that ICP34.5 was key for maintenance of HSV-1 latency. While interaction of ICP34.5 with the autophagy regulator Beclin 1 was important for maintaining latency, this was not due to modulation of bulk autophagy. Our work from primary mouse neurons suggested that the major effect of ICP34.5 on latency maintenance occurs in an IRF3/7-dependent manner. Notably, the role of ICP34.5 in regulating latency and reactivation differs between neurons derived from human iPSCs (iNeurons) and primary mouse trigeminal (TG) neurons. This highlights the importance of selecting an appropriate neuronal model and validating experimental outcomes in multiple models.

Herpes simplex virus (HSV-1) has been associated with molecular and cellular signatures associated with Alzheimer’s disease (AD). We explored the use of both recent single-cell and bulk transcriptomics technologies in dissecting the molecular and cellular virus-human interactions with HSV-1 infected cerebral organoids (2D and 3D). We compared the results with our previous observations from bulk RNA sequencing and discovered novel insights into HSV-1 induced AD-associated molecular pathology that were made possible by each transcriptomics technology. We used recent emerging technologies such as single-cell spatial RNA sequencing (STOmics Stereo-seq) and single-cell non-spatial RNA sequencing (Parse Evercode). In addition, we have data generated from our previous work using bulk RNA sequencing. Previously, we found that differentially expressed genes in HSV-1 infected dissociated cells from cerebral organoids (2D cOrgs) using bulk RNA sequencing data were exclusively enriched for AD-associated genes implicated through genome-wide association studies (GWAS), but not for genes associated with other neurodegenerative or autoimmune diseases. UMAP analyses on single-cell non-spatial RNA sequencing data with GFP-tagged HSV-1 infected 2D cOrgs clustered by viral transcripts [Fig. 1] revealed huge differences in the proportions of true late viral transcripts but not of leaky late viral transcripts between clusters, while similar clustering of 3D cOrgs [Fig. 2] showed distinct proportions of both true late and leaky late transcripts. Pseudobulk analyses showed no enrichment for AD-associated GWAS genes among the differentially expressed genes for HSV-1 infected 2D cOrgs versus uninfected 2D cOrgs, but revealed significant enrichment for GWAS genes associated with autoimmune diseases such as Type 1 diabetes and multiple sclerosis. Similar analyses of HSV-1 infected 3D cOrgs versus uninfected 3D cOrgs did not show enrichment in GWAS gene lists across the 21 common neurodegenerative, neuropsychiatric or autoimmune diseases that we had surveyed. Taken together, our use of multi-transcriptomics technologies has enabled us to gain course to fine molecular granularity into neuroinflammation-induced AD viral-human gene interactions and a careful dissection of the multi-transcriptomics data enables us to gain insights into biomarkers and gene targets for AD therapeutics development in the near future.

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

The application of gene therapy to the treatment of human disease with complex etiology and pathology will often require the delivery of large payloads exceeding 10 kbp in size. This is generally not possible with the most popular vectors such as adeno-associated viruses (AAVs), lentiviruses (LVs), retroviruses (RVs), and many nonviral delivery systems. There is a high likelihood that the correction of many human gene defects such as those associated with neurodegenerative diseases and inflammatory processes will require single large genes or complex genetic payloads that will often necessitate precise regulatory control of the specificity, timing, and duration of corrective gene expression. The regulation of cellular gene products typically depends on genomic promoter systems and splicing-driven transcription variants, necessitating a delivery vector with substantial payload capacity. Replication-defective herpes simplex virus (rdHSV) mutants lack at least one essential viral gene product and are propagated in host cells that supply the missing gene product. This review explores next-generation rdHSV vectors, which do not express viral genes, offer high payload capacity, and can be engineered for safe, long-term transgene expression. These advanced vectors enable the correction of complex diseases affecting neurons and other tissues, paving the way for large or intricate gene replacement strategies.

Viral infections leading to inflammation have been implicated in several common diseases, such as Alzheimer’s disease (AD) and type 1 diabetes (T1D). Of note, herpes simplex virus 1 (HSV-1) has been reported to be associated with AD. We sought to identify the transcriptomic changes due to HSV-1 infection and anti-viral drug (acyclovir, ACV) treatment of HSV-1 infection in dissociated cells from human cerebral organoids (dcOrgs) versus stem cell-derived pancreatic islets (sc-islets) to gain potential biological insights into the relevance of HSV-1-induced inflammation in AD and T1D. We observed that differentially expressed genes (DEGs) in HSV-1-infected sc-islets were enriched for genes associated with several autoimmune diseases, most significantly, T1D, but also rheumatoid arthritis, psoriasis, Crohn’s disease, and multiple sclerosis, whereas DEGs in HSV-1-infected dcOrgs were exclusively enriched for genes associated with AD. The ACV treatment of sc-islets was not as effective in rescuing transcript perturbations of autoimmune disease-associated genes. Finally, we identified gene ontology categories that were enriched for DEGs that were in common across, or unique to, viral treatment of dcOrgs and sc-islets, such as categories involved in the transferase complex, mitochondrial, and autophagy function. In addition, we compared transcriptomic signatures from HSV-1-infected sc-islets with sc-islets that were infected with the coxsackie B virus (CVB) that had been associated with T1D pathogenesis. Collectively, this study provides tissue-specific insights into the molecular effects of inflammation in AD and T1D.

Productive replication of herpes simplex virus (HSV) relies upon a well-ordered transcriptional cascade flowing from immediate-early (IE) to early (E) to late (L) gene products. While several virus-encoded transcriptional activators are involved in this process, IE and E gene promoters also contain multiple binding sites for the ubiquitously expressed cellular transcription factor Sp1. Sp1 has been previously implicated in activating HSV-1 gene transcription downstream of these sites, but why Sp1-binding sites are maintained in the promoters of genes activated by virus-encoded activators remains unclear. We hypothesized that Sp1 enables continued HSV-1 transcription and replication when viral transactivators are limited. We used a depletion-based approach in human foreskin fibroblasts to investigate the specific contribution of Sp1 to the initiation and progression of the HSV-1 lytic gene cascade. We found that Sp1 increased viral transcript levels, protein expression, and replication following infection with VP16- or ICP0-deficient viruses but had little to no effect on rescued viruses or during wild-type (WT) HSV-1 infection. Moreover, Sp1 promoted WT virus transcription and replication following interferon treatment of fibroblasts and thus may contribute to viral immune evasion. Interestingly, we observed reduced expression of Sp1 and Sp1-family transcription factors in differentiated sensory neurons compared to undifferentiated cells, suggesting that reduced Sp1 levels may also contribute to HSV-1 latent infection. Overall, these findings indicate that Sp1 can promote HSV-1 gene expression in the absence of key viral transactivators; thus, HSV-1 may use Sp1 to maintain its gene expression and replication under adverse conditions.IMPORTANCEHerpes simplex virus (HSV) is a common human pathogen that actively replicates in the epithelia but can persist for the lifetime of the infected host via a stable, latent infection in neurons. A key feature of the HSV replication cycle is a complex transcriptional program in which virus and host-cell factors coordinate to regulate expression of the viral gene products necessary for continued viral replication. Multiple binding sites for the cellular transcription factor Sp1 are located in the promoters of HSV-1 genes, but how Sp1 binding contributes to transcription and replication of wild-type virus is not fully understood. In this study, we identified a specific role for Sp1 in maintaining HSV-1 gene transcription under adverse conditions, as when virus-encoded transcriptional activators were absent or limited. Preservation of Sp1-binding sites in HSV-1 gene promoters may thus benefit the virus as it navigates diverse cell types and host-cell conditions during infection.

DNA polymerases are important drug targets, and many structural studies have captured them in distinct conformations. However, a detailed understanding of the impact of polymerase conformational dynamics on drug resistance is lacking. We determined cryoelectron microscopy (cryo-EM) structures of DNA-bound herpes simplex virus polymerase holoenzyme in multiple conformations and interacting with antivirals in clinical use. These structures reveal how the catalytic subunit Pol and the processivity factor UL42 bind DNA to promote processive DNA synthesis. Unexpectedly, in the absence of an incoming nucleotide, we observed Pol in multiple conformations with the closed state sampled by the fingers domain. Drug-bound structures reveal how antivirals may selectively bind enzymes that more readily adopt the closed conformation. Molecular dynamics simulations and the cryo-EM structure of a drug-resistant mutant indicate that some resistance mutations modulate conformational dynamics rather than directly impacting drug binding, thus clarifying mechanisms that drive drug selectivity.

In 2015, the oncolytic herpes simplex virus 1 (HSV-1) T-VEC (talimogene laherparepvec) was approved for intratumoral injection in non-resectable malignant melanoma. To determine whether viral replication is required for oncolytic activity, we compared replication-deficient HSV-1 d106S with replication-competent T-VEC. High infectious doses of HSV-1 d106S killed melanoma (n = 10), head-and-neck squamous cell carcinoma (n = 11), and chondrosarcoma cell lines (n = 2) significantly faster than T-VEC as measured by MTT metabolic activity, while low doses of T-VEC were more effective over time. HSV-1 d106S and, to a lesser extent T-VEC, triggered caspase-dependent early apoptosis as shown by pan-caspase inhibition and specific induction of caspases 3/7, 8, and 9. HSV-1 d106S induced a higher ratio of apoptosis-inducing infected cell protein (ICP) 0 to apoptosis-blocking ICP6 than T-VEC. T-VEC was oncolytic for an extended period of time as viral replication continued, which could be partially blocked by the antiviral drug aciclovir. High doses of T-VEC, but not HSV-1 d106S, increased interferon-β mRNA as part of the intrinsic immune response. When markers of immunogenic cell death were assessed, ATP was released more efficiently in the context of T-VEC than HSV-1 d106S infection, whereas HMGB1 was induced comparatively well. Overall, the early oncolytic effect on three different tumour entities was stronger with the non-replicative strain, while the replication-competent virus elicited a stronger innate immune response and more pronounced immunogenic cell death.

Herpes simplex virus 1 (HSV-1) latent infection entails repression of viral lytic genes in neurons. By functional screening using luciferase-expressing HSV-1, we identify ten neuron-specific microRNAs potentially repressing HSV-1 neuronal replication. Transfection of miR-9, the most active candidate from the screen, decreases HSV-1 replication and gene expression in Neuro-2a cells. Ectopic expression of miR-9 from lentivirus or recombinant HSV-1 suppresses HSV-1 replication in male primary mouse neurons in culture and mouse trigeminal ganglia in vivo, and reactivation from latency in the primary neurons. Target prediction and validation identify transcription factors Oct-1, a known co-activator of HSV transcription, and all three Onecut family members as miR-9 targets. Knockdown of ONECUT2 decreases HSV-1 yields in Neuro-2a cells. Overexpression of each ONECUT protein increases HSV-1 replication in Neuro-2a cells, human induced pluripotent stem cell-derived neurons, and primary mouse neurons, and accelerates reactivation from latency in the mouse neurons. Mutagenesis, ChIP-seq, RNA-seq, ChIP-qPCR and ATAC-seq results suggest that ONECUT2 can nonspecifically bind to viral genes via its CUT domain, globally stimulate viral gene transcription, reduce viral heterochromatin and enhance the accessibility of viral chromatin. Thus, neuronal miR-9 promotes viral epigenetic silencing and latency by targeting multiple host transcription factors important for lytic gene activation.

The herpes simplex virus 1 DNA polymerase contains a highly conserved structural motif found in most family B polymerases and certain RNA-binding proteins. To investigate its importance within cells, we constructed a mutant virus with substitutions in two residues of the motif and a rescued derivative. The substitutions resulted in severe impairment of plaque formation, yields of infectious virus, and viral DNA synthesis while not meaningfully affecting expression of the mutant enzyme, its co-localization with the viral single-stranded DNA binding protein at intranuclear punctate sites in non-complementing cells or in replication compartments in complementing cells, or viral DNA polymerase activity. Taken together, our results indicate that the RNA binding motif plays a crucial role in herpes simplex virus 1 DNA synthesis through a mechanism separate from effects on polymerase activity, thus identifying a distinct essential function of this motif with implications for hypotheses regarding its biochemical functions.

ABSTRACT Neuroinflammation is a central process in the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease (AD), and there are active efforts to target pathways involved in neuroinflammation for molecular biomarker discovery and therapeutic development in neurodegenerative diseases. It was also proposed that there may be an infectious etiology in AD that is associated with viruses such as herpes simplex virus (HSV-1) and influenza A virus (IAV), leading to neuroinflammation-induced AD pathogenesis or disease progression. We sought to develop high-throughput, quantitative molecular biomarker assays using dissociated cells from human cerebral organoids (dcOrgs), that can used for screening compounds to reverse AD-associated neuroinflammation. We found that HSV-1 infection, but not IAV infection, in dcOrgs led to increased intracellular Aβ42 and phosphorylated Tau-Thr212 (pTau-212) expression, lower ratios of secreted Aβ42/40, as well as neuronal loss, and increased proportions of astrocytes and microglia, which are hallmarks of AD. Among the glia cell-type markers, Iba1 (microglia) and GFAP (astrocyte) expression were most strongly correlated with HSV-1 expression, which further supported that these biomarkers are perturbed by glia-mediated neuroinflammation. By performing large-scale RNA sequencing, we observed that differentially expressed transcripts in HSV-1 infected dcOrgs were specifically enriched for AD-associated GWAS genes, but not for genes associated with other common neurodegenerative, neuropsychiatric or autoimmune diseases. Immediate treatment of HSV-1 infected dcOrgs with anti-herpetic drug acyclovir (ACV) rescued most of the cellular and transcriptomic biomarkers in a dosage-dependent manner, indicating that it is possible to use our high-throughput platform to identify compounds or target genes that can reverse these neuroinflammation-induced biomarkers associated with AD.

DNA virus infection triggers an antiviral type I interferon (IFN) response in cells that suppresses infection of surrounding cells. Consequently, viruses have evolved mechanisms to inhibit the IFN response for efficient replication. The cellular cGAS protein binds to double-stranded DNA and synthesizes the small molecule cGAMP to initiate DNA-dependent type I IFN production. We showed previously that cGAMP production is relatively low during HSV-1 infection compared to plasmid DNA transfection. Therefore, we hypothesized that HSV-1 produces antagonists of the cGAS DNA sensing pathway. In this study, we found that the HSV-1 ICP8 protein is required for viral inhibition of the cGAS pathway by reducing cGAMP levels stimulated by double-stranded DNA transfection. ICP8 alone inhibited the cGAMP response and may inhibit cGAS action by direct interaction with DNA, cGAS, or other infected cell proteins. Our results reveal another cGAS antiviral pathway inhibitor and highlight the importance of countering IFN for efficient viral replication.

Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV.

Tumors in immune equilibrium are held in balance between outgrowth and destruction by the immune system. The equilibrium phase defines the duration of clinical remission and stable disease, and escape from equilibrium remains a major clinical problem. Using a non-replicating HSV-1 vector expressing interleukin-12 (d106S-IL12), we developed a mouse model of therapy-induced immune equilibrium, a phenomenon previously seen only in humans. This immune equilibrium was centrally reliant on interferon-γ (IFNγ). CD8+ T cell direct recognition of MHC class I, perforin/granzyme-mediated cytotoxicity, and extrinsic death receptor signaling such as Fas/FasL were all individually dispensable for equilibrium. IFNγ was critically important and played redundant roles in host and tumor cells such that IFNγ sensing in either compartment was sufficient for immune equilibrium. We propose that these redundant mechanisms of action are integrated by IFNγ to protect from oncogenic or chronic viral threats and establish IFNγ as a central node in therapy-induced immune equilibrium.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals.

Summary Cancer therapeutics can lead to immune equilibrium in which the immune response controls tumor cell expansion without fully eliminating the cancer. The factors involved in this equilibrium remain incompletely understood, especially those that would antagonize the anti-tumor immune response and lead to tumor outgrowth. We previously demonstrated that continuous treatment with a non-replicating herpes simplex virus 1 expressing interleukin (IL)-12 induces a state of cancer immune equilibrium highly dependent on interferon-γ. We profiled the IL-12 virotherapy-induced immune equilibrium in murine melanoma, identifying blockade of innate inflammatory cytokines, tumor necrosis factor alpha (TNFα), IL-1β, or IL-6 as possible synergistic interventions. Antibody depletions of each of these cytokines enhanced survival in mice treated with IL-12 virotherapy and helped to overcome equilibrium in some tumors. Single-cell RNA-sequencing demonstrated that blockade of inflammatory cytokines resulted in downregulation of overlapping inflammatory pathways in macrophages, shifting immune equilibrium towards tumor clearance, and raising the possibility that TNFα blockade could synergize with existing cancer immunotherapies.

Herpes simplex virus 1 (HSV-1) has been associated with pathogenesis of Alzheimer’s Disease (AD) through several landmark studies. We explored the transcriptomics landscape of HSV-1 infection and anti-viral treatment using acyclovir on AD-associated genes using large-scale bulk RNA sequencing to identify biomarkers, cell types and cell type specific interactions. We used recent technological advances in human 3D brain organoids and transcriptomics sequencing tools. We found that HSV-1 infection in cerebral organoids from a single individual resulted in transcriptomic perturbations were enriched for AD-associated genes. ACV treatment restored HSV-1 induced host transcript perturbations in cerebral organoids for most genes in a dosage-dependent manner, demonstrating that immediate anti-viral treatment of HSV-1 infection is effective in our human cellular model. However, we also observed off-target transcriptomic effects from ACV treatment that were enriched for AD-associated genes and genes associated with autoimmune diseases. HSV-1 infection in cerebral organoids also resulted in significantly decreased Abeta42/40 and Abeta42/38 ratios detected from the supernatant, compared to uninfected control cerebral organoids. HSV-1 infection followed by immediate ACV treatment rescued the Abeta42/40 ratios detected from the supernatant, but did not rescue the Abeta42/38 ratios. We found that influenza A (IAV) infection did not result in an enrichment of AD-associated transcriptomic perturbations and both Abeta42/40 and Abeta42/38 ratios were not significantly changed in the supernatant between IAV-infected cerebral organoids and uninfected cerebral organoids. We explored cell type specific effects of HSV-1 infection and ACV treatment in cerebral organoids through cell type enrichment analyses of the bulk RNA sequence data and flow cytometry experiments. We found complex cell type interactions perturbed by HSV-1 infection with varying degrees of rescue by ACV, such as increased proportions of reactive astrocytes and decreased proportions of live neurons. Finally, we correlated the transcriptomic signature of HSV-1 infected cerebral organoids with transcriptomes of post-mortem brain samples from LOAD patients and discovered a high correlation with subtype A (31% of patients). Our work is an unbiased study to identify biomarkers and novel therapeutic pathways, as well as potential off-target effects associated with HSV-1 infection and ACV treatment in AD.