Journal of Virology

GC-rich sequences are abundant in human herpesviruses genomes. GC-rich regions can form three-stranded RNA:DNA hybrid structures called R-loops. Though these hybrid structures serve important biological roles at telomeres or during cellular DNA synthesis, unscheduled or prolonged R-loop formation causes DNA damage and genome instability. For this reason, several mechanisms exist to resolve R-loops including endoribonucleases RNaseH1 (constitutively expressed) and RNaseH2A (cell cycle-regulated) which degrade the RNA portion of the R-loop. The Kaposi's sarcoma-associated herpesvirus (KSHV) origins of lytic replication (OriLyts) contain multiple cis-acting elements that are required for viral DNA replication including the production of GC-rich and repetitive transcripts, T1.4 (OriLyt-L) and kaposin (OriLyt-R). We previously showed that R-loops form at both OriLyts and that deleting kaposin repeats or decreasing their GC-rich content prevented R-loop formation at OriLyt-R, reduced genome amplification after primary infection and caused defects in latency establishment. To define the contribution that R-loops play in KSHV replication, we overexpressed RNaseH1, reasoning that excess RNaseH1 would resolve both OriLyt R-loops. However, RNaseH1 protein levels decreased following KSHV reactivation in both iSLK and BCBL-1 cell lines. Using co-transfection, we discovered that the KSHV viral replication and transcription activator protein, RTA, mediated RNaseH1 protein decreases in a E3 ligase domain-dependent manner without impacting levels of its cognate RNA transcript. We attempted to construct an RTA-resistant yet functional version of RNaseH1 by site-directed mutagenesis of lysine residues individually or in combination, yet these constructs remain susceptible to RTA-mediated protein decreases. An amino terminally tagged RNaseH1 displayed reduced susceptibility to RTA, suggesting that RTA may target the N-terminus of RNaseH1 for ubiquitination. However, overexpression of the cell-cycle regulated endonuclease, RNaseH2, exhibited RTA resistance, suggesting RNaseH2 may be a tool that will effectively resolve R-loops during KSHV infection. KSHV is not the only herpesvirus to encode a protein that reduces RNaseH1 levels, as co-expression of RTA homologs from the related gamma-herpesviruses EBV and MHV-68 likewise decreased steady-state levels of RNaseH1 protein. We propose that RTA-mediated RNaseH1 degradation is conserved strategy to ensure R-loop persistence during gamma-herpesvirus infection, underscoring the importance of these structures.

Mareks disease virus (MDV) UL47 tegument protein 5 (UL47_TEG5) is a major tegument protein essential for viral replication and horizontal transmission in chickens. The protein undergoes dynamic nucleocytoplasmic shuttling, likely regulated by host and viral factors, including post-translational modifications (PTMs). PTMs, in particular phosphorylation, on homologous herpesviral UL47_TEG5 proteins regulate nucleocytoplasmic shuttling through nuclear localization (NLS) and export (NES) motifs. Mass spectrometry-based proteomics of feather follicle epithelial skin from chickens infected with MDV recently showed that MDV UL47_TEG5 is phosphorylated. In silico analysis identified putative NLS and NES motifs near or within these phosphorylation sites. To test whether these motifs act as predicted, we systematically deleted the predicted NLS and NES motifs and assessed subcellular localization in transient transfection assays. Individual NLS deletions had minimal effect on nuclear localization, whereas NES deletions caused cytoplasmic accumulation and abolished nucleocytoplasmic shuttling. Next, we generated recombinant MDV lacking UL47_TEG5 (v{Delta}47) or the NES2 motif (aa 664-678) of UL47_TEG5 (v47{Delta}NES2) using two-step Red recombination. In experimentally infected chickens, replication and MD incidence were not different from those of wild-type MDV. However, in contact chickens, only wild-type MDV transmitted between birds, whereas both v{Delta}47 and v47{Delta}NES2 failed to transmit horizontally. These results demonstrate that nucleocytoplasmic shuttling of UL47_TEG5 is essential for horizontal transmission. In addition, our studies suggest that UL47_TEG5 expression, or lack thereof, in cells may be related to protein stability. Our findings link the nucleocytoplasmic shuttling of UL47_TEG5 to its essential function during natural infection and suggest this tegument protein may be a promising target for next-generation MDV vaccines.

Recombination can generate genetic diversity among RNA viruses; yet, mechanisms governing recombination for segmented, double-stranded RNA viruses remain poorly understood. Reovirus genomes contain ten segments of double-stranded RNA. Previous analyses suggested low rates of reovirus RNA recombination and led to a proposed mechanism of recombination during minus-strand synthesis. These analyses focused on recombination events detected in genomic double-stranded RNA extracted from purified viral particles. In the current study, we looked deeper into the origins of the observed recombination events by assaying total cellular material, which comprises viral genomic material, as well as replication intermediates, mRNAs, and products of recombination that may not be eligible for packaging into virus particles. Intracellular viral RNA, which is almost exclusively plus-strand RNA, exhibited substantially higher recombination frequency than particle-associated double-stranded RNA. The diversity and frequency of identified junctions were more similar among samples of a given type (intracellular or virion-encapsidated) from distinct infections than between distinct sample types from the same infection. Although potential recombination sites are widely distributed across the genome, a more limited set was used in junctions in packaged viral RNA than in intracellular viral RNA. Together, these observations suggest recombination may occur more frequently than previously recognized during reovirus infection, and many such events likely occur during plus-strand synthesis. However, recombination products generated during viral transcription may be selectively incorporated into viral particles.

Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in transplant patients and is a major cause of congenital disease. Understanding the role of viral genes during infection, viral replication, and viral latency establishment and reactivation is key to the development of new antivirals, which are currently limited. The viral RL11 is a conserved, but understudied viral locus, with previously described roles in specific phases of the viral lifecycle. Antisense to and within the RL11 region are two unrelated genes, including UL2. In this study, we investigate the role of UL2 during viral infection and find that UL2 is not required for lytic replication nor required for viral infection, latency establishment (or persistence) and reactivation in either THP-1 monocytes or primary human hematopoietic progenitor cells (HPCs). As viral genes are evolutionarily optimized for viral fitness, this study suggests that HCMV UL2 has a role outside of viral fitness and may be a unique target or contributor to HCMV-mediated processes including immune response or other viral co-factor regulation.

Viral infection, including for the prototypical betaherpesvirus human cytomegalovirus (HCMV) is typically studied in two-dimensional monocultures, which provide experimentally tractable systems for measuring viral replication but do not reproduce the biologically accurate multicellular organization or interactions, nor the three-dimensional (3D) architecture of stratified epithelial tissues. Here, we evaluated a biofabricated 3D skin model containing fibroblasts and keratinocytes as a system to study HCMV infection. The model is generated using a fibrin-based matrix and spatially organization deposition of keratinocytes in combination with fibroblasts, followed by cellular differentiation controlled by calcium conditions. Cultures were infected with a GFP-expressing clinical strain of HCMV (TB40/E-GFP) and compared to traditional monolayer cultures or single cell type 3D cultures. HCMV infection was detectable by GFP expression in an MOI-dependent and longitudinal manner. Infectious virus was recovered from both the cellular associated and released into the extracellular space of the model, demonstrating that the model supports productive viral infection. Transit of infectious virus is reduced in both multicellular and single type 3D cultures, suggesting that the matrix composition influences viral kinetics. Treatment with the antiviral Ganciclovir suppressed virus production in both fibroblast monolayer cultures and 3D skin cultures. These findings establish a tractable, longitudinal observable, multicellular 3D system that supports productive HCMV infection and provides a reliable platform for investigating viral replication in a spatially organized tissue context.

The live, attenuated vaccine for Varicella-Zoster Virus (VZV) protects against chickenpox in children and adults. However, the vaccine strain Oka (vOka) establishes latency and may reactivate. The molecular basis of vOka attenuation remains unknown. We investigated whether three high-frequency single-nucleotide polymorphisms (SNPs) that differentiate vOka from parental strain Oka (pOka) are responsible: S628G and R958G in the immediate-early regulatory protein IE62, and *130R in the membrane protein ORF0. We evaluated how these fixed SNPs mediate the attenuated growth phenotype of VZV and diminish skin pathology by creating recombinant pOka VZV carrying these SNPs in the wild-type background. These were assessed in cells and human skin organ cultures (SOCs). In epithelial and fibroblast cell lines, the individual IE62 mutations slightly delayed viral growth at 8 - 24 hours post-infection but not in human epidermal keratinocytes (htert-HEK). The fixed ORF62 SNPs also affected ORF62 transcription, IE62 protein abundance at early times, and delayed IE62 accumulation in the cytoplasm later during infection. The *130R mutation in ORF0 conferred a growth advantage in culture, suggesting it is an adaptation to cell culture. While there was donor-to-donor variability and differences in overall virus spread in SOC, the R958G and *130R conferred an attenuated growth phenotype similar to that of vOka in SOC. Histopathologic analysis of VZV-infected skin sections revealed that, similar to vOka, S628G and R958G in IE62 limited the spread of VZV skin lesions. Our findings underscore the critical roles of conserved vOka SNPs in understanding the attenuation of the live vaccine strain vOka.

Avian influenza A virus (IAV) is a major global health threat. Not only does it cause significant economic losses to the agricultural industry, but it also presents a risk of spillover into the human population. However, several obstacles must be overcome for a successful cross-species transmission event to occur. One of these obstacles is the innate immunity of the host. While the innate immune response to IAV in humans has been well-studied, the innate immune response to IAV in birds is not as well understood. Here, we explore the interferon-induced immune response by identifying interferon-stimulated genes (ISGs) in chicken, quail, and duck cells, and find that they are largely species-specific. Using this data, we performed a CRISPR knockout ISG screen against two strains of IAV, the mouse-adapted PR8, and the low pathogenicity avian IAV isolate WF10. We find that for PR8, IFITM3 is the dominant restrictor of viral replication with several additional hits with modest effects, consistent with previous work in mammals. For avian WF10, IFITM3 was also a hit however there were two additional hits absent for PR8, the HSP70 co-chaperone protein BAG5 and deubiquitinase OTUD4. Overall, these findings confirm the divergence of interferon-mediated immunity between species and establish a screening system that can be used to identify ISGs important for viral restriction in avian species. IMPORTANCEWild birds, especially waterfowl and shore birds, are the primary natural reservoir for influenza A viruses. These birds can transmit IAV to domestic poultry, where the spillover risk to humans increases. Chickens are the most common species of domestic poultry world-wide and are frequently infected with avian influenza viruses. Despite this, relatively little is known about how their immune system responds to influenza infection. This lack of knowledge creates a barrier to finding ways of preventing or mitigating IAV infection in chicken flocks. In this study, we characterized the interferon-induced immune response to IAV in chickens and found that only a small subset of ISGs were responsible for restricting IAV in chicken cells.

The human MxA (myxovirus resistance protein A) host restriction factor inhibits orthomyxoviruses, such as Thogotovirus (THOV) and influenza A virus (IAV), by binding to their nucleoproteins. Despite being discovered over six decades ago, how MxA interacts with viral targets remains unclear. Earlier studies using evolutionary analysis and mutagenesis showed that the MxA L4 loop, especially a hydrophobic aromatic amino acid at residue 561, is crucial for binding THOV nucleoprotein (NP) and restricting THOV. Here, we combined previous insights with structure prediction methods, molecular dynamics simulations, and experimental validation to define the human MxA L4 loop binding interface to THOV NP. We also evaluated the stability of MxA L4-NP binding through classical all-atom molecular dynamics simulations. Our model revealed MxA L4 binding to a surface-exposed site on THOV NP, including residues previously linked to viral escape from MxA restriction, even though this information was not used to guide our modeling efforts. This MxA-THOV NP interface is distinct from NPs RNA-binding or oligomerization surfaces. Our molecular dynamics simulations also agree with earlier data indicating that F561Y enhances MxA binding to THOV NP, whereas F561W reduces it and F561V ablates it entirely. Based on this model, we predicted specific variants in human MxA or THOV NP that could result in increased host restriction or viral escape. We tested these predictions using a viral minireplicon assay to validate our model. Our efforts will guide vital viral surveillance studies and the development of MxA-based antivirals. (240) Significance StatementThe interferon-stimulated MxA protein encodes a critical barrier to zoonotic spillover of orthomyxoviruses, restricting infection by binding viral nucleoprotein (NP). Despite being among the best-studied interferon-stimulated genes (ISGs), the specific host-viral interaction surface involved in binding or restriction is poorly understood. We address this hurdle using structural modeling and molecular dynamics simulations to generate a model of the biochemical interactions between MxA and Thogotovirus NP. Our model is consistent with previous work identifying mutations that strengthen or weaken the binding interaction. We validate this model using in vitro analysis of predicted mutations at the host-virus interface. Our model provides a framework for additional mutational studies, surveillance of viruses poised for zoonosis, and rational antiviral design. (115)

To date, twenty-seven pathogenic human viruses require host-catalyzed de novo fatty acid biosynthesis for replication. This pathway is driven by fatty acid synthase (FASN), which produces palmitate. Palmitate is a precursor for various functions during viral infection, including lipid droplet formation for assembly, beta-oxidation for ATP generation, and post-translational modification of proteins. Whether Mayaro virus (MAYV), an emerging alphavirus that causes debilitating arthritogenic disease, required FASN for infection was unknown. Using genetic and pharmacological approaches in a human cell line and primary cell model, we found that MAYV requires FASN-dependent palmitate synthesis for virion production. To determine how palmitate contributes to infection, we pharmacologically inhibited pathways downstream of FASN and found that only 2-bromopalmitate (2-BP), a protein palmitoylation inhibitor, led to a 94% reduction in MAYV infection. S-palmitoylation is a post-translational modification in which palmitate is attached to sulfur atoms in cysteine residues. In chikungunya virus, a related alphavirus, FASN-dependent palmitoylation of nonstructural protein 1 (nsP1) is essential for membrane association and replication. Consequently, we hypothesized that MAYV nsP1 is palmitoylated in a FASN-dependent manner. Using an alkyne acetate analog, Alk-4, metabolized by FASN into alkyne palmitate, we observed specific labeling of wild-type nsP1 at conserved cysteine residues (C417-419), but not of a cysteine-to-alanine triple mutant. Treatment with TVB-2640 or 2-BP abrogated Alk-4 labeling of wild-type nsP1 during active infection, reinforcing that MAYV protein palmitoylation is a FASN-dependent process. Our findings reveal a conserved mechanism of FASN-dependent protein palmitoylation in alphaviruses and highlight FASN as a potential anti-viral target. ImportanceMayaro virus (MAYV) is a neglected, mosquito-borne tropical virus that causes debilitating pathologies, such as chronic joint pain that can last from months to years. Currently, MAYV transmissions are endemic in sylvatic and peri-urban regions in Central and South America and the Caribbean. However, MAYV has been detected in urban-adapted mosquitos like Aedes aegypti (Ae. aegypti) and is a concern for potential global spread. Consequently, investigating the mechanisms of MAYV infection is critical to uncover opportunities for antiviral drug development. In this study, we report that MAYV infection requires host fatty acid synthase (FASN) derived palmitate for palmitoylation of the viral non-structural protein 1 (nsP1). In addition, we report that inhibiting FASN with the clinically advanced small molecule TVB-2640 significantly reduced MAYV infection and nsP1 palmitoylation. This study highlights FASN as an essential host factor for MAYV replication and establishes it as a promising therapeutic target for MAYV and related alphaviruses.

Herpes simplex virus-1 (HSV-1) has infected more than 67% of the world population causing diseases that range in severity from benign cold sores to encephalitis. To infect and persist in the host, HSV-1 has evolved sophisticated strategies to counteract antiviral responses. The viral immediate early protein ICP0 (Infected Cells Protein 0) plays a fundamental role in this process. ICP0 is an E3 ubiquitin ligase that is required for successful onset of lytic infection and productive reactivation of viral genomes from latency. Most ICP0 studies have been focused on its nuclear functions, but late gene expression drives accumulation of ICP0 to the cytoplasm where its functions are unknown. We recently demonstrated that cytoplasmic ICP0 localizes on the surface of endosomes through its interaction with CIN85 (Cbl-interacting protein of 85 kDa), a scaffolding protein for factors involved in endocytosis. This interaction is required for extrusion of host antiviral factors and immunoevasion. Here, we report that the E3 ubiquitin ligase activity of ICP0 is also required for these effects, as disruption of ICP0 RING finger domain, which disrupts its catalytic activity, resulted in an increase in the number of CIN85 endosomes that cannot be exocytosed. These endosomes colocalize with autophagosome components, but they do not enter the lysosomal pathway for degradation. Mutant viruses that cannot exocytose CIN85 endosomes produce EVs that are more potent at activating antiviral responses.

RNA polymerase III (Pol III) activity is upregulated during herpesvirus infection, yet the functional consequences of this response remain poorly understood. To investigate the role of host Pol III transcription during murine gammaherpesvirus 68 (MHV68) infection, we depleted the Pol III transcription factor Brf1, an essential component of the TFIIIB complex required for transcription from Type I and Type II Pol III promoters. Brf1 depletion enhanced MHV68 replication during low multiplicity of infection (MOI) conditions, resulting in increased viral gene expression, viral protein accumulation, infectious virion production, and extracellular viral genome copies. These effects were confirmed to be Brf1-specific through rescue with an siRNA-resistant Brf1 construct. In contrast, Brf1 depletion produced only modest effects during high-MOI infection, suggesting that Brf1-dependent antiviral activity is most important during multistep viral spread. Transcriptomic analysis revealed accelerated induction of interferon-responsive genes early during infection in Brf1-deficient cells, followed by enhanced host transcript depletion at later stages, consistent with amplified host shutoff. Genetic disruption of the RIG-I/MAVS signaling pathway failed to abolish Brf1 antiviral activity, indicating that this phenotype is independent of MAVS-dependent interferon signaling. Finally, plaque assays demonstrated that Brf1-deficient cells supported larger plaques and increased plaque numbers, suggesting enhanced viral spread and/or entry. Together, our results reveal an unexpected role for Brf1-dependent Pol III activity in controlling gammaherpesvirus spread and suggest that virus-induced Pol III activation contributes to host antiviral defense.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus. Common marmosets, a potential non-human primate model for vaccine development and infection studies, have previously been shown to support KSHV infection. We established infection of marmosets with BAC-derived, recombinant KSHV to analyze the role of individual glycoproteins (GPs) in tissue tropism. Three groups of four animals were infected intravenously with BAC-derived KSHV wild type (wt), EphA2-receptor-binding-deficient KSHV gHASAELAAN, or KSHV{triangleup}K8.1, lacking glycoprotein K8.1. Viral DNA was detected in multiple tissues of all animals, with the highest loads in spleen, liver, heart, and bone marrow. In most tissues, viral DNA loads did not significantly differ between groups. However, KSHV{triangleup}K8.1-infected animals exhibited significantly lower KSHV DNA levels in liver and bone marrow. KSHV wt infection induced activation of CD4+ and CD8+ T-cells. In animals infected with KSHV wt, KSHV gHASAELAAN or KSHV{triangleup}K8.1, different B cell subpopulations expanded after infection, which suggests that the glycoproteins themselves or the cell tropism imparted by their presence or absence might shape the host response to KSHV. In summary, our findings demonstrate that neither the Eph receptor interaction nor GP K8.1 is essential for intravenous infection, but K8.1 critically contributes to KSHV tissue tropism in vivo.

Respiratory syncytial virus (RSV) is an enveloped virus that assembles and buds at the plasma membrane, producing filamentous infectious particles. Unlike many other enveloped viruses, a substantial fraction of RSV virions remains associated with the surface of infected cells, limiting our understanding of the membrane-scission mechanisms responsible for particle release. Here, we identified the actin-binding protein drebrin as a cellular interactor of the RSV matrix (M) protein, both in vitro and in infected cells. We demonstrated that RSV infection induces the relocalization of drebrin to actin-rich structures at the cell periphery associated with viral filaments. Using a Transwell-based assay to quantify infectious particles release without mechanically perturbing cells, we confirmed that RSV release is independent of the endosomal sorting complex required for transport (ESCRT) pathway and showed that drebrin depletion reduced RSV release, whereas drebrin overexpression enhanced it. Since drebrin depletion did not significantly affect overall infectious virus production, our results suggest that drebrin may act during the final step leading to membrane scission and RSV particle release. Finally, proteomics analysis identified Transmembrane 9 superfamily member 2 (TM9SF2) as a drebrin-associated protein specifically during infection. TM9SF2 depletion reduced RSV particle release and impaired the RSV-induced relocalization of drebrin to the plasma membrane. Together, these findings identify drebrin and TM9SF2 as host factors regulating RSV release and support a model in which TM9SF2 promotes the recruitment of drebrin to viral budding sites.

Interferon gamma (IFN-{gamma}) is a potent cytokine secreted following primary varicella-zoster virus (VZV) infection. Our previous studies demonstrated that pretreatment with IFN-{gamma} completely inhibited VZV replication in lung fibroblast MRC-5 and retinal epithelial ARPE-19 but not in melanoma MeWo cells, suggesting that IFN-{gamma}-stimulated protein(s) inhibit viral replication. Our microarray analysis revealed that 34 interferon-stimulated genes (ISGs) were upregulated by greater than 2.5-fold at 8 h post-treatment in both ARPE-19 and MRC-5 cells compared to those of MeWo cells. The depletion of CTSS, GBP4, MX1, IFIT1, IFITM1 and IRF1 by siRNA in IFN-{gamma}-treated cells significantly increased VZV yields. Ectopic overexpression of interferon-induced myxovirus resistance 1 (MX1) or transmembrane protein 1 (IFITM1) significantly reduced VZV yield both in ARPE-19 and MeWo cells. IFITM1 abrogated viral major immediate-early IE62 protein-mediated trans-activation. IFITM1 reduced the level of VZV immediate-early IE62 protein, but not the IE62 mRNA, suggesting that IFITM1 expression reduces the expression level of IE62 by post-transcriptional regulation. Cell-free VZV failed to infect human primary epidermal keratinocytes, in which the basal level of endogenous MX1 gene expression is very high. Pharmacologic inhibition of JAK signaling reduced MX1 expression and enhanced VZV replication in calcium chloride-induced differentiated keratinocytes. Collectively, these findings identify MX1 and IFITM1 as key IFN-{gamma}-induced restriction factors that inhibit VZV replication and suggest that suppression of JAK-STAT signaling can promote VZV replication in differentiated keratinocytes.

Newborns and infants are particularly vulnerable to influenza, largely due to the immaturity of their immune systems. As such, maternal immunization is a valuable strategy to enhance influenza-neutralizing antibodies in both blood and breast milk, ensuring optimal passive immunity transfer to the infant. In our pregnant and lactating ferret maternal-neonatal model, we evaluated antibody responses to an adenovirus type 5-vectored H1N1 hemagglutinin vaccine delivered enterically, intranasally, or intramuscularly. While all three routes elicited a robust antibody response compared to saline-treated controls, intranasal vaccination significantly increased influenza-specific antibodies in dam serum, upper respiratory tract secretions, and milk, and subsequent transfer to suckling kits. Our results suggest that intranasal immunization was the most effective route for inducing functional influenza-specific antibodies in both dam serum and milk, with efficient passive transfer to suckling kits. Leveraging mucosal immune induction may enhance maternal antibody responses, transfer to offspring, and downstream neonatal protection.

Infectious bronchitis virus (IBV) is an economically important avian gammacoronavirus. IBV uses sialic acids and heparan sulfate for cellular attachment, but the host proteins governing its infection remain poorly defined. Here, we investigated the roles of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) during IBV Beaudette (IBV BD) infection. ACE2 knockout impaired IBV replication in DF-1 cells, whereas ACE2 reconstitution restored infection. Furthermore, ACE2 overexpression enhanced infection of DF-1 and primary chicken embryo kidney cells, supporting the role of chicken ACE2 as a proviral host factor. In contrast, TMPRSS2 exerted a restrictive function on IBV BD infection as demonstrated in TMPRSS2 KO DF-1 cells and upon pharmacological inhibition, which significantly increased IBV BD infection in DF-1 and Vero cells. RNA sequencing revealed significant changes in lipid metabolism and membrane-related processes following TMPRSS2 knockout and pharmacological inhibition. These findings highlight the opposing roles of ACE2 and TMPRSS2 in IBV BD infection, emphasizing the combined impact of proviral host factors, protease activity, and the cellular membrane environment during gammacoronavirus infections.

Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) nonstructural protein 1 (Nsp1) dampens the host immune response by shutting off host gene expression, a strategy that has remained evolutionary conserved among a diverse range of coronaviruses (CoVs). Residues important for SARS-CoV-2 Nsp1 mediated host shutoff have been incompletely defined. We have generated and characterized the ability of four Nsp1 mutants to inhibit host gene expression in both plasmid-based overexpression assays and utilized reverse genetic approaches to generate recombinant (r)SARS-CoV-2 expressing each Nsp1 mutant to investigate their impact on viral infection. Infection of K18-hACE2 transgenic mice with the rSARS-CoV-2 Nsp1 mutants resulted in reduced pathogenicity as determined by body weight maintenance and survival, attenuated viral replication in lung and nasal turbinate, distinct immune signatures in lung, and less severe lung pathology in comparison to wild-type (WT) virus-infected mice. Our data suggests amino acid residues in the C-terminal domain, in addition to the linker domain, of SARS-CoV-2 Nsp1 are critical determinants of viral pathogenicity as a result of their role in disrupting host gene expression. The reduced pathogenicity of rSARS-CoV-2 Nsp1 mutants highlights the potential for targeting Nsp1 for rational design of viral inhibitors and development of live-attenuated vaccine strategies as effective prophylactic and therapeutic treatments, respectively, to combat SARS-CoV-2 and possibly other CoV infections. IMPORTANCETo mitigate the ongoing public health threat posed by Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) and prepare for future coronavirus (CoV) outbreaks, there is an urgent need for effective prophylactic and therapeutic strategies, including vaccines and antivirals. The nonstructural protein 1 (Nsp1) is a conserved CoV virulence factor that suppresses host gene expression and disrupts immune responses. However, the contribution of specific Nsp1 residues to CoV pathogenesis remains unclear. Here, we identify residues within the C-terminal and linker regions of Nsp1 as critical determinants of SARS-CoV-2 pathogenicity in vivo. These findings advance our understanding of CoV host shutoff mechanisms and support Nsp1 as a promising target for the development of live-attenuated vaccines and antiviral therapeutics.

Human gammaherpesviruses (gHVs) including Epstein-Barr Virus and Kaposis sarcoma associated herpesvirus are linked to cancer and inflammatory disease development. The outcome of gHV infection is highly regulated by cell type, with macrophages identified as an important early infection target that can also serve as a latency reservoir. Here, we used the mouse gHV model, murine gammaherpesvirus (MHV68), to identify a dose-dependent outcome of MHV68 infection of macrophages, with high dose infection resulting in extensive morphological changes, viral gene expression, and a pronounced inflammatory cell death not observed under conditions of lytic replication occurring at lower infectious dose. Virion envelope and tegument components were not sufficient for this dose-dependent cell death, which required intact viral DNA and virus transcription, yet occurred independently of viral DNA replication and late gene transcription. Necrostatin-1 (Nec-1) treatment, an anti-inflammatory therapeutic, limited cell death and reduced cell morphology alterations, with no impact on virus replication, uncoupling cell death from lytic replication. Activation of autophagy profoundly limited high dose infection outcomes, with increased cell survival and reduced viral gene expression. Despite the improved cell survival observed in cells treated with Nec-1, biochemical analysis of infected cultures identified robust apoptosis induction with subsequent activation of gasdermin E, suggesting a multi-modal cell death mechanism culminating in pyroptosis. Our findings suggest that high dose macrophage infection byMHV68 is a major driver of inflammatory cell death to potentially shape downstream inflammation and immune responses.

Epstein-Barr virus (EBV) establishes distinct latency programs that drive B-cell transformation through coordinated viral and host gene regulation. However, the contribution of host long noncoding RNAs (lncRNAs) to EBV-mediated pathogenesis remains poorly understood. Here, we performed transcriptome-wide lncRNA profiling comparing Type I and Type III EBV latency and identified differentially expressed host lncRNAs, among which we characterized a previously unannotated natural antisense transcript (NAT) of IRF4, designated IRF4-AS1. IRF4-AS1 is markedly upregulated in Type III latency and positively correlates with IRF4 expression across EBV-transformed lymphoblastoid cell lines (LCLs) established from diverse clinical backgrounds. Functional studies indicate a context-dependent positive regulatory relationship between IRF4-AS1 and IRF4. While overexpression of IRF4-AS1 increased IRF4 expression and IRF4 overexpression increased IRF4-AS1 levels, loss-of-function analyses suggest that this relationship is modulated by additional regulatory inputs in established transformed cells. In contrast to IRF4, IRF4-AS1 transcription is not regulated by the LMP1- NF{kappa}B signaling, indicating that the two transcripts are regulated through partially distinct mechanisms. In addition, we identified MIR3142HG as a highly induced latency-associated lncRNA and found that its embedded miRNAs, miR-146a and miR-3142, exhibited divergent expression patterns, suggesting differential post-transcriptional regulation. Together, these findings identify IRF4-AS1 as a NAT lncRNA for IRF4 and reveal extensive remodeling of host lncRNA networks during EBV latency, providing a foundation for future studies of lncRNA- mediated host-virus interactions in EBV pathogenesis. ImportanceEBV drives B-cell transformation through extensive reprogramming of host transcriptional networks, yet the contribution of host long noncoding RNAs (lncRNAs) to this process remains poorly defined. Transcriptome-wide lncRNA profiling, together with functional analysis, identified IRF4-AS1 as a previously unannotated natural antisense transcript of IRF4. IRF4- AS1 is upregulated in Type III latency and positively correlates with IRF4 expression across EBV-transformed cells. Together, this study provides a foundation for future mechanistic studies of lncRNA-mediated host-virus interactions in EBV pathogenesis.

The Gamak virus (GAKV) is a recently identified shrew-borne paramyxovirus belonging to the genus Parahenipavirus, which also includes the zoonotic Langya virus (LayV). Despite the growing recognition of shrew-associated paramyxoviruses, the host pathways that detect infection and regulate antiviral responses remain poorly understood. In this study, we characterized host responses to GAKV infection using integrated in vitro and in vivo approaches. GAKV infection induced robust innate immune responses in A549 cells, characterized by activation of interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 1 (STAT1), together with induction of type I interferon (IFN) and interferon-stimulated genes (ISGs). Transcriptomic analysis further revealed coordinated enrichment of antiviral and intrinsic apoptosis-associated pathways, suggesting a link between innate immune signaling and apoptosis during GAKV infection. Genetic analyses identified retinoic acid-inducible gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS) as essential mediators of antiviral signaling and apoptosis during GAKV infection. Furthermore, disruption of type I IFN-STAT1 signaling attenuated apoptosis. NOXA knockdown reduced apoptosis and enhanced viral replication, identifying NOXA as a downstream effector linking innate immune activation to apoptosis. Consistent with these in vitro findings, intranasal GAKV infection in six-week-old female wild-type BALB/c mice was associated with lung-restricted viral RNA detection and induction of antiviral responses without overt disease. Together, these findings identify a RIG-I-MAVS-IFN-NOXA signaling axis that integrates antiviral and apoptotic responses during GAKV infection, providing a mechanistic framework for understanding host defense against parahenipaviruses.