Journal of Virology

The Hippo pathway is conserved across eukaryotes and controls key biological processes, including cell growth and organ development. Notably, humans with biallelic loss-of-function mutations in the Hippo kinase gene MST1 suffer from combined immunodeficiency, including recurrent infections of herpes simplex virus (HSV), implicating the pathway in host immune regulation. We investigated the role of MST1 and its homolog MST2 in HSV-1 infection. We found that human epithelial cells HEp-2 proteolytically converted full-length MST1/2 into smaller N-terminal fragments (MST1/2-NT) to enhance cell apoptosis in response to the HSV-1 infection. Moreover, while infection by mutants lacking ICP0 or US3 elevated the production of MST1/2-NT and apoptosis, the overexpression of MST1-NT significantly reduced HSV-1 replication, revealing anti-viral properties of MST1/2 cleavage and the viral counteractions by ICP0 and US3. Consistently, we discovered that MST1/2-NT production, which was high in {Delta}ICP0-infected HEp-2 cells, was completely diminished in cells permissive to the {Delta}ICP0 infection, linking the counteraction against host MST1/2 cleavage to ICP0 functions. In addition, host caspases cleaved MST1/2 with differential preferences toward MST1 or MST2 in different infection contexts, indicating multiple regulations on the MST1/2-NT production during HSV-1 infection. While double-knockouts of MST1/2 in HEp-2 cells had marginal effects on wild type HSV-1 replication, it substantially reduced the early intake of {Delta}ICP0 DNA, suggesting a role of full-length MST1/2 in early infection. Altogether, these results uncover the novel and distinct roles of full-length and cleaved Hippo kinases during HSV-1 infection and their multifaceted interactions with ICP0. ImportanceHSV poses serious threats to human health, ranging from cold sores to fatal brain infection. As a successful human pathogen, it deploys various viral proteins to counteract host defenses and subjugate host machinery, but mechanisms underlying these complex HSV-host interactions are not fully understood. For the first time, we report multifaceted interactions between the Hippo kinases and viral proteins during HSV-1 infection. We show that the Hippo kinases are converted to smaller fragments through protein cleavage in HSV-1 infected cells, and the cleaved Hippo fragments are accompanied by host cell death to execute their anti-viral activities. Moreover, multiple viral proteins contribute to counteracting this host defense, including ICP0, which executes a complex interplay with the Hippo kinases to promote infection. Understanding this new layer of virus-host interaction may pave the road to developing novel treatments for herpetic diseases.

Orthologs of the herpes simplex virus (HSV) pUL16 tegument protein are conserved throughout the Orthoherpesviridae family. During HSV infection, pUL16 functions in the nuclear egress of nascent nucleocapsids from the nucleus to the cytoplasm, prevents the docking of nascent cytoplasmic nucleocapsids to nuclear pore complexes, promotes the final envelopment of cytoplasmic nucleocapsids, and enhances cell-to-cell spread of virus infection. How pUL16 performs these diverse functions is poorly understood. To gain further insight into the mechanisms by which pUL16 mediates its activities, we utilized a BioID approach to identify cellular and viral proteins in proximity to pUL16 during the infection of human keratinocytes. By comparing proteins in proximity to pUL16 during infection with proteins in proximity to its well-known virus-encoded binding partner, pUL21, we provide new insight into the activities of pUL16 that likely occur in complex with pUL21 and those that are independent of pUL21. A key function of pUL21 is to deliver protein phosphatase 1 (PP1) to viral and cellular substrates to mediate their dephosphorylation. Intriguingly, the findings presented suggest that pUL16 interactions with pUL21 may regulate the isoform of PP1 that is bound to pUL21 and thereby regulate the specificity of substrate dephosphorylation. ImportanceHSV-1 and HSV-2 are important human pathogens that currently infect roughly 3.8 billion and 520 million people, respectively. These viruses cause lifelong, recurrent, infections and cause a variety of diseases including vesicular lesions of the oral and genital mucosa, corneal blindness, meningitis, encephalitis and devastating neonatal infections. The HSV pUL16 tegument protein performs a number of critical functions for the virus that influence virion assembly and the spread of infection between cells. In this study we have identified cellular and viral proteins that are proximal to pUL16 during infection of human keratinocytes, providing new insight into the mechanisms used by pUL16 to perform its activities.

Zika virus (ZIKV) is an arthropod-borne flavivirus of international public health impact. ZIKV has a positive-sense, single-stranded RNA genome and remodels intracellular membranes to form replication complexes (RCs). The objective of this study was to isolate and characterize the RCs from ZIKV-infected cells and to identify host-cell components recruited to participate in viral replication. Here, we isolated the RCs from ZIKV-infected Vero cells by detergent treatment and flotation centrifugation. Fractional flotation analysis demonstrated that ZIKV proteins NS2B, NS3, and NS5, and ZIKV RNA were present in the detergent-resistant membranous fraction. In contrast, the ER-resident protein calnexin and a mitochondrial protein were present in the detergent-soluble fractions. The isolated RCs were functional for ZIKV RNA synthesis, as shown by quantitative PCR. To determine the components of the RCs, we conducted mass spectrometry analysis and identified numerous cellular proteins. Among them is the replication factor C subunit 2 (RFC2), an accessory protein of DNA polymerase. RFC2 is involved in ATP binding and hydrolysis and may promote cell survival. ZIKV infection increased the RFC2 protein level and induced its relocation to the cytoplasm. RNAi-mediated silencing of RFC2 reduced ZIKV replication. Together, our results provide insights into ZIKV replication and virus-cell interaction.

Capsid stability is vital for virion survival as the capsid must withstand varying environmental challenges such as pH and temperature to allow the virus to reach a target cell. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of importance to human health globally, with no approved vaccine or antiviral available. Despite this, the molecular mechanisms behind norovirus capsid stability and capsid rearrangement prior to RNA translocation are understudied. Using murine norovirus as a model, we utilised thermal stress to create a thermally stable virus population. By introducing three identified substitutions in the major capsid protein VP1 from this virus population into an infectious clone, we were able to create a heat and pH stable virus that had delayed viral uncoating during the infectious lifecycle. Cryo-EM reconstructions of the triple substitution virus demonstrated that enhanced inter-chain hydrogen bonding was vital for increased capsid stability. Finally, mutagenesis to remove the enhanced inter-chain hydrogen bonding reverted capsid stability back to wild-type levels. This work contributes to fundamental calicivirus biology by demonstrating areas of importance in capsid stability down to amino acid resolution. Furthermore, this work could inform vaccine design for a thermostable norovirus vaccine in the future.

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.

Interferon-induced transmembrane (IFITM) proteins are broad-spectrum antiviral restriction factors that inhibit viral entry of diverse enveloped viruses. Comparative genomic studies have revealed extensive lineage-specific diversification of IFITM genes, yet the functional consequences of this diversification remain poorly understood. We previously identified an expanded IFITM repertoire in macaques consisting of the canonical IFITM proteins, IFITM1 and IFITM3, a duplicated IFITM3 paralog (IFITM3A), and two retrotransposed IFITM3-derived genes (IFITM3-R1 and IFITM3-R2). Here, we thoroughly characterized the antiviral activities, intracellular localization, and mechanisms of regulation of canonical and non-canonical macaque IFITMs against vesicular stomatitis virus (VSV), influenza A virus (IAV), Sendai virus, and HIV-1. IFITM3A exhibited enhanced antiviral activity relative to IFITM3, particularly against VSV and HIV-1. Comparative mutational analyses identified amino acid substitutions in IFITM3 that contribute to the enhanced antiviral phenotype of IFITM3A. In contrast, the retrocopy IFITM3-R1 exhibited markedly reduced expression due to lysosome-dependent protein turnover mediated by a PPxY motif and a unique lysine residue (K51). Alteration of these determinants increased IFITM3-R1 expression and selectively enhanced restriction of VSV and IAV. Although several macaque IFITMs reduced HIV-1 infectivity when expressed in producer cells, none significantly inhibited HIV-1 infection in target cells. Furthermore, differential incorporation of IFITMs into HIV-1 virions did not consistently correlate with antiviral activity, indicating that virion incorporation alone is insufficient to explain HIV-1 restriction. Together, these findings demonstrate that gene duplication and retrotransposition have generated a functionally diverse IFITM repertoire in macaques and provide insight into how evolutionary diversification expands innate antiviral defenses in primates. ImportanceIFITM proteins are broad-spectrum antiviral restriction factors that inhibit infection of numerous enveloped viruses. Although IFITM genes have undergone extensive diversification during mammalian evolution, the functional consequences of this diversification remain poorly understood. Here, we show that expansion of the macaque IFITM locus through gene duplication and retrotransposition generated proteins with distinct antiviral activities, intracellular localization patterns, and regulatory mechanisms. We identify amino acid determinants that contribute to the enhanced antiviral activity of the duplicated paralog IFITM3A and demonstrate that lysosome-dependent turnover mediated by a PPxY motif and a unique lysine residue limits expression and antiviral activity of the retrocopy IFITM3-R1. We further show that macaque IFITMs inhibit HIV-1 predominantly through producer-cell-dependent mechanisms and reveal that IFITM virion incorporation alone does not predict antiviral potency. These findings provide mechanistic insight into how restriction factor diversification expands innate antiviral defenses and shapes host-virus interactions in primates.

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.

Mammalian hantaviruses are RNA viruses that cause hantavirus cardiopulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome in Eurasia. The cellular entry mechanisms of most hantaviruses remain poorly defined. To examine entry by phylogenetically distinct hantaviruses, we generated replication-competent recombinant vesicular stomatitis viruses (rVSVs) bearing Gn/Gc proteins from Necocli, Sangassou, Thottapalayam, Kenkeme, Nova, Oxbow, and Tula viruses. All these Gn/Gc proteins except Kenkeme supported infection of primary human endothelial cells, indicating that endothelial cell entry is permissive for a broader range of hantaviruses than previously appreciated. Except for rVSV-Kenkeme, these rVSVs did not acquire additional mutations beyond pre-engineered rescue-enhancing changes during rescue and passaging. Genetic studies in human cells lacking protocadherin-1 (PCDH1) showed that Necocli, Tula, and Nova viruses use PCDH1 for efficient infection, although the Nova phenotype was weaker. These three Gn/Gc proteins bound soluble PCDH1 with different apparent avidities, and infection by the corresponding rVSVs was inhibited by soluble PCDH1; Necocli and Tula, but not Nova, were also blocked by a PCDH1-targeting monoclonal antibody. Authentic Tula virus infection was similarly reduced in PCDH1 knockout endothelial cells. Finally, the broadly reactive anti-Gn/Gc human monoclonal antibody ADI-42898 efficiently neutralized Necocli, Nova, Sangassou, and Kenkeme rVSVs but showed weak or undetectable activity against Oxbow, Tula, and Thottapalayam rVSVs. Together, these findings expand the range of hantavirus glycoproteins capable of mediating infection of human endothelial cells, broaden the phylogenetic scope of PCDH1-dependent entry, and identify receptor-targeted and viral glycoprotein-targeted strategies with differential activity. ImportanceMany newly discovered hantaviruses are known only from sequence data, leaving their ability to enter human cells and their receptor usage unresolved. Using a BSL2-compatible rVSV system, we show that glycoproteins from several divergent hantaviruses can mediate infection of primary human endothelial cells, indicating that endothelial cell entry is permissive for a broader range of hantaviruses than previously appreciated. We also show that protocadherin-1 (PCDH1), previously linked mainly to New World hantaviruses, is used by Necocli, Tula, and Nova viruses but not universally across the panel, revealing broader but heterogeneous receptor usage. An authentic Tula virus experiment supports this conclusion beyond the surrogate system. Finally, a broadly reactive anti-Gn/Gc antibody neutralizes several, but not all, of these viruses, highlighting both the promise and the limits of broadly protective countermeasures and the utility of these rVSVs for evaluating entry inhibitors.

Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus with an unusually broad host and vector range. Despite increasing mosquito-to-human spillover, the viral determinants governing host adaptation remain poorly defined. We examined changes in JCV replication during serial passage in mosquito cells and sought to link adaptive changes in viral fitness to specific genetic mutations. In mosquito-derived C6/36 cells, JCV exhibited a distinct lag-burst phenotype in which viral replication remained nearly undetectable for 10 days before abruptly increasing. Strikingly, following reinfection of fresh C6/36 cells, JCV that had been passaged once in mosquito cells exhibited immediate, robust replication with no detectable lag phase. Sequencing before and after passage identified multiple M segment mutations associated with enhanced replication. Using a plasmid-based reverse genetics system, individual mutations were introduced into recombinant JCV and evaluated for their effects on replication in mosquito cells. All tested mutations independently enhanced replication efficiency, demonstrating that adaptation can arise through multiple independent genetic pathways. However, no individual mutation fully reproduced the phenotype acquired naturally through mosquito cell passage. Together, these findings demonstrate that JCV rapidly adapts to mosquito cells under minimal selective pressure and highlight the potential for emergence of increasingly well-adapted viral variants.

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.

Human alpha-defensins are antimicrobial peptides abundantly expressed in neutrophils and the small intestine. They block infection of several families of non-enveloped DNA viruses by binding to and stabilizing the viral capsid during entry, thereby preventing the genome from reaching the nucleus to initiate replication. It is unclear if a similar mechanism also applies to RNA viruses. To study this further, we investigated the interaction of human alpha-defensin 5 (HD5) with enterovirus A71 (EV-A71). We found that HD5 disrupts EV-A71 infection in cell culture and blocks viral entry. HD5 binds directly to the EV-A71 capsid and disrupts key conformational changes essential to the initiation of in vitro uncoating as well as downstream viral genome release. Using a suite of HD5 point mutants, we found that these two uncoating blocks are separable, and HD5 must achieve both to fully neutralize EV-A71 infection. This work advances our understanding of alpha-defensin antiviral action and demonstrates several conserved features of HD5 inhibition that expand to a clinically important RNA virus.

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.

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.

Type-I IFNs mediate the principle antiviral response of cells by controlling the expression of hundreds of IFN-regulated genes (IRGs), many of which have antiviral functions. The best understood mediators of IFN signalling are STAT1 and STAT2, and STAT1/2-dependent gene induction is conventionally viewed as the primary outcome of type-I IFN signalling. To overcome the IFN response, viruses express proteins called IFN-antagonists, which target IFN signalling pathways (e.g. rabies virus P-protein (RABV-P) binds and inhibits IFN-activated STAT1/2) and so are typically considered to mediate shutdown of the IFN response. However, IFN signalling is not exclusively antiviral, with many IRGs reported to be required for or to facilitate infection by certain viruses. How viruses coordinate the apparent need to suppress certain antiviral IRGs, while presumably permitting the expression of others, including proviral IRGs is poorly defined. However, it has been shown that type-I IFN can activate multiple pathways other than classical STAT1/2, so discriminatory targeting of specific pathways by IFN-antagonists may enable highly selective regulation of distinct IRGs, dependent on the requirements of the specific virus. Here, we analyse the global effects of RABV-P protein on the IFN-regulated transcriptome. We confirm that IFN not only stimulates (IFN-stimulated genes, ISGs) but also represses (IFN-repressed genes, IRepGs) a large number of IRGs. Notably, our data indicate that RABV-P protein can antagonize both the IFN-dependent stimulation and repression of certain ISGs and IRepGs, without significantly impacting the expression of large proportion of IRGs. Antagonized ISGs included classical antiviral genes, while non-antagonized ISGs include genes with pro-viral effects on RABV. Transcription factor analysis indicated that RABV-P antagonizes STAT1/2-regulated IRGs, but not IRGs regulated by other pathways including MAP-kinase pathways, which are important to process such as cell survival and inflammatory response. These data indicate that selective modulation rather than global inhibition of IFN-signalling has beneficial outcomes for replication. The data also support the significance of IRepGs, and their modulation by IFN antagonists in viral infection. Significance StatementThe ability of viruses to evade immunity is critical to disease and so presents targets for the development of interventions. The principle antiviral response of cells is mediated by interferons (IFNs), which activate STAT proteins to induce hundreds of genes including antiviral genes. Viruses counter this by expressing IFN-antagonist proteins, many of which directly inhibit STATs. IFN-antagonists are typically considered to shut down IFN responses, but many IRGs have pro-viral functions. How viruses coordinate the apparent need to antagonise some IRGs but not others are poorly understood. Using a well-characterised viral IFN-antagonist, we find that by selectively targeting certain IFN-activated pathways, IFN-antagonists can inhibit effects of IFN on specific subsets of IRGs (including antiviral genes) without affecting others (including proviral genes); thus, IFN-antagonists may be redefined as selective IFN-modulators.

Kaposis sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that causes Kaposis sarcoma and several lymphoproliferative disorders. The KSHV open reading frame (ORF) 16 encodes viral Bcl-2 (vBcl-2), which inhibits apoptosis and autophagy. vBcl-2 is required for efficient lytic replication and production of infectious progeny; however, this essential role is independent of its established functions regulating cell death. To identify host factors that may contribute to vBcl-2 function, we employed proteomic analysis of HA-vBcl-2 immunoprecipitates from lytically reactivated KSHV-infected cells. This analysis identified the BTB-Kelch protein KLHL9, a substrate-specific adaptor for Cullin 3-RING ligase (CRL3) complex, as a candidate vBcl-2-associated factor. We validated the vBcl-2-KLHL9 association by reciprocal co-immunoprecipitation from infected cells, and by ectopic expression in uninfected cells. During lytic reactivation, KLHL9 redistributed from a predominantly cytoplasmic/perinuclear pattern to a mitochondrial pattern that overlapped with HA-vBcl-2. Alanine-scanning mutagenesis mapped a KLHL9-interaction determinant to the N-terminal region of vBcl-2, and vBcl-2 mutants defective in binding failed to colocalize with KLHL9. Conversely, KLHL9 deletion analysis implicated the C-terminal Kelch-repeat region in efficient association with vBcl-2. AlphaFold modeling supported the interface regions. However, vBcl-2 did not show detectable KLHL9-dependent ubiquitination, suggesting that it is unlikely to be a substrate of a KLHL9-containing complex. Functionally, CRISPR/Cas9-mediated KLHL9 disruption reduced lytic viral protein accumulation and infectious progeny production, while re-expression of sgRNA-resistant KLHL9 partially restored these phenotypes. Together, these findings suggest that vBcl-2 may engage host CRL3 complexes during productive infection, and identify KLHL9 as a vBcl-2-associated host factor that supports efficient KSHV lytic replication. Author SummaryKaposis sarcoma-associated herpesvirus (KSHV) is a cancer-associated virus that alternates between a latent cycle and a productive lytic cycle, during which new infectious virus particles are made. The viral protein vBcl-2 is best known for regulating cell-death and autophagy pathways, but earlier studies showed that it is required for efficient virus production through additional, non-canonical functions. In this study, we identify the cellular protein KLHL9 as a host factor that associates with KSHV vBcl-2. KLHL9 normally functions as a specific adaptor in protein ubiquitination pathways that regulate the turnover, localization, or activity of target proteins through ubiquitination. We show that vBcl-2 and KLHL9 associate with one another, and that KLHL9 redistributes toward the mitochondria in cells expressing vBcl-2. Importantly, loss of KLHL9 impairs the expression of viral lytic proteins and reduces the production of new infectious viruses. This defect resembles the phenotype caused by loss of vBcl-2, suggesting that KSHV employs vBcl-2 to engage KLHL9-dependent host machinery during productive infection. Understanding this interaction may help reveal how herpesviruses reprogram infected cells to create an environment that supports virus production.

Mareks disease virus (MDV) is an oncogenic herpesvirus that establishes latency in CD4 T cells, from which it reactivates to initiate productive replication and dissemination. To define host mechanisms governing the transition from latency to early and late lytic replication, we developed recombinant MDV expressing early RLORF4mRFP and late UL47eGFP, which was used to isolate latent (Lo; mRFP low), early lytic (Hi; mRFP high), and late lytic (DP; mRFPeGFP) populations from MDV-induced lymphoblastoid cell lines (LCLs). Two independent LCLs (Lines 62 and 82) were subjected to Illumina RNA sequencing after sorting-purified populations. Viral transcription increased progressively from Lo to Hi to DP populations; however, initiation of viral gene expression differed markedly between cell lines. During the early transition (Lo to Hi), Line 82 exhibited robust induction of 17 viral genes, whereas Line 62 showed only two differentially expressed viral genes. However, both lines converged on a highly conserved transcriptional program during the transition from latency (Lo) to late-lytic replication (DP), sharing 118 viral transcripts, most of which were structural and assembly-associated genes. Among the host transcriptional responses, Line 82 exhibited activation of TP53-associated stress signaling, chromatin remodeling factors, and RNA biogenesis pathways, consistent with a permissive cellular state. In contrast, Line 62 displayed enhanced inflammatory, metabolic, and proteostasis-associated signatures, suggesting a restrictive environment that limits early viral induction. Collectively, these findings demonstrate that MDV reactivation is a host-gated process in which the cellular state governs the initiation of the lytic switch, whereas downstream replication proceeds through a conserved viral program. IMPORTANCEThis study provides critical insights into the host-virus dynamics governing herpesvirus reactivation from latency. By developing a dual-reporter system using recombinant Mareks disease virus (MDV) in MDV-transformed lymphoblastoid cell lines (LCLs), an oncogenic herpesvirus model, reactivating cells were sorted for latent, early-lytic, and late-lytic populations from two independent LCLs. The initial transition from latency to early-lytic replication was highly variable and strongly influenced by cellular state: one line showed rapid viral gene induction linked to stress, chromatin remodeling, and permissive conditions, while the other exhibited a more restrictive, inflammatory/metabolic response. However, progression to late-lytic replication converged on a highly conserved viral transcriptional program dominated by structural genes. These findings establish that host cellular context gates the lytic switch in herpesvirus reactivation, offering a powerful framework for understanding latency control across oncogenic herpesviruses and potential therapeutic targets to prevent reactivation and tumor formation.

Tacaribe virus (TCRV) was the first arenavirus discovered in the New World and was isolated from Artibeus bats in Trinidad and Tobago in the 1950s. One isolate, TRVL-11573, remains but it was passaged by intracranial inoculation of newborn mice 22 times that likely changed its biology. This isolate has been extensively used for arenavirus research, including our previous work that showed it can cause fatal neurological disease in Jamaican fruit bats (Artibeus jamaicensis). Another divergent TCRV, DOM2014, was recently identified from a Jamaican fruit bat captured in the Dominican Republic that contained TCRV genome. A kidney fragment homogenate from this bat was inoculated into Jamaican fruit bats and all became infected with signs of mild liver disease. Experimental challenge of Jamaican fruit bats with DOM2014 led to nonfatal infection that persisted through the end of the study on day 21 and with contact transmission to naive bats. Histopathology, immunohistochemistry and serum chemistry confirmed infection and mild liver disease, but none of the bats produced neutralizing antibodies. B cell receptor transcripts suggested limited somatic hypermutation that could explain the lack of detectable neutralizing antibodies. Transcriptome profiling of livers and spleens showed signatures of a typical innate antiviral response; however, evidence of adaptive immune suppression was also present. Similarly, liver transcriptome analysis showed signatures of an expected innate antiviral response and metabolic dysfunction. The isolation of TCRV-DOM2014 provides a relevant model for the study of a bat reservoir host, and which may challenge the extensive work previously conducted with TRVL-11573. IMPORTANCESeveral New World arenaviruses cause disease in humans and many are BSL-4 agents. TCRV strain TRVL-11573 has been used since the 1950s to study arenavirus biology; however, because it was intracranially passaged in newborn mice and Vero cells, it likely accumulated mutations that changed its biology. This assertion has been reinforced in recent years with discovery of divergent TCRV sequences in wild Artibeus bats that are substantially different than TRVL-11573, thus the prototype strain is unlikely to represent wildtype TCRV. The isolation of a new TCRV strain that has retained its genome fidelity allows a better understanding of TCRVs biology and pathogenic potential. The availability of pathogen-free Jamaican fruit bats and cell lines that are permissive for TCRV-DOM2014 will also help retain the biological features of the virus. Collectively, this is among the most tractable bat reservoir host models developed and provides a system for dissection of how bats host viruses. Moreover, it may be a suitable model for the study of therapeutics and vaccines for New World arenaviruses.