Journal of Virology

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.

Viruses make long (>40 bp) double stranded RNA (LdsRNA) during replication, which stimulates the innate antiviral immune response. In vertebrates, LdsRNA can induce the type I interferon response (IFN) or the antiviral RNA interference response (dsRNAi) to limit viral replication. Extracellular vesicles (EVs) have previously been shown to carry a variety of nucleic acids for intercellular signaling, and insects and plants have been shown to package LdsRNA in EVs as part of their antiviral immune response. We hypothesized that a similar phenomenon occurs in vertebrates in which EVs traffic LdsRNA between cells during viral infection to induce an antiviral response in naive cells. In this study we showed that both vesicular stomatitus virus (VSV)-derived and in vitro transcribed (ivt)-LdsRNA can be packaged into EVs. LdsRNA was detectable by immunoblots in EVs extracted by both differential ultracentrifugation from ivt-LdsRNA treated U937 and ExoQuick-TCTM precipitation from VSV-infected U937 cells (dsRNA-EVs) but not uninfected controls (control EVs). Isolated EVs were roughly 100 nm in diameter and were able to protect LdsRNA from degradation by RNase III. LdsRNA delivery by dsRNA-EVs was visualized in HEL-299 cells via immunocytochemistry (ICC). The LdsRNA-EVs protected against infection from HCoV-229E, while control EVs did not. Together these results indicate EVs can package and deliver long dsRNA to provide antiviral protection in naive vertebrate cells. Author SummaryWhen viruses infect cells, they produce double-stranded RNA, a molecule that alerts the body to the presence of infection and triggers antiviral defenses. Previous studies have shown that cells can release small membrane-bound packages called extracellular vesicles, which carry biological messages to other cells. However, it was not known whether antiviral double-stranded RNA could be transported in these vesicles and shared with neighboring cells. In this study, we investigated whether human cells package double-stranded RNA into extracellular vesicles and whether this cargo helps protect other cells from viral infection. We found that both synthetic and virus-derived double-stranded RNA were incorporated into extracellular vesicles and shielded from degradation. These vesicles successfully delivered double-stranded RNA to untreated cells, substantially protecting these cells from infection with a human coronavirus. Our findings suggest that cells can communicate antiviral warnings to neighboring cells by packaging double-stranded RNA into extracellular vesicles. This work reveals a previously unrecognized way that antiviral protection may spread through tissues during infection. By extending antiviral signals beyond directly infected cells, extracellular vesicles may help coordinate a broader host defense response. Understanding this natural communication system could also inform the development of new RNA-based antiviral therapies.

The conserved herpesvirus protein kinase (CHPK) is encoded by all members of the Orthoherpesviridae and contributes to replication in cell culture but is not strictly required. Mareks disease virus (MDV) CHPK is dispensable for replication in cultured cells yet essential for horizontal transmission in chickens. To elucidate its role during natural infection, we performed RNA sequencing (RNA-seq) and mass spectrometry (MS)-based phosphoproteomics on spleen and feather follicle epithelial skin cells from chickens infected with wild-type or CHPK-null MDV. RNA-seq detected only a limited number of viral transcripts in the spleen--including latency-associated transcripts (LATs) and the major oncogene Meq--with minimal differences between wild-type and CHPK-null infections. In feather follicle epithelial skin cells, the full repertoire of viral genes was expressed, but only seven genes showed differential expression between wild-type and CHPK-null viruses. In striking contrast, MS-based phosphoproteomics identified many differentially phosphorylated proteins, including 21 viral proteins. These findings indicate that CHPKs critical functions in skin replication and subsequent horizontal transmission are primarily mediated through post-translational modifications (PTMs) rather than transcriptional regulation. Among the CHPK-targeted viral proteins were three MDV-unique proteins, eight conserved within the Alphaherpesvirinae, and ten conserved across the Orthoherpesviridae. In silico analysis revealed that many differentially phosphorylated serine and threonine residues lie near or within predicted nuclear localization signals (NLS) and nuclear export signals (NES). Functional validation confirmed that several of these motifs actively control nucleocytoplasmic shuttling of the respective viral proteins. Collectively, these data suggest that MDV CHPK orchestrates the subcellular localization of multiple viral proteins in epithelial skin cells via phosphorylation, thereby enabling efficient replication and horizontal transmission in the natural host. AUTHOR SUMMARYUnderstanding the mechanisms by which herpesviruses replicate and spread within their natural hosts and identifying the viral genes essential for these processes are fundamental to developing effective antiviral strategies. Mareks disease virus (MDV), a highly contagious alphaherpesvirus, remains a major economic threat to the global poultry industry while serving as a powerful natural animal model for studying herpesvirus pathogenesis and transmission in vivo. Using an established in vivo enrichment method for infected cells, we conducted a comprehensive analysis of viral gene expression, protein abundance, and post-translational modifications (PTMs) during natural infection. Remarkably, RNA sequencing revealed virtually no differences in viral transcription between wild-type and CHPK-null viruses in either spleen or feather follicle epithelial skin cells. In contrast, phosphoproteomics showed that CHPK extensively regulates the phosphorylation of multiple viral proteins specifically in skin epithelial cells. In silico and functional analyses further indicate that these CHPK-mediated phosphorylations occur near or within nuclear localization (NLS) and nuclear export (NES) signals, directly controlling the nucleocytoplasmic shuttling of key viral proteins. This work suggests CHPK as a master regulator of viral protein subcellular localization during replication in the natural host and highlights CHPK orthologs as promising broad-spectrum therapeutic targets against herpesviruses.

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.

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.

Hepatitis C virus (HCV) depends on host lipid metabolism and lipid droplets (LDs) for genome replication, assembly, and particle production, yet how LD structure and lipid utilization change over the course of infection remains incompletely understood. Here, we investigated the temporal remodeling of LD-associated metabolic pathways during HCV JFH-1 infection of human hepatoma Huh7 cells. HCV infection transiently increased LD fluorescence intensity at 24 hours post-infection (hpi), followed by normalization or relative loss of LD signal at later time points. Concomitantly, LDs became progressively clustered and enlargement during late infection, despite reduced protein levels of the canonical LD fusion proteins CIDEA, CIDEB, and CIDEC, suggesting that HCV-induced LD enlargement occurs through CIDE-independent mechanisms. Transcriptomic, RT-qPCR, and immunoblot analyses revealed time-dependent regulation of genes and proteins involved in LD structure, triglyceride synthesis, lipolysis, lipid uptake, and mitochondrial fatty acid utilization. Subcellular fractionation demonstrated preferential accumulation of fatty acids in mitochondrial fractions at 24-72 hpi. This redistribution was accompanied by increased oxygen consumption rate, elevated extracellular acidification, and progressive reactive oxygen species accumulation, indicating infection-associated metabolic activation and oxidative stress. Pharmacological inhibition of DGAT1-dependent LD biogenesis, LIPA-dependent lysosomal lipid hydrolysis, LIPE/HSL-dependent lipolysis, or CPT1-dependent mitochondrial fatty acid transport markedly reduced mitochondrial fatty acid accumulation and suppressed HCV-induced respiratory activity. Inhibition of LIPA or LIPE/HSL reduced both HCV RNA and core protein levels, whereas inhibition of CPT1 or DGAT1 had more pronounced effects on core protein than on viral RNA. Together, these findings support a model in which HCV dynamically remodels LDs, mobilizes LD-associated fatty acids, and redirects them toward mitochondria to support infection-associated metabolism and downstream stages of the viral life cycle. Lipid hydrolysis and mitochondrial fatty acid trafficking therefore represent potential host-directed targets for limiting HCV infection. SIGNIFIGANCEHepatitis C virus depends on host lipid metabolism for replication, assembly, and production of infectious particles, but how it uses lipid droplets over time remains incompletely understood. This study shows that hepatitis C virus dynamically remodels lipid droplets, causing an early increase in lipid storage followed by droplet enlargement and mobilization of fatty acids during later infection. The released fatty acids preferentially accumulate in mitochondria, where they are associated with increased cellular respiration and oxidative stress. Blocking lipid droplet formation, lipid breakdown, or fatty acid transport to mitochondria reduced this metabolic response and decreased viral RNA or core protein accumulation. Inhibition of lysosomal acid lipase and hormone-sensitive lipase suppressed both viral RNA and protein levels. These findings identify lipid droplet breakdown and mitochondrial fatty acid trafficking as important host processes used by hepatitis C virus and as potential targets for host-directed antiviral intervention.

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.

Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide, yet no effective antiviral therapeutics are currently available. Although environmentally induced capsid conformational changes associated with infectivity have been reported in murine noroviruses (MNVs), comparable conformational switching has not been demonstrated in HuNoVs. In this study, we generated HuNoV GII.3 virus-like particles (VLPs) using a baculovirus expression system and identified two distinct T = 3 particle populations coexisting within VLP preparations derived from a single strain through cryo-electron microscopy single-particle analysis. Comparative structural analysis revealed that these two T = 3 capsid conformations correspond to the resting and rising states of the protruding (P) domain. Rearrangement of the P domain alters intermolecular interactions between adjacent capsid subunits, resulting in distinct capsid surface architectures. In the resting state, intermolecular contacts were mediated predominantly by the P2 subdomain, with limited contribution from the P1 subdomain. In contrast, the rising state exhibited a shift toward an alternative interaction interface primarily involving the P1 subdomain. These findings demonstrate previously unrecognized structural polymorphism in the HuNoV capsid and provide evidence that conformational switching may occur in HuNoVs. Our results offer new insights into norovirus capsid dynamics and may inform future structure-based vaccine and antiviral drug development.

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.

Bunyamwera virus (BUNV) is the prototypical member of the Peribunyaviridae family of arthropod-borne viruses and possesses a genome comprising three segments of negative-sense RNA, named small, medium and large. The medium segment encodes a polyprotein that is processed to form Gn and Gc spikes and a non-structural protein, NSm. The role of NSm during replication in mammalian cells is poorly characterized, although it associates with a Golgi-derived structure called the virus factory (VF), the site of BUNV genome replication and virion assembly. To further define NSm function, we generated an epitope-tagged BUNV and used co-immunoprecipitation and quantitative proteomics to identify host interacting partners. NSm interacted with BCL-2 interacting protein 1 (BNIP1), a SNARE protein involved in COPI vesicle trafficking, with the importance of this interaction demonstrated by siRNA-mediated knockdown of BNIP1 expression, which significantly reduced BUNV gene expression and virion production. Interestingly, NSm also interacted with components of the NRZ complex, involved in COPI vesicle tethering in association with BNIP1, and inhibition of COPI complex formation resulted in loss of NSm expression. Taken together, our results identify BNIP1 as a host cell factor necessary for efficient BUNV replication and suggest the cellular localization of NSm at the VF is COPI-dependent.

Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality after both solid organ and hematopoietic stem cell transplant, due to bone marrow stem cell engraftment failure, myelosuppression, and immunosuppression. While direct infection and viral replication lead to disease, latent infection in the CD34+ hematopoietic progenitor cell (HPC) pool has direct and indirect effects on hematopoiesis. Studies from several laboratories support the hypothesis that HCMV latency and reactivation are intrinsically linked with the state of the cell. We, and others, have previously demonstrated roles for different viral gene products in regulating both cellular differentiation and the balance between viral latency and reactivation, including the viral RL11 proteins UL7 and UL8. In this study, we show that UL4 is expressed during latency and required for reactivation in HPCs, THP-1 monocytes, and humanized mice. describe both a cell-specific and viral lifecycle-specific role for the RL11 gene UL4 during HCMV infection. Additionally, we demonstrate that UL4 plays specific roles in controlling essential cellular functions including aspects of proliferation and differentiation and immune signaling to assist in establishing a virus-favorable environment in progenitor cells. This study identifies a novel viral reactivation factor that has implications for both viral control and alteration of hematopoiesis in transplant patients.

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.

Orthoflaviviruses undergo significant structural changes through maturation and infection, yet the molecular mechanisms remain incompletely understood. Using St. Louis encephalitis virus (SLEV) as a model, a reemerging mosquito-borne orthoflavivirus endemic in the Americas, we elucidated the structures of immature and mature SLEV particles at resolutions of 4.4 [A] and 3.3 [A], respectively, using cryo-EM. SLEV is characterized by glycosylated E and prM proteins, the presence of lipid pockets, and is stabilized by an intricate network of inter- and intra-protein interactions between E and (pr)M across maturation stages. Several interactions were mediated by histidines that play different roles depending on SLEV maturation and pH. Non-lethal single mutations of H285R and H443R in E protein affect SLEV replication in mammalian and mosquito cell lines, and delay death in mouse models of infection. These histidine residues are conserved across orthoflaviviruses, illustrating the complexity of orthoflavivirus particles and indicating a possible strategy for attenuation.

The 2022 Monkeypox virus (MPXV) outbreak renewed interest in vaccines for orthopoxviruses. Initial development efforts focused on well-established antigen targets, especially A35, B6, and M1. However, orthopoxvirus surfaces are complex, displaying many antigens across two infectious forms, mature virions (MV) and extracellular virions (EV) and targets relevant to protection remain to be comprehensively defined. We leveraged advances in orthopoxvirus protein biology and mRNA vaccine technology to compare immunity to all feasible targets. Mice were immunized with mRNAs encoding each antigen, or antigen complex, and neutralizing antibody responses were measured prior to heterologous challenge with vaccinia virus. Among MV antigens, A28 induced potent complement-mediated neutralizing antibodies, and the A17:G10 complex induced neutralizing antibodies and protected from challenge. For EV antigens, A36 induced neutralizing antibodies and protected from challenge. Our results affirm the consensus strategy focusing on key antigens while highlighting additional targets that could enhance updated MPXV mRNA vaccines. SIGNIFICANCEMonkeypox virus, a member of the Orthopoxvirus genus along with variola virus, has been associated with two recent outbreaks of mpox disease leading to a renewed focus on orthopoxvirus vaccine development. We report an agnostic screen of all monkeypox virus surface antigens where we combined recent advances in structural biology and mRNA technology to evaluate these potential new vaccine targets. We confirmed that historically prioritized antigens M1, A35 and B6 were protective but also discovered new antigens of interest including A28, the A17:G10 complex and A36 that can be the targets of protective immune responses. These findings are critical to inform next-generation vaccine designs should novel orthopoxviruses emerge as human pathogens.

Herpes simplex virus 1 (HSV-1) is a highly prevalent DNA virus with a major impact on human health. The HSV-1 genome is assembled into silenced stable chromatin and minimally transcribed during latency or assembled into permissive highly dynamic chromatin and highly transcribed during lytic infections. It is unclear how HSV-1 genomes transition between chromatin states, but epigenetics, including chromatin dynamics, have been proposed to play a major role. Chromatin remodeling complexes regulate cellular chromatin dynamics and contribute to DNA transcription, replication, and repair. The BAF family of chromatin remodeling complexes includes three ubiquitously expressed complexes (cBAF, PBAF, and GBAF) and several cell type-specific ones. Some common BAF subunits interact with two HSV-1 proteins, VP16 or ICP8. Three subunits shared by all BAF complexes and a unique subunit from each cBAF, PBAF, and GBAF were enriched in herpes nuclear domains (HND), the novel nuclear domains formed during lytic infection in which HSV-1 genomes are transcribed, replicated, and packaged. The shared ATPase SMARCA4 bound, directly or indirectly, to HSV-1 genomes. Bromodomains bind to acetylated histones and may thus be involved in this binding. However, none of four structurally unrelated inhibitors of BAF bromodomains drastically affected the recruitment of BAF subunits to HND, and neither of four commonly acetylated histone residues recognized by BAF bromodomains was enriched in the HND. BAF complexes are thus recruited to the HND by their interactions with VP16, which activates viral transcription, and ICP8. Surprisingly, the BAF complexes recruited by VP16 and ICP8 participate in inhibition of immediate-early, early, and early-late HSV-1 transcription, but not DNA replication or late transcription. We propose that BAF complexes are recruited to the HND by VP16 and ICP8, independently of their bromodomains, to inhibit viral transcription early in infection, thus contributing to the regulated cascade of gene expression. These findings also have implications to epigenetic anticancer drugs, in that it should be considered whether their use may reactivate latent herpes simplex viruses. Author SummaryHerpes simplex virus 1 (HSV-1) infects over two-thirds of the world population. HSV-1 establishes latency in neurons, resulting in life-long infection. Although most infections are asymptomatic, reactivation can produce a wide range of clinical manifestations, including cold sores, stromal keratitis, and encephalitis. Available treatments do not prevent reactivation or eliminate latent viral reservoirs, as no viral proteins are expressed during latency. Epigenetic regulation plays a role during the lytic and latent cycles. Lytic HSV-1 chromatin is highly dynamic whereas latent chromatin is stable. Chromatin dynamics are regulated by multiple factors, including the chromatin remodeling complexes. Here we show that the BAF chromatin remodeling complexes regulate HSV-1 transcription during lytic infection in primary fibroblast and transformed epithelial human cells. Although these complexes are recruited to the viral genomes by viral proteins, they counterintuitively downregulate viral transcription before the onset of DNA replication. We propose that BAF complexes play a major role in the regulation of the orchestrated cascade of viral gene expression and propose to consider the potential for reactivation of herpes simplex viruses when using epigenetic inhibitors in the treatment of cancer.

RNA molecules form specific 3D structures that facilitate a variety of functions through interactions with other macromolecules. Many RNA viral genomes maintain these structures to interact with and evade host immunity machinery. One such element, the competitive inhibitor RNA (ciRNA), discovered in the protein coding region of the poliovirus serotype 1 (PV1) genome, inhibits a host antiviral protein, ribonuclease L (RNase L). Although some functionally essential structural motifs of the PV1 ciRNA have been studied, the extent of its evolutionary conservation and other structural requirements remained unexplored. Here we combined bioinformatic and biochemical techniques to further define the requirements of a functional ciRNA and assess its phylogenetic distribution. We systematically mutated ciRNA structural features, verifying that ciRNA inhibitory activity requires a conserved loop E motif and a long-range base-pairing interaction, but its peripheral stems are dispensable and in fact a circularly permuted version maintains function. A structure-based homology search identified potential ciRNAs across the Picornaviridae family, but only a subset of those tested were functional - all are in Enterovirus coxsackiepol. When structural features needed for function were transposed from PV1 ciRNA to an RNA unable to inhibit RNase L, the chimeric RNAs did not gain wild-type function, and chemical probing data revealed that these nonfunctional RNAs are unable to form the correct secondary structure. Overall, the dual constraints of encoding a protein and forming a specific functional structure appear to not only limit the sequence diversity, but also the phylogenetic distribution, of ciRNAs.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that undergoes lytic replication in epithelial and other permissive cell types and can establish latency in peripheral neurons. ICP22 is a multifunctional HSV-1 immediate-early protein that localizes to the nucleus of infected cells; however, its interactions with host cellular factors remain incompletely understood. Here, ICP22 was demonstrated to interact with the human antisense function 1 protein (ASF1), including both ASF1a and ASF1b, in transfected cells and HSV-1-infected cells, respectively. ICP22 also colocalized with ASF1 in the nucleus. ICP22 amino acids 213 to 340 are important for the interaction of ICP22 with ASF1, whereas amino acids 37 to 153 of ASF1a and ASF1b are critical for their interactions with ICP22. Furthermore, ICP22 expression was associated with reduced ASF1-H3.1 co-immunoprecipitation under the tested conditions. ASF1 knockdown also reduced HSV-1-BAC-Luc luciferase output, indicating that ASF1 contributes to efficient infection-associated reporter activity in this study. Collectively, these results indicate that the interaction of HSV-1 ICP22 with ASF1 might help regulate the transcription of viral or cellular genes during HSV-1 infection. Keywords: HSV-1, ICP22, ASF1, histone H3.

Nucleotide-binding leucine-rich repeat (NLR) immune receptor genes are of significant value in disease resistance breeding and the control of viral diseases. Soybean mosaic virus (SMV) poses a serious threat to soybean production and the Rsv1 locus in soybean cultivar Suweon 97 confers broad-spectrum resistance against SMV strains G1 to G7; however, this locus harbors no fewer than 18 NLR genes, and thus the broad-spectrum antiviral mechanisms underlying the Rsv1 locus remain poorly understood to date. Here, we established a rapid and highly efficient screening system for cloning NLR genes from soybean Rsv1 locus and identified a broad-spectrum antiviral NLR gene 13g184900 from this highly complicated locus. The NLR encoded by 13g184900 can recognize viral P3 protein from all SMV strains (G1-G7) and another potyvirus Bean common mosaic virus (BCMV). The coiled-coil (CC) domain of this NLR directly interacts with viral P3 protein. Additionally, we showed that this NLR originated from wild soybean accession in East China and has been introduced into several soybean cultivars during domestication. Collectively, we developed a high-throughput screening system for identifying NLR genes in soybean and our study provides new mechanistic perspective on how the Rsv1 locus mediates the broad-spectrum resistance to all SMV G1-G7 strains.

Lone star virus (LSV) is a bandavirus first isolated from Amblyomma americanum ticks in the United States (U.S.) and is phylogenetically related to severe fever with thrombocytopenia syndrome virus (SFTSV), Heartland virus (HRTV), and Bhanja virus, each of which has been associated with severe human disease. In contrast to these better-characterized bandaviruses, LSV remains poorly studied, and its pathogenic potential is not well defined. Recent detection of LSV RNA in cerebrospinal fluid from an immunocompromised patient in Idaho, U.S., with fatal meningoencephalitis further highlights the need for experimental systems to investigate LSV biology. Here, we rescued recombinant (r) LSV from cloned cDNA and used it to characterize LSV. rLSV replicated similarly to the parental isolate in mammalian cells and caused rapid, systemic, and lethal disease in IFNAR-/- mice, with widespread detection of viral (v) RNA across multiple tissues, hepatic and splenic pathology, and induction of inflammatory cytokines. In contrast, C57BL/6J mice controlled infection and exhibited no clinical disease. To place LSV within a broader comparative framework, we generated rSFTSV from cloned cDNA and compared rLSV, rSFTSV, and HRTV in cell culture and IFNAR-/- mice. Our studies revealed distinct disease kinetics among these related tick-borne bandaviruses and showed that HRTV-induced immunity protected against homologous HRTV rechallenge and heterologous rSFTSV challenge, but not rLSV challenge. Together, these findings establish reverse-genetics platforms and small-animal models for comparative bandavirus studies, define key features of LSV pathogenesis, and place this neglected virus within a framework of related bandaviruses that differ in virulence and immunological overlap.

Given their rapid evolutionary dynamics, viruses offer a powerful system to investigate the mechanisms underlying host jumps. Here, we experimentally evolved endive necrotic mosaic virus (ENMV) in five plant hosts within the family Asteraceae: two putative ancestral hosts (Lactuca sativa and Tragopogon pratensis), and three alternative crop or weed species (Cichorium endivia, Zinnia elegans and Calendula arvensis). The resulting evolved viral populations, together with the ancestral strain, were then evaluated in a reciprocal cross-inoculation experiment across all five host species. ENMV exhibited clear adaptive responses in two hosts, Z. elegans and C. arvensis, with increased infection success and higher systemic viral accumulation compared to the ancestral virus. In contrast, no evidence of adaptation was detected in L. sativa, T. pratensis and C. endivia. Strikingly, strong cross-adaptation emerged between Z. elegans and C. arvensis: viral populations evolved in either host consistently outperformed those evolved in other hosts, as well as the ancestral strain, when infecting the reciprocal host. Sequencing of the VPg cistron in adapted populations revealed multiple nonsynonymous mutations, several of which arose independently across evolutionary lineages and in both Z. elegans and C. arvensis selection regimes. Functional assays using an infectious ENMV cDNA clone demonstrated that seven of these substitutions, individually or in combination, significantly increased the infection rate in both Z. elegans and C. arvensis. Notably, several of these substitutions also enhanced infectivity across four additional Asteraceae species among the eleven tested, without a clear relationship to host phylogenetic distance. Remarkably, all identified substitutions map to amino acid positions or adjacent residues in VPg previously implicated in the breakdown of recessive resistance genes against potyviruses in both crop and model plant systems. Together, these results suggest that adaptation to host resistance and host range expansion in potyviruses may rely, at least in part, on shared molecular pathways.