Journal of Virology

Infection with Human Cytomegalovirus (HCMV) can result in a significant burden of disease in those that are immunocompromised or immunonaive. HCMV encodes a repertoire of glycoproteins that facilitate its extensive viral tropism, some of which remain to be characterized. Currently, there is no effective vaccine or cure for HCMV, therefore emphasizing the need to identify viral proteins of critical function. UL14 was selected as an open reading frame of interest due to its high scoring on an in-silico prediction algorithm, as well as its conservation amongst CMVs. Our goal was to elucidate the function of this uncharacterized viral open reading frame. We hypothesized that UL14 functions in the establishment of infection in epithelial cells, due to its predicted structural similarity to UL141. This study demonstrates that HCMV UL14 is a glycosylated viral protein packaged with the virion. Importantly, the deletion of UL14 resulted in a significant reduction of viral growth in epithelial cells, whereas no growth defect was observed in fibroblasts. Mechanistically, we found this defect to be a result of post entry, pre-IE transcription in the establishment of infection, consistent with a defect endosomal escape. Taken together, our results suggest that UL14 functions in the establishment of infection in an epithelial cell-specific manner and may be a novel target for future vaccines or antiviral therapies. Author SummaryHCMV is found in a wide variety of human cells during the course of viral infection. As such, HCMV encodes several glycoprotein complexes that dictate tropism. In this work we report the identification of a novel glycoprotein, UL14, that is involved in establishing productive infections of epithelial cells, a common site of HCMV induced disease. We report that deletion of UL14 from the viral genome impacts its ability to infect ARPE19 cells at a stage indicative of viral events post viral entry but prior to viral transcriptional activation. Further, trans complementation of UL14 by expansion of mutant virus in cells expressing the viral glycoprotein, restore viral infectivity suggesting that UL14 mediates events early in viral infection. Importantly, the characterization of this viral envelope protein provides key insights into viral tropism and identifies a novel target for vaccine design and antiviral therapies.

Three consecutive deoxyguanosine residues (the GGG-tract) in the U3/R junction of the 5 long terminal repeat (LTR) are strictly conserved among all HIV-1 subtypes. Each deoxyguanosine within the tract has been reported to function as transcription initiation site for HIV-1 RNA. Furthermore, RNAs whose transcription initiates from the third deoxyguanosine in the tract (1G form RNAs) are predominant in virus particles and serve as the primary templates for reverse-transcription. In this study, we generated mutant HIV-1s by replacing the tracts in both the 5 and 3 LTR with other nucleotides to elucidate their functional significance. We identified several proviral sequences containing unexpected mutations near the 5 tract after infection with the mutant, but not the wild-type virus. Five-prime rapid amplification of cDNA end (5 RACE) analyses of RNAs purified from mutant virus particles revealed multiple RNA variants with 5 terminal sequences differing from the plasmid used for producing the particles. Some of the unexpected proviral sequences likely arise directly from these variants during reverse-transcription. We also found that replacing all three nucleotides in the 3 tract with deoxyadenosines decreased the proportion of the 1G form RNAs in particles to 32.6%. Nevertheless, up to 88% of provirus was likely generated with the 1G form RNAs, although they were no longer absolutely predominant in particles. Our results demonstrate that the GGG-tracts in the 5 and 3 LTR are conserved to maintain the integrity of reverse-transcription for LTR sequence generation by controlling multiple steps of HIV-1 replication, including transcription, RNA packaging and reverse-transcription. ImportanceHIV-1 is highly mutable, yet certain conserved sequences remain consistent across most strains. These sequences are thought to be maintained because mutations in these regions typically impair viral fitness, leading to the elimination of such variants through natural selections. This study suggests that HIV-1 has a unique mechanism to autonomously prevent acquisition of mutations in certain regions. Specifically, the GGG-tracts in the 5 and 3 LTR ensure accurate transcription of HIV-1 RNAs, selective packaging of the 1G form RNAs, and preferential use of these 1G forms as a template for reverse-transcription. GGG preservation minimizes unwanted mutations in this region, allowing progeny virus to inherit intact LTR sequences. Interestingly, the preferential usage of 1G form RNAs as template for reverse-transcription is not due to their predominance in virus particles. This suggests that genome packaging independently contributes to reverse-transcription as preparatory stage by concentrating the most suitable RNAs for reverse-transcription in particles.

The guinea pig with guinea pig cytomegalovirus (GPCMV) is the only small animal model for congenital CMV (cCMV). GPCMV cell entry is dictated by specific viral gH/gL-based complexes: gH/gL/gO trimer (direct entry); pentamer complex, PC (endocytic entry). GPCMV gB as the fusogenic protein is also essential for all entry pathways. PDGFRA and NRP2 are receptors for direct and endocytic virus entry respectively based on strain 13 animal fibroblast ATCC cell line studies. All non-fibroblast guinea pig cell lines are derived from Dunkin-Hartley animals, the focus of cCMV studies. GPCMV infection of Dunkin-Hartley embryo fibroblasts (GEFh) and epithelial cells were compared. Knockout of PDGFRA on GEFh cells prevented GPCMV(PC-) direct entry but not endocytic GPCMV(PC+) infection, demonstrating both pathways of infection. Fibroblast generated virus poorly infected epithelial cells compared to epithelial virus stock, which exhibited full tropism to all cell types. Guinea pig epithelial cell lines are NRP2-positive and PDGFRA-negative requiring PC for GPCMV infection. Epithelial and GEFh cells, but not strain 13 fibroblasts, additionally expressed ThBD. In immunoprecipitation assays, PC and ThBD interacted unlike CD46 receptor candidate targeting gH/gL. Double-knockout of NRP2/ThBD in epithelial cells impaired infection unlike single knockouts. Individual ectopic species-specific receptor expression restored infection on double-knockout epithelial (NRP2/ThBD) and fibroblast (PDGFRA/NRP2) cell lines. Knockout of NRP2/ThBD receptors did not enhance GPCMV neutralization by gB antibodies on PDGFRA-negative cells demonstrating a limitation of a gB vaccine strategy. Overall, GPCMV and HCMV similarity for receptors and cell tropism maintains the translational importance of this model.

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.

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.

Human adenovirus serotype 4 (Ad4) is used as a replication-competent oral vaccine that safely and effectively prevents Ad4 respiratory illness in US military personnel. Recombinant Ad4 vaccine candidates elicit mucosal and systemic immune responses against respiratory viruses in hamsters, nonhuman primates, and humans. Although evaluation of Ad4 vaccine candidates in mice would be extremely useful given the large number of immunologic tools available, this has been limited by concerns about a lack of viral replication in these animals. Here we generated recombinant Ad4 vectors that express either luciferase (Ad4-Luc) or herpes simplex virus type 2 (HSV-2) glycoprotein D (Ad4-gD2) to identify transgene expression kinetics, the presence of Ad4 vector replication, and HSV-2 immune responses and protection against HSV-2 infection. Local luciferase activity was observed from 7 hours to 20 days after intranasal inoculation of BALB/c and humanized mice. Subsequent inoculations with Ad4-Luc showed reduced luciferase expression in BALB/c mice, but robust expression in humanized mice, suggesting an immune response to the vector in wild-type mice. Ad4 DNA, but not luciferase activity, was reduced in the lungs of BALB/c mice treated with cidofovir before inoculation with Ad4, implying that Ad4 replicated, albeit at a low level, in the lungs. Intranasal vaccination of mice with Ad4-gD2 resulted in HSV-2 neutralizing antibody in the serum, and after HSV-2 intravaginal challenge reduced disease scores, increased survival, and reduced shedding. Overall, the BALB/c mouse model is semi-permissive to Ad4 mucosal infection, but transgene expression is sufficient for the study of Ad4-based vaccine candidates. ImportanceMucosal surfaces serve as the primary site of infection and shedding for many viral pathogens. Immune responses at mucosal sites provide protection, but few mucosal vaccines are licensed. The oral replication-competent adenovirus serotype 4 (Ad4) vaccine is used to prevent respiratory illness in military recruits, has an extraordinary record of safety and efficacy and has been tested as a recombinant platform for other viruses. Further development of this vaccine platform has been partially hindered by the perceived inability to evaluate vaccine candidates in mice. Here we characterize recombinant Ad4 transgene expression kinetics and viral replication after inoculation at various sites and show protection against herpes simplex virus type 2 (HSV-2) genital disease in mice after intranasal vaccination. We show that Ad4 can induce protective efficacy, even in a semi-permissive mouse model, suggesting this is a promising vector for HSV-2 and potentially other viral pathogens.

Many mammalian cells restrict viral replication by utilizing various host restriction factors. We recently demonstrated that CCHC-type zinc-finger-containing protein 3 (ZCCHC3) suppresses human immunodeficiency virus type 1 (HIV-1) replication through multiple mechanisms. We also revealed that single-nucleotide polymorphisms (SNPs) in human ZCCHC3 affect its antiviral function; however, whether similar genetic and functional diversity is present in other species remains unknown. In this study, we investigated the genetic and functional diversity of ZCCHC3 in cynomolgus macaques, a critical animal model for HIV-1-related research. Sequencing analysis of eight independent ZCCHC3 clones per animal revealed substantial amino acid diversity among cynomolgus macaques. We selected 12 representative variants and examined their antiviral activity against several retroviral vectors derived from HIV-1, simian immunodeficiency virus, feline immunodeficiency virus, and murine leukemia virus. Moreover, using replication-competent HIV-1, we showed that selected cynomolgus macaque ZCCHC3 variants can affect both viral production and viral infectivity. These results suggest that the genetic and functional diversity of ZCCHC3 is not limited to humans and underscore the importance of considering ZCCHC3 variation in cynomolgus macaques when using them as animal models for HIV-1-related research.

The matrix protein VP40 of orthoebolaviruses coordinates virion release and downregulates viral RNA synthesis through distinct oligomeric states, including dimers, octamers, and filamentous assemblies. To dissect the contributions of two oligomeric interface residues, L117 and W95, in the Sudan virus (SUDV) VP40 (sVP40), we created variants carrying alanine substitutions and assessed their structural and functional properties. sVP40 L117A failed to form dimers and was predominantly monomeric showing increased structural flexibility, reduced thermal stability together with loss of plasma membrane transport, budding activity, and the ability to regulate viral RNA synthesis. VP40 W95A preserved dimerization but also exhibited increased structural flexibility and reduced thermal stability. Functionally, sVP40 W95A more strongly inhibited viral RNA synthesis and markedly enhanced budding. However, in a transcription- and replication-competent virus-like particle (trVLP) assay, trVLPs produced with sVP40 W95A induced substantially reduced reporter activity in target cells, indicating impaired particle infectivity or functionality and suggesting possible defects in minigenome packaging, entry, or early post-entry steps. These results demonstrate that mutations at key oligomerization interfaces exert distinct structural and functional effects and highlight the requirement for precise oligomerization in coordinating sVP40s dual roles in genome regulation and virion release. By defining the contributions of L117 and W95, this study advances mechanistic understanding of sVP40 function and identifies processes that may serve as targets for antiviral intervention. ImportanceSudan virus (SUDV) causes regular outbreaks in Sub-Sahara Africa with unusually high lethality rates. However, in contrast to the more often occurring Zaire ebolavirus (EBOV), no monoclonal antibodies or vaccines are available and SUDV is generally understudied. The matrix protein VP40 is responsible for the downregulation of viral genome replication and transcription as well as budding. Here, we present structural and functional characterization of the SUDV VP40 interface residues L117 and W95 and show that while both amino acids are crucial for VP40s structural integrity, their functional effects are dramatically different ranging from complete abolishment to improving regulatory and budding activities.

Immediate-early protein IE1 is an essential protein of baculoviruses that is involved in transcription and replication. In the present study, we provide several lines of evidence for how ancient IE1 evolved into its current version. Using progressive truncations and site-directed mutations coupled with fluorescence microscopy, surprisingly, we showed that the previously identified nuclear localization sequence (NLS), basic domain II, was essential for sequence non-specific DNA binding, but not required for IE1 nuclear import, and demonstrated that, BmNPV IE1 (BmIE1) possesses not only a unique bipartite NLS that contains a monopartite NLS but also a cryptic non-canonical NLS that is correlated with sequence non-specific DNA binding. The non-canonical NLS alone was sufficient to launch infection. We also found that N-terminally truncated IE1 can enter the nucleus through its sequence non-specific binding ability. Remarkably, the monopartite NLS, when fused to the N-terminus of EGFP, can form a novel NLS that is also functional in a mammalian cell line. Moreover, residues 58 to 151 of BmIE1 were shown to be dispensable, and residue 152 was found to be critical for launching a productive infection. To gain insight into how ancient IE1 acquired its multi-functionality, we reorganized the N-terminal 23 aa and 132 aa of BmIE1 with EGFP in a variety of manners and found that both fragments are separable and transferable. Notably, we observed that the nuclear levels of BmIE1 should reach certain thresholds to initiate infection. Additionally, we found that BmNPV could launch infection more efficiently in a BmN cell line over another BmN cell line by increasing transcription levels of immediate early genes. Collectively, these findings suggest a hypothesis where ancient IE1 might have evolved the two additional NLSs and acquired the N-terminal 132 aa through gene fusion so as to reach infection-initiating thresholds at a faster pace. Author SummaryBasic domain II was previously shown to be the NLS of AcMNPV IE1 protein. In the present study, we demonstrated that it is essential for sequence non-specific DNA binding, but not required for IE1 nuclear import, and found that BmNPV IE1 (BmIE1) possesses not only a unique bipartite NLS that contains a monopartite NLS but also a cryptic non-canonical NLS that is correlated with sequence non-specific DNA binding. We also found that N-terminally truncated BmIE1 can enter the nucleus through its sequence non-specific binding ability. Remarkably, the monopartite NLS, when fused to the N-terminus of EGFP, can form a novel NLS that is also functional in a mammalian cell line. Moreover, the domains of BmIE1 was shown to be separable and transferable. We also demonstrated that higher expression of functionally impaired BmIE1 achieved by higher MOIs or higher transfection efficiency can partially complement its compromised functions. Consistently, we found that BmNPV could launch infection more efficiently in a BmN cell line over another BmN cell line by increasing transcription levels of immediate early genes.

Development of effective immune responses against any pathogen requires efficient activation of the innate immune system followed by induction of an appropriate adaptive immune response using specific signaling pathways tailored to each infection. The sole innate immune receptor implicated in the induction of adaptive immunity to retroviral infection is Toll like receptor 7 (TLR7). We have found that germinal center responses, neutralizing antibody production, and clearance of murine leukemia virus (MLV) infection in BALB.J and C57BL/6N (B6N) mice occurs in the absence of TLR7 signaling. This suggests that a previously unknown alternative sensing pathway exists for the activation of protective immune responses upon retroviral infection. Genetic crosses indicate that the ability to control retroviral infection in the absence of TLR7 signaling is determined by the same recessive mechanism in both B6N and BALB.J mice, suggesting that TLR7-independent responses do not result from a gain-of-function of an alternative pattern recognition receptor. Additionally, we observed that TLR7-deficient BALB.J mice produce neutralizing antibodies against mouse mammary tumor virus (MMTV) infection, indicating that this alternative sensing pathway is active against retroviruses of multiple genera. Finally, we determined that the alternative sensing pathway is also independent of both MyD88 and STING signaling. The ability of mice of two genetic backgrounds to control retroviral infection in the absence of TLR7 signaling provides a valuable tool for the identification of a novel mechanism of retrovirus control. The dissection of this pathway has the potential to alter our understanding of the requirements for the stimulation of antigen-specific neutralizing immunity. Significance StatementInnate immune sensors are required for induction of pathogen-specific immune responses, and the development of novel vaccines requires an understanding of the basic mechanisms by which the immune system detects and responds to pathogens. While multiple innate immune receptors have been implicated in the sensing of retroviral infection, only Toll-like receptor 7 (TLR7) has been found to upregulate adaptive immune responses. Here, we demonstrate that an additional TLR7-independent mechanism for the activation of antiretroviral antibody responses is present in inbred mice from two genetic backgrounds. This finding has implications for the selection of mouse models for the study of antiviral immune responses and has the potential to alter our understanding of the requirements for the stimulation of antigen-specific neutralizing immunity.

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.

How host functions affect resistance to antiviral drugs is poorly understood. Ganciclovir, a chain-terminating nucleoside analog, is a first-line therapy against human cytomegalovirus, a widespread herpesvirus that causes life-threatening disease in immunocompromised individuals and newborns. Ganciclovir resistance, which is caused by mutations that affect the viral kinase, UL97 and/or the viral polymerase, UL54, can cause treatment failures. Among these mutations, those reducing the exonuclease activity of the viral DNA polymerase permit ganciclovir incorporation without chain termination. However, the fate of DNA strands containing the incorporated nucleotide analog is unknown. We show here that template DNA containing ganciclovir fails to support DNA synthesis of the complementary strand by exonuclease-mutant polymerase. Moreover, while DNA synthesis and ganciclovir incorporation are limited in drug-treated fibroblasts infected by virus with wild-type polymerase, an exonuclease-resistant mutant virus can better synthesize full-length genomes and incorporate substantially more ganciclovir into DNA. Notably, ganciclovir is lost from DNA when drug is removed, suggesting that ganciclovir-containing templates are repaired. We identify the host nucleotide excision repair component, XPA, and the repair enzyme, polymerase kappa, as each being necessary for mutant virus ganciclovir resistance and polymerase kappa as being required for the mutants cidofovir resistance, demonstrating a role for host DNA repair machinery in a mechanism of antiviral resistance. We propose a model for this mechanism, which has relevance for at least one other antiviral drug and likely other nucleoside analog therapeutics, and highlights the participation of host DNA repair machinery during human cytomegalovirus DNA replication. IMPORTANCENucleoside analogues such as ganciclovir, which is a leading drug for preventing and treating human cytomegalovirus, are a critical defense against viral diseases, but antiviral resistance often results in treatment failures. This study reveals a critical role for host DNA repair in a mechanism of resistance to ganciclovir, and identifies at least one specific repair pathway that permits viral DNA synthesis in the presence of ganciclovir, defining a mechanism by which cellular DNA repair pathways conspire to enable antiviral drug resistance. This mechanism is relevant to at least one other antiviral drug and may apply to other antiviral and anticancer agents. The study also showcases the participation of host DNA repair machinery during human cytomegalovirus DNA synthesis.

Flaviviruses like Zika virus (ZIKV), contain RNA tertiary structures within the 3 untranslated region (UTR) that halt the 5-to-3 RNA exonuclease, Xrn1. Halting of Xrn1 at the two RNA structures, termed exonuclease-resistantRNA1 and 2 (xrRNA1 and xrRNA2), results in the formation of subgenomic flavivirus RNAs (sfRNA) that support viral pathogenesis. While the role of the flavivirus xrRNA1 in pathogenesis is well characterized, the role of the flavivirus xrRNA2 structure is not well studied. Using xrRNA crystal structure data, we inserted structure-informed mutations in ZIKV xrRNA2 to disrupt tertiary folding independent of significant sequence changes, evaluate sfRNA production, and define pathogenesis in a murine model of ZIKV infection. Compared to our prior work with ZIKV xrRNA1, we found that ZIKV xrRNA2 is under increased selection pressure to maintain sfRNA production resulting in multiple targeted mutations in xrRNA2 junctional region to induce a stable mutant. Using three targeted xrRNA junctional mutations termed ZIKV X2.L1, we found that the resulting ZIKV clone exhibits attenuated cell death in cultures and decreased viral growth in tissue cultures. In a murine model of ZIKV infection, mice inoculated with ZIKV X2.L1 exhibit significantly decreased symptomatic infection, improved survival, decreased end-organ infection in the brain, and continued robust neutralizing antibody responses to ZIKV. Despite attenuation, serum from ZIKV X2.L1-infected mice or mice vaccinated with ZIKV X2.L1, exhibited 100% protection from lethal ZIKV challenge. These studies show that RNA structure-informed mutations provide a robust model for flavivirus attenuation and vaccine design. Additional studies defining the mechanisms of robust neutralizing antibody responses and flavivirus-specific vaccine development are needed to continue the development of this novel vaccine platform approach for medically important flavivirus infections. Author summaryZika virus is a member of the Orthoflavivirus (referred to as flavivirus) genus that exhibit conserved RNA structures in the 3 untranslated region of the viral RNA genome. Two concerned RNA structures, termed exonuclease-resistant RNA 1 and 2, are important to support the ability of the virus to cause disease. While the first RNA structure is well studied, less is known about the role of exonuclease-resistant RNA 2 in the flavivirus infection. Using reverse genetics, we made mutations in the Zika virus exonuclease-resistant RNA 2 structure and studied how this mutant Zika virus was weakened or attenuated. We found that the mutant Zika virus clone exhibits reduced virus replication, reduced ability to kill cells, and decreased virulence in mouse models of Zika virus disease. Using this mutant virus as a potential vaccine candidate, we found that Zika virus with mutations in the exonuclease-resistant RNA 2 structure provide complete protection from lethal Zika virus challenge. These data suggest that targeting the second exonuclease resistant RNA structure in flaviviruses is a viable platform for the development of vaccine candidates for this important group of viruses.

Viruses alter host gene expression to create a proviral environment while the host simultaneously regulates gene expression to restrict the virus spread. Owing to RNA virus complete reliance on the host translational machinery, it is important to assess the translational control during virus infection. Therefore, we used ribosome profiling (ribo-seq) paired with RNAseq to observe how red clover necrotic mosaic virus (RCNMV) infection of Arabidopsis plants alters cellular gene expression at the levels of mRNA abundance and translation efficiency. We determined that at 5 days post-inoculation (dpi), the translational response to RCNMV infection is enriched in genes of the innate immune system. Expression of a tumor necrosis factor receptor-associated factor (TRAF)-like protein, a regulator of development and immune response, was translationally but not transcriptionally upregulated early in systemic infection. By 8 dpi, many pathways were regulated/dysregulated, and unfolded protein response (UPR) genes were transcriptionally upregulated but with reduced translation efficiency. Ribosome profiling of RCNMV RNAs revealed (i) -1 programmed ribosomal frameshifting at 7.5-8.0%, the first direct measurement of frameshift efficiency in infected cells for any plant virus; (ii) that coat protein is translated at extremely high efficiency, while the RNA-dependent RNA polymerase is translated least efficiently, and (iii) an unexpected extremely strong ribosomal pause site in the open reading frame that encodes the movement protein. To our knowledge, this is the first genome-wide study that assesses the translational control of gene expression in plants infected with a virus from the large and diverse Tombusviridae family. ImportancePositive strand RNA viruses usurp the hosts translation machinery to synthesize viral proteins. Moreover, translation of host mRNAs is altered by virus infection, both as part of the host immune response and by the virus to inhibit host defenses. To assess all these changes globally, we used ribosome profiling of plants infected with a member of the large and ubiquitous Tombusviridae family. We identified key host genes and pathways that were differentially altered in translation efficiency, giving us an understanding of host responses not detectable by conventional RNA sequencing. Moreover, ribosome profiling revealed (i) the most accurate calculation of efficiency of ribosomal frameshifting during infection for any plant virus, (ii) the extremely high level of translation of viral coat protein, and (iii) an unexpected strong ribosomal pause site in the movement protein gene. This work provides understanding of a new dimension of gene expression control in plant-virus interactions.

Most adenovirus (Ad) vectors are based on the genome of human Ad type 5 (Ad5), which targets their entry to cells that express the Coxsackie and Adenovirus Receptor (CAR or CXADR). However, certain human Ads do not use CAR, for example Ad35 interacts with cell-surface CD46 and Ad3 uses desmoglein 2 (DSG2) for cell entry. In this study, a comparison of different Ad receptors using transcriptomic and proteomic databases showed that CD46 is widely expressed across human cells and tissues whereas CAR and DSG2 are more restricted to epithelial cells. We have used a hybrid virus, Ad5F35, that comprises an Ad5 genome in which the Ad5 fibre was replaced with that of Ad35, thus retargeting the virus from CAR- to CD46-expressing cells and enabling transduction of primary human NK and T cells. However, lymphocytes required approximately 10 to 20-fold more Ad5F35 particles per cell (ppc) compared to A549 epithelial cells to achieve a similar level of transduction. Consistent with this, quantitation of the cell-surface density of CD46 molecules revealed approximately 100 CD46 molecules per {micro}m2 in primary NK cells compared with approximately 2000 CD46 per {micro}m2 in HeLa cells, a 20-fold difference. Cell-surface CD46 density was reduced by approximately 95% by RNA interference in HeLa cells to levels that approximate those found on NK cells. Lower CD46 density reduced transduction by Ad5F35 but this could be compensated for with increased MOI. Our results identify the density of cell surface CD46 as a critical determinant of Ad5F35 transduction and demonstrate that Ad5F35 is an efficient vector for gene delivery in primary human NK cells.

Human adenoviruses (HAdVs) are double-stranded DNA viruses that cause a wide range of diseases, including respiratory, ocular, and gastrointestinal infections. HAdVs rely extensively on alternative splicing to expand their coding capacity and regulate gene expression during infection. Using replication-competent epitope-tagged HAdV-C5 viruses, we investigated the function of the newly identified alternatively spliced viral transcript, 19K/IX, derived from E1B19K and pIX genes. We show that 19K/IX binds to and stabilizes the minor capsid protein IX (pIX), thereby preventing its proteasomal degradation and promoting the production of infectious viral progeny. Mechanistically, we demonstrate that pIX degradation is mediated by a non-canonical ubiquitination of conserved tyrosine residues, which also partially mediate pIX interaction with the PSMC3 subunit of the 26S proteasome. Collectively, these findings identify 19K/IX as a novel regulator of HAdV-C5 infection and suggest that non-canonical tyrosine ubiquitination may represent a mechanism by which HAdV-C5 modulates protein degradation during infection. Author summaryHuman adenoviruses (HAdVs) are clinically important pathogens and widely used as therapeutic tools. Because of their compact genomes, HAdVs use their genetic information very efficiently through alternative pre-mRNA processing. Here, we describe and characterize 19K/IX, a novel fusion protein generated by alternative splicing of two virus genes during infection. We demonstrate that 19K/IX stabilizes the capsid protein IX by preventing its degradation, revealing a previously unrecognized strategy by which HAdVs ensure efficient production of new virus particles. These findings deepen our understanding of HAdV-C5 gene regulation and protein homeostasis and highlight the functional importance of non-canonical viral transcripts.

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.