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.

It has been suggested that the hemagglutinin of the human-infecting cattle-derived 2.3.4.4b virus A/Texas/34 (H5TX) requires only one mutation, namely Q226L, to switch from binding avian-type to human-type receptor preference. In this study, we examined the binding of H5TX Q226L, along with other key mutations, to sections of human trachea. We conclude that, while H5TX Q226L can bind human-type receptors, more than a single mutation is required for this protein to bind to human respiratory tract tissue. We also report changes in receptor-binding specificity of another 2.3.4.4b HA mutant, H5FR Q226L, associated with the presence of a multibasic cleavage site. This study offers insight into the determinants of evolution towards human-type receptor binding in currently circulating H5Nx viruses. It also emphasizes the importance of testing individual strains using additional methods, including tissue-based approaches, alongside synthetic glycans. ImportanceCurrently, H5N1 influenza A viruses are responsible for numerous zoonotic spillover events, from infecting birds to other mammals, including dairy cattle. Although no human-to-human transmission has been observed, several people have been infected. This host range expansion is typically linked to changes in one of the viral surface proteins, hemagglutinin, which can switch its preference from avian-type to human-type receptors. To better understand the potential of the currently circulating H5N1 virus to transmit among humans, we evaluated the effects of the Q226L mutation, in combination with other amino acid substitutions, on binding to the human trachea. We also studied the effect of the multibasic cleavage site, a specific motif present in highly pathogenic influenza strains, on receptor binding properties. These findings provide insight into the role of receptor binding in influenza infections.

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and herpes zoster, while herpes simplex virus 1 (HSV-1) causes oral and genital herpes. Both infections manifest with skin blisters from which the viruses are transmitted to new hosts either via aerosol (VZV) or skin microabrasions (HSV-1). VZV reaches the skin through the blood route, and in the skin epidermis it first infects undifferentiated keratinocytes of the basal layer. Conflicting evidence exists for HSV-1, making it unclear whether HSV-1 infects undifferentiated or differentiated keratinocytes. Here, we developed in vitro models of primary human epidermal keratinocytes differentiation to recapitulate infection of distinct layers of the epidermis by VZV and HSV-1. Our data show that replication of both viruses is restricted, VZV more than HSV-1, if initial infection occurs in differentiated keratinocytes, but not if initial infection occurs in basal undifferentiated keratinocytes. Like VZV, HSV-1 downregulates expression of proteins associated with keratinocyte differentiation, such as the suprabasal keratin K10. However, whereas downregulation of K10 occurs soon after VZV infection and before the virus has replicated, HSV-1-mediated K10 downregulation appears to require full viral replication. These observations provide insights into the potential for VZV and HSV-1 interactions with epidermal differentiation to yield strategies for developing host and pathogen-directed antiviral agents.

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.

Highly pathogenic avian influenza H5N1 2.3.4.4b genotype D1.1 lineage continues to predominate in the United States wild bird population and has spilled over into dairy cattle three independent times. To assess the transmission risk of this sublineage, we performed direct-contact transmission experiments for three distinct D1.1 strains in ferrets. Two of these strains were isolated from humans and one from a lethal cat infection. We found that only one human isolate (A/NV/10/2025) was able to transmit efficiently between ferrets. Compared to the other strains, this isolate harbored the mammalian adaptive PB2 D701N mutation, suggesting this mutation may be critical for D1.1 transmission as opposed to the PB2 E627K substitution present in the lethal cat isolate. Based on these data we conclude that the transmission fitness of D1.1 strains is modest but that special attention should be paid to emergence of adaptation at the PB2 701 position.

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.

High-risk human papillomaviruses (HPV), including HPV16, produce circular RNA that encompass the E7 oncogene (circE7). CircE7 can be detected in HPV16-positive cells and tumors, is preferentially localized to the cytoplasm, is N6-methyladenosine (m6A) modified and can be translated to produce the E7 oncoprotein. Here, we explored the regulation and function of circE7. Mutation of m6A motifs flanking the backsplice junction revealed a single essential m6A motif to be essential for circE7 formation. Mutation of this m6A motif promoted linear splicing of the E6*I splice site (226^409), suggesting that linear and circular E7 splicing are inversely regulated. Additionally, mutation of an IRES-like motif in circE7 significantly decreased E7 protein expression, without having significant effects on circE7 RNA levels. Knockdown of YTHDC1, but not other m6A-binding proteins, decreased both circE7 RNA and protein expression. BaseScope ISH was used to confirm the expression of circE7 in HPV positive head and neck squamous cell carcinoma cell lines. Using both qRT-PCR and BaseScope ISH, we found that serum and amino acid starvation significantly increased circE7 levels. Finally, we generated a HPV16 genome with two point mutations in the circE7 m6A motif (Mut2). Stable transduction of primary keratinocytes with Mut2 confirmed the loss of circE7 and increased expression of E6*I. The Mut2 HPV16 genome exhibited significantly decreased viral replication but an increased ability to transform primary keratinocytes. Our studies reveal that the precise regulation of circE7 and E6*I by m6A are critical for the ability of HPV16 to infect and transform keratinocytes.

Type I interferon (IFN) induction is a central component of the innate immune response to viral infection, and the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) has been identified as a key mediator of IFN production during human cytomegalovirus (HCMV) infection. However, how cGAS detects HCMV remains unresolved, as the viral genome is encapsidated and trafficked directly to the nucleus, limiting cytoplasmic exposure. Here, we show that IFN induction during HCMV infection of primary fibroblast cells is predominantly driven by cGAS recognition of exogenous DNA present in standard laboratory virus preparations rather than the encapsidated viral genome. DNase treatment of AD169 and low-passage TB40/E-GFP viral stocks substantially reduced total DNA content without affecting infectivity, yet markedly abrogated IFN induction, IFN-stimulated gene expression and IRF3 nuclear translocation. Immunofluorescence analysis further revealed cytoplasmic accumulation of DNA in cells infected with untreated virus stocks, which was absent following DNase treatment. Together, these findings demonstrate that contaminating DNA in viral preparations is sufficient to activate cGAS and drive IFN responses during HCMV infection in vitro, highlighting a critical confounding factor in studies of innate immune sensing. Author SummaryHuman cytomegalovirus (HCMV) is a common herpesvirus that establishes lifelong infection and can cause serious disease in immunocompromised individuals and newborns. When cells detect viral infection, they produce type I interferons (IFNs), antiviral molecules that help limit virus spread. Previous studies have suggested that HCMV is sensed by a cellular DNA sensor called cGAS, which detects viral DNA in the cytoplasm and triggers IFN production. However, how cGAS gains access to the HCMV genome has remained unclear, because the viral DNA is enclosed within a protective capsid and transported directly to the nucleus during infection. In this study, we show that most IFN production observed during HCMV infection of fibroblast cells in vitro is driven not by sensing of the viral genome itself, but by contaminating DNA present in standard laboratory virus preparations. Treating virus stocks with DNase to remove this exogenous DNA abolished IFN induction without affecting viral infectivity. These findings highlight the importance of controlling for exogenous nucleic acids when interpreting how host cells detect viral infection.

Influenza D virus (IDV), the most recently identified member of the Orthomyxoviridae, was first isolated from pigs but cattle have been identified as the reservoir host. To date, IDV has not been confirmed to cause human disease. Like the haemagglutinin (HA) of influenza A virus (IAV) and the haemagglutinin-esterase fusion (HEF) protein of influenza C virus (ICV), the IDV HEF is produced as a precursor protein (HEF0) that must be proteolytically cleaved by host cell proteases (into HEF1 and HEF2) to gain its fusion capacity. The proteases that activate IAV HA have been extensively studied, but those responsible for activation of IDV HEF were unknown. Identifying these proteases is key to understanding early virus-host interactions and host restriction. Therefore, we generated ICV and IDV pseudotyped viruses (PVs) in HEK 293T producer cells with or without co-transfection of plasmids expressing different type II serine proteases. Subsequent transduction of swine testicular (ST) cells indicated strong activation of both ICV and IDV PVs by the human airway trypsin-like protease (HAT) and its swine homologue (swAT). Furthermore, like influenza A/Puerto Rico/8/34 (H1N1) virus, addition of exogenous protease is not essential for IDV replication in MDCK II cells, most likely due to endogenous expression of matriptase. In conclusion, our data unveil new information on host cell proteases that activate ICV and IDV HEF proteins. Importantly, the data suggest that protease specificity is not a factor in restriction of IDV replication in the human upper respiratory tract.

Retrovirus replication requires coordinated interplay between viral proteins and host cellular machinery, including reverse transcription of the viral RNA genome into DNA and its subsequent integration into the host genomes. SOX2-dependent retrotransposon dynamics have been reported for endogenous retrovirus HERV-K; however, whether a similar intracellular pathway exists for exogenous retroviruses remains unclear. To address whether infection-independent intracellular reverse transcription and partial integration can occur, that is, whether retrovirus can exhibit retrotransposon-like activity, we utilized HeLa and 293T cells, which express no HIV-1 entry receptors. We engineered an Env-deficient HIV-1 NL4-3-based reporter encoding a reverse-oriented, intron-disrupted nanoluciferase cassette that becomes expressible only after splicing followed by reverse transcription. We found that reporter activation depends on reverse transcriptase and protease activities. While integrase is dispensable for early expression, it is essential for long-term maintenance of the nanoluciferase signal. Integration site mapping using next-generation sequencing further confirmed that stable reporter activity requires integrase-dependent proviral insertion. Functional analysis of Gag revealed that membrane binding, multimerization, and budding are prerequisite steps for reporter activation. Concentrated virus preparations from culture supernatants failed to activate the reporter in 293T cells, ruling out a role for reinfection. Electron and confocal microscopy suggested that Gag or viral particles traffic through endosomal compartments. Furthermore, inhibition of dynamin- and clathrin-dependent endocytic pathways reduced reporter activity, indicating that these pathways contribute to efficient reporter activity. Collectively, these finding support the conclusion that HIV-1 can undergo intracellular reverse transcription and partial integration in an infection-independent manner, prompting a reconsideration of the boundary between exogenous retroviruses and endogenous retroelements. Author summaryEnv-independent, infection-independent intracellular reverse transcription and integration in HIV-1 may contribute to integration-site diversity within the same cell. More broadly, this phenomenon suggests continuity between the retroviral life cycle and retrotransposition dynamics, therefore informing our understanding of host-virus coevolution, mechanisms of long-term persistence, and the redesign of therapeutic strategies targeting pre-integration steps.

Cell cycle manipulation is critical to oncogenesis, including cancers associated with oncogenic gammaherpesviruses, Epstein-Barr Virus and Kaposis Sarcoma-associated Herpesvirus. Infection with these viruses can result in various cancers, including lymphomas and carcinomas. In healthy individuals, gammaherpesvirus infections result in lifelong latent infections with occasional reactivation. The cell cycle plays a critical role in infection, particularly in reactivation from quiescent latency to lytic virus replication. A number of cyclin-dependent kinase (CDK) inhibitors are clinically available but with little investigation thus far for virus-associated cancers. Using the mouse gammaherpesvirus model, we assessed the impact of CDK inhibitors on virus reactivation. First, we tested chemical inducers of reactivation, and found that optimal reactivation occurred with a combination of PMA and sodium butyrate. Application of optimal reactivation triggers demonstrated distinct stage-specific outcomes of reactivation, distinguished using flow cytometry to measure expression of GFP (early reactivation) and vRCA, a late viral protein (late reactivation). Following chemical induction of reactivation, we used flow cytometry to demonstrate that the early effects of induction were unaffected by CDK inhibitors. However, all broad spectrum CDK inhibitors tested, Dinaciclib, Alvocidib, and Seliciclib, decreased both reactivation from latency and primary lytic replication. In contrast, the impact of targeted CDK 4/6 inhibitors, Palbociclib, Ribociclib, and Abemaciclib, was more nuanced, with decreased reactivation when given concurrently, but increased reactivation when administered prior to induction. These findings were consistent for both murine gammaherpesvirus and Epstein-Barr Virus. Overall, our data indicate that CDK inhibitors may be useful for targeted treatment of gammaherpesvirus-associated cancers, but optimal use of targeted CDK 4/6 inhibitors requires careful consideration of cell state and order of therapies.

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.

The accumulation of subgenomic flavivirus RNAs (sfRNAs) modulates viral fitness and pathogenicity in culture and in vivo. These noncoding RNAs are produced by incomplete digestion of the flavivirus genome by the cellular 5-3 exoribonuclease (XRN1). Diverse flaviviruses have conserved RNA structural elements (RSEs) that map to their 3-untranslated region (3-UTR): Xrn-resistant RNA structures, dumbbell structures, and a 3-stem loop (3SL). Despite the importance of the 3-UTR RSEs for flavivirus replication, the structural dynamics of sfRNA during flavivirus infection are understudied. Here, we use digital droplet PCR to quantify sfRNA levels during infection for a panel of mosquito-borne flaviviruses (MbFV) including dengue virus serotypes 1 (DENV1), 2 (DENV2), and 4 (DENV4), and Zika virus (ZIKV). We then used SHAPE-MaP on XRN1-digested, in vitro-transcribed sfRNAs from each virus to determine their secondary structures compared to the corresponding sfRNAs obtained from flavivirus-infected A549 cells. Results seen in-cell and in vitro were largely similar; however, motifs within the dumbbell, the small hairpin (sHP) directly upstream of the 3-SL, and 3-SL regions showed significant differences in the extent of nucleotide reactivity. These differences were consistent among the four flaviviruses examined and may indicate regions of sfRNA that are shielded by interaction with proteins or other nucleic acids during infection. However, strong protection indicative of sustained interaction was not apparent. Our findings suggest that sfRNA interactions with viral and host factors within the cell are few, occur via base-paired regions, or are highly transient. ImportanceFlaviviruses are highly prevalent human pathogens. The flavivirus genome contains RNA structural elements (RSEs), including those encoded in the 3-UTR, that are necessary for viral replication. Subgenomic flavivirus RNAs (sfRNAs) are produced by incomplete digestion of flavivirus genomic RNA due to the cellular exoribonuclease XRN1 encountering 3-UTR RSEs that promote its stalling and disassociation. Viruses unable to produce sfRNAs are highly attenuated, underlining their biological importance. sfRNA secondary structure has been investigated previously but little information is available on sfRNA secondary structure dynamics in infected cells. By comparing SHAPE-MaP reactivities in vitro and in cells, we determined that previously inferred structures are likely maintained within infected cells. We also identified differences in the extent of SHAPE reactivity between in vitro and in-cell environments that were common to multiple mosquito-borne flaviviruses. These differences suggest that sfRNAs may engage in transient interactions within the cell that may be important for their function.

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.

SFTSV is an emerging tick-borne pathogen associated with high case fatality rates, particularly in elderly patients. While severe pathogenicity has been reported in aged ferrets, lethal or clinically significant infection in younger animals and genotype-dependent differences in pathogenicity remain insufficiently defined. In this study, we established a ferret infection model using two Korean clinical isolates representing genotypes B and F and systematically compared disease progression between one-year-old and three-year-old ferrets. Three-year-old ferrets exhibited rapid fever onset, marked body weight loss, early clinical deterioration, severe thrombocytopenia and leukopenia, significant elevations in AST and ALT levels, and earlier peak viremia with higher tissue viral loads, indicating impaired early viral control and accelerated systemic dissemination. Notably, one-year-old ferrets also developed measurable pathogenic manifestations, including febrile responses, progressive weight loss, detectable viremia, and multiorgan viral distribution, although disease progression was delayed and less severe compared with older animals. Within the same age group, differences in pathogenicity between genotypes B and F were limited. These findings demonstrate that host age is a major determinant of SFTSV disease severity and support the use of an age-stratified ferret model for preclinical evaluation of vaccines and antiviral therapeutics. ImportanceSFTS is an emerging tick-borne disease that can cause high fever, thrombocytopenia, and multi-organ failure, with particularly severe outcomes in older adults. Currently, no approved vaccines or specific antiviral treatments are available. Reliable animal models that recapitulate human disease are therefore essential for the development of effective countermeasures. Ferrets have recently been proposed as a useful model for SFTS, especially in aged animals, but the susceptibility of younger ferrets and the impact of viral strain differences remain unclear. Here we show that host age strongly determines disease severity in ferrets infected with two genetically distinct SFTS virus strains, establishing a flexible animal model for evaluating vaccines and antiviral therapies.

Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) are emerging tick-borne bandaviruses. They have high case fatality rates (10%), and no FDA-approved vaccines exist for disease prevention. SFTSV and HRTV are therefore identified as priority pathogens. A recombinant vesicular stomatitis virus (rVSV) vaccine, which replaces the original VSV glycoprotein with the SFTSV glycoproteins, shows early promise for SFTSV as it induces strong immune responses that are protective against lethal challenge. However, rVSV-SFTSV is highly attenuated in cell culture, which may be due to incompatibility between the assembly sites of SFTSV (the Golgi and ERGIC) and that of VSV (the plasma membrane). In this study, we identify a noncanonical COPI binding motif found in the cytoplasmic tail of SFTSV glycoproteins and demonstrate that an amino acid substitution in this motif (K1071A) inhibits binding to COPI. This mutation results in increased surface expression of SFTSV glycoproteins, improved incorporation onto VSV virions, and enhanced replication of rVSV-SFSTV in vitro. A mutation in a homologous site (K1074A) of HRTV has similar results, and rVSV-HRTV K1074A exhibits increased replication in vitro and in vivo. We show that vaccination with rVSV-HRTV K1074A results in improved induction of neutralizing antibody responses in immunocompetent C57BL/6 mice, and neutralizing antibodies elicited by vaccination are protective when administered to severely immunocompromised mice via passive transfer. Overall, our study identifies a mutation that improves the efficacy of the rVSV-SFTSV vaccine candidate and introduces the first vaccine candidate directly addressing HRTV infections. ImportanceSevere fever with thrombocytopenia syndrome virus (SFTSV) and Heartland bandavirus (HRTV) are emerging tick-borne viruses with high fatality rates. FDA-approved vaccines and antiviral drugs are unavailable but critically needed. We identify an important mutation in the SFTSV glycoprotein that disrupts a previously unreported COPI binding site. The mutation improves the efficacy of the previously described recombinant vesicular stomatitis virus vaccine candidate for SFTSV (rVSV-SFTSV). We also develop an rVSV-HRTV vaccine and show potent induction of neutralizing antibodies and protection from lethal challenge. This is the first study directly addressing the lack of vaccines specifically targeting HRTV.

Andes virus (ANDV), a highly pathogenic orthohantavirus, enters host cells through low pH-triggered membrane fusion mediated by the Gc glycoprotein, a class II fusion protein containing a single C-terminal transmembrane domain (TMD). While the ectodomain has been extensively characterized, the role of the TMD in late-stage fusion remains unclear. Here, we investigated the minimal functional length and sequence requirements of the ANDV Gc TMD using site-directed mutagenesis. C-terminal deletion mutants and serine-to-alanine substitutions were evaluated for protein expression, virus-like particle production, cell-cell fusion, pseudotyped vector entry, and hemifusion activity. Deletion of the Gc cytoplasmic tail (CT) or a single C-terminal TMD residue was tolerated, whereas deletion of two or more residues impaired particle production and fusion, indicating that at least 21 of the 22 TMD residues are required for efficient membrane fusion and viral entry. Hemifusion assays showed that deletion of two or three residues, or substitution of the strictly conserved S1121, allowed lipid mixing but blocked progression to full fusion, while deletion of four residues also abolished hemifusion. In contrast, mutation of the less conserved S1126 had minimal effect. These results identify a precise TMD length and a conserved polar TMD residue as critical determinants of fusion pore formation in ANDV.

The Capripoxviruses (CaPV) comprise three species: goatpox virus (GTPV), sheeppox virus (SPPV) and lumpy skin disease virus (LSDV). They are large double-stranded DNA viruses with highly conserved core genomes and variable terminal regions. Previous studies have described variation in CaPV gene content, their broader population structure and the contribution of non-coding and structural variation remains opaque. This study investigated the genomic diversity and evolutionary history of GTPV and SPPV using an integrative framework combining phylogenetics, pangenome variation graphs (PVGs), and gene-specific analyses. We found marked differences in population structure between the two viruses. GTPV comprised three deeply divergent and genetically stable lineages with limited evidence of recent gene flow, whereas SPPV had weaker clade separation consistent with an ancestral bottleneck followed by recent population expansion. PVG-based analyses indicated that GTPV has a comparatively closed pangenome, while SPPV remains open, particularly at the genome termini. Structural and haplotypic variation was concentrated at the inverted terminal repeats (ITRs), which moderate host immunity and specificity. In several lineages, extended putative ORFs spanning adjacent terminal genes were observed, indicating recurrent structural plasticity at the genome ends. Patterns of gene-specific conservation and divergence highlighted loci under strong constraint and lineage-specific structural changes that may contribute to host specificity. Together, these results demonstrate how graph-based genome models complement gene-based analyses in resolving poxvirus genome evolution and provide a resource for improved comparative and population genomic studies of large DNA viruses. SignificanceCapripoxviruses are economically important livestock pathogens, yet the genomic mechanisms underlying their diversification and host specificity remain poorly resolved. By applying pangenome variation graphs alongside phylogenetic and gene-level analyses, this study reveals fundamental differences in how goatpox and sheeppox viruses have evolved. Goatpox virus had a deeper, more stable lineage structure, whereas sheeppox virus was more recent and diverse. Importantly, structural variation at the inverted terminal repeats emerged as a major driver of genomic diversity, including lineage-specific haplotypes and variable gene structures. These findings demonstrated the value of graph-based genome representations for resolving complex variation in large DNA viruses and provides a framework for improving genomic surveillance, comparative analyses, and future investigations into host range, virulence and tropism.

High-risk human papillomavirus (HPV) E7 proteins bind and inactivate host cellular tumor suppressors and are essential for the immortalization of primary human keratinocytes. E7 proteins from high- and low-risk HPV genotypes bind directly to at least two tumor suppressors, RB1 and PTPN14, and inactivate both. We previously characterized mutations in high-risk HPV E7 proteins that selectively abrogate the ability of E7 to bind either RB1 or PTPN14. Here, we established a genetic complementation system using the E7 mutants defective for binding to RB1 or PTPN14. Neither mutant alone could extend the lifespan of primary keratinocytes. When expressed together, the mutants could, like wild-type high-risk HPV E7, extend keratinocyte lifespan. Both high- and low-risk E7 reduced PTPN14 protein levels and reduced expression of keratinocyte differentiation genes, whereas only high-risk E7 reduced steady-state RB1 levels and induced E2F-dependent genes. Depletion of either RB1 or PTPN14 could cooperate with low-risk HPV6 E7 to extend keratinocyte lifespan, prompting the observation that PTPN14 depletion and RB1 inactivation by HPV E7 acted synergistically to induce certain cell cycle regulatory genes. Our findings advance the model that inactivation of at least two tumor suppressors is required for the carcinogenic activity of high-risk HPV E7. Although RB1 and PTPN14 regulate distinct signaling pathways, their combined inactivation may also contribute to the biological activity of HPV E7. SignificanceInactivation of the retinoblastoma tumor suppressor (RB1) is necessary but insufficient for HPV E7-mediated immortalization of human cells. In addition to inactivating RB1, HPV E7 proteins also target for degradation PTPN14, a tumor suppressor and inhibitor of the YAP1 oncoprotein. We report genetic complementation experiments demonstrating that RB1 inactivation and PTPN14 inactivation are separate activities of E7. Either depletion of RB1 or PTPN14 can confer lifespan extension activity on a low-risk HPV E7 and reduced levels of either tumor suppressor increases the expression of certain cell cycle genes. These findings redefine our understanding of the transforming activity of the E7 oncoprotein. Inactivation of two tumor suppressors is required for E7 activity and our findings support that targeting either E7/RB1 or E7/PTPN14 would be of therapeutic benefit. We propose that synergistic control of cell cycle gene expression by E2F and YAP1-dependent transcription is essential for the transforming activity of oncogenic HPV.

Respiratory viral-bacterial co-infections cause severe disease across species, yet the molecular mechanisms underlying enhanced pathogenesis remain poorly understood. This study characterised H3N8 equine influenza A virus (IAV) and Streptococcus equi subspecies zooepidemicus (SEZ) co-infections using complementary ultrastructural and transcriptomic approaches. Transmission electron microscopy demonstrated direct physical binding between spherical (A/equine/Miami/63) and filamentous (A/equine/Sussex/89 and A/equine/Newmarket/5/2003) IAV isolates and SEZ, including when SEZ was heat-inactivated ({theta}SEZ). Lectin staining revealed that SEZ expresses predominantly 2,3-linked sialic acids, the receptor for equine IAV. However, virus-bacteria binding persisted despite neuraminidase treatment. Scanning electron microscopy quantification demonstrated that viral pre-infection significantly enhanced bacterial adherence to cells of the DH82 canine macrophage-like cell line (2-fold increase, p<0.01) but not ExtEqFL (equine lung-derived) cells, revealing cell-type-specific enhancement. RNA-sequencing analysis showed that bacterial infection drove most transcriptional changes during co-infection with little difference in the number of differentially expressed genes (DEGs) between infection with SEZ alone (146 DEGS) or after pre-infection with either A/equine/Sussex/89 (166 DEGS) or A/equine/Newmarket/5/2003 (149 DEGS). Validation of upregulation of selected cytokines by RT-qPCR and ELISA demonstrated that SEZ infection drives dramatic cytokine upregulation compared to mock or {theta}SEZ controls. Viral pre-infection did not alter the SEZ-induced pro-inflammatory cytokine responses (IL-6, IL-8, TNF-) but significantly reduced IFN-{beta} expression compared to SEZ infection alone. These findings suggest that direct virus-bacteria physical interactions may drive cell-type-specific enhancement of bacterial colonisation, fundamentally advancing our understanding of respiratory co-infection pathogenesis.