Journal of Virology最新文献

Treatment options remain limited for human cytomegalovirus (HCMV). Host telomerase has been implicated in the pathogenesis and oncogenesis of multiple herpesviruses, most recently including HCMV. In this study, we investigated the effect of telomerase inhibition on HCMV replication, as well as the mechanism of the interaction between HCMV and host telomerase in vitro. We found that lytic HCMV infection increases host telomerase activity, at least in part, through modulation of hTERT expression during earlier phases of the HCMV replication cycle. We found telomerase inhibition strongly reduced viral titer for two HCMV strains in a dose-specific manner. Both post-translational pharmaceutical telomerase inhibition and siRNA-mediated knockdown of hTERT reduce HCMV yield. Telomerase inhibition results in both reduction of viral gene and protein expression across the HCMV replication cycle, and suppressed viral genome replication and viral infectivity, suggesting interference with at least early steps of the HCMV viral life cycle. Altogether, our findings indicate telomerase plays an important, perhaps non-canonical role in lytic HCMV infection which includes the support of viral replication and infectivity.

Importance: Human cytomegalovirus (HCMV) seroprevalence and morbidity in immunocompromised patients and neonates infected in utero remain high globally. Host telomerase has been implicated in the success of multiple infection-induced pathologies, including the success of both lytic infection and oncogenesis in certain herpesviruses. The results of this study suggest a similar biologically important role for host telomerase in lytic HCMV infection. Furthermore, these results may provide the potential for a novel, adjunctive anti-viral treatment for HCMV infection as well as insight into the viral products likely to be involved with HCMV regulation of telomerase.

RAF1 is a key player in growth factor receptor signaling, which has been linked to multiple viral infections, including human cytomegalovirus (HCMV) infection. Although HCMV remains latent in most individuals, it can cause acute infection in immunocompromised populations, such as transplant recipients, neonates, and cancer patients. Current treatments are suboptimal, highlighting the need for novel therapies. Multiple points in the growth factor signaling pathway are important for HCMV infection, but the relationship between HCMV and RAF1, a component of the mitogen-activated protein kinase (MAPK) cascade, is not well understood. The AMP-activated protein kinase (AMPK) is a known regulator of RAF1, and AMPK activity is induced by HCMV infection, which is important for productive HCMV replication. Our data indicate that HCMV infection induces AMPK-specific changes in RAF1 protein phosphorylation, including increasing phosphorylation at RAF1-Ser621, a known AMPK phospho-site, which results in increased binding to the 14-3-3 scaffolding protein, an important aspect of RAF1 protein activation. Inhibition of RAF1, either pharmacologically or via shRNA or CRISPR-mediated targeting, inhibits viral replication and spread in both fibroblasts and epithelial cells. Collectively, our data indicate that HCMV infection and AMPK activation modulate RAF1 activity, which is important for viral replication.

Importance: Human cytomegalovirus (HCMV) infection is a widespread infection impacting approximately 60-90% of the global population. Although latent in healthy individuals, acute infection in immunocompromised populations, such as neonates, transplant recipients, and cancer patients, can result in retinal and gastrointestinal problems, hearing loss, and even death. Current antivirals are suboptimal due to the development of viral resistance or toxicity in patients, highlighting the need for novel treatments. Our research suggests a new potential target, RAF1, which is a regulator of cellular growth and proliferation. We find that RAF1 is phosphorylated by AMP-activated protein kinase, and that inhibition of RAF1 negatively impacts viral infection. Furthermore, drugs currently used to treat certain cancers also inhibit RAF1 and may have an additional anti-HCMV therapeutic effect in HCMV-susceptible cancer patients.

A successful strategy employed by RNA viruses to achieve replication is to evade host cell RNase degradation. However, the mechanisms through which plus-strand RNA viruses effectively shield viral RNA from cellular ribonuclease degradation remain unclear. In this study, we identified the phenomenon whereby plus-strand RNA viruses, including avian leukosis virus subgroup J (ALV-J), reticuloendotheliosis virus (REV), chicken astrovirus (CAstV), and porcine epidemic diarrhea virus (PEDV), hijacked host cellular Musashi homolog 1 (MSI1). These viruses upregulated MSI1 expression and facilitated its translocation from the cytoplasmic periphery to a position proximal to and within the nucleus, thereby protecting viral RNA from degradation. Mechanistic analyses revealed that these viruses use distinct regions, the unique (U3) region or three prime untranslated region (3'UTR), to engage with MSI1, consequently shielding their viral RNA from cytoplasmic ribonuclease degradation. These results offer significant implications for understanding the replication tactics used by plus-strand RNA viruses, thereby advancing our understanding of their biological behaviors.IMPORTANCEThe intricate interplay between RNA viruses and host cell RNA regulation encompasses viral mechanisms designed to circumvent RNase-mediated degradation. However, the specific strategies employed by plus-strand RNA viruses to shield their RNA from host ribonucleases remain inadequately characterized. In this study, Musashi homolog 1 (MSI1) is predominantly localized in the cytoplasm of normal cells, distinct from the nucleus. Following infection by plus-strand RNA viruses such as avian leukosis virus subgroup J (ALV-J), reticuloendotheliosis virus (REV), chicken astrovirus (CAstV), and porcine epidemic diarrhea virus (PEDV), these viruses hijack MSI1 to relocate near and within the nucleus. This hijacking is facilitated by specific regions, including unique or three prime untranslated regions, thereby preventing viral RNA from degradation by cytoplasmic ribonucleases. These findings have significant implications for elucidating the replication strategies of plus-strand RNA viruses, thereby advancing our understanding of their biological mechanisms.

Bovine mastitis, primarily caused by Staphylococcus aureus, significantly affects the dairy industry by reducing milk production and quality. The rise of antibiotic-resistant bacteria has prompted the need for alternative treatments. The three newly isolated bacteriophages, OPT-SA02, OPT-SC01, and OPT-SX11, were isolated from chicken fecal and sewage samples in South Korea. These bacteriophages were characterized via physiological and genomic analyses, identifying their therapeutic potential against S. aureus-induced mastitis. The bacteriophages were identified as members of the Herelleviridae family, exhibiting stability across broad pH (2-12) and temperature (37-70°C) ranges, as well as strong antibacterial activity at low multiplicity of infection (MOI) levels. Genomic analysis revealed that the conservation of lysis-related genes (holin and endolysin) is responsible for their lytic capabilities. Additionally, protein structural predictions revealed multi-domain structures in their endolysins, enhancing their lytic potential. These findings suggest that OPT-SA02, OPT-SC01, and OPT-SX11 show significant promise as alternative treatments for bovine mastitis.IMPORTANCEBovine mastitis, caused by pathogens such as Staphylococcus aureus and Staphylococcus xylosus, remains a major challenge in dairy farming, leading to significant economic losses and reduced milk quality. The increasing prevalence of antibiotic-resistant strains further complicates treatment, emphasizing the need for alternative strategies. This study identifies three newly isolated bacteriophages with effective antibacterial activity against these pathogens and provides comprehensive genomic and structural insights into their mechanisms. Genomic characterization revealed conserved lytic cassettes and genetic diversity within related bacteriophages, offering a deeper understanding of their evolutionary relationships and potential applications. Furthermore, protein structure analysis of the endolysin derived from these bacteriophages identified multi-domain architectures with preserved catalytic cores, underscoring their lytic efficacy against bacterial cell walls. These findings advance the understanding of the genetic and structural mechanisms of bacteriophage-mediated lysis and highlight their potential as sustainable tools for managing bovine mastitis and improving milk quality in dairy farming.

Recent avian-origin H3N8 influenza A virus (IAV) that have infected humans pose a potential public health concern. Alterations in the viral surface glycoprotein, hemagglutinin (HA), are typically required for IAVs to cross the species barrier for adaptation to a new host, but whether H3N8 has adapted to infect humans remains elusive. The observation of a degenerative codon in position 228 of HA in human H3N8 A/Henan/4-10/2022 protein sequence, which could be residue G or S, suggests a dynamic viral adaptation for human infection. Previously, we found this human-isolated virus has shown the ability to transmit between ferrets via respiratory droplets, with the HA-G228S substitution mutation emerging as a critical determinant for the airborne transmission of the virus in ferrets. Here, we investigated the receptor-binding properties of these two H3N8 HAs. Our results showed H3N8 HAs have dual receptor-binding properties with a preference for avian receptor binding, and G228S slightly increased binding to human receptors. Cryo-electron microscopy structures of the two H3N8 HAs with avian and human receptor analogs revealed the basis for dual receptor binding. Mutagenesis studies reveal that the Q226L mutation shifts H3N8 HA's receptor preference from avian to human, while the G228S substitution enhances binding to both receptor types. H3N8 exhibits distinct antigenic sites compared to H3N2, prompting concerns regarding vaccine efficacy. These findings suggest that the current H3N8 human isolates are yet to adapt for efficient human-to-human transmission and further continuous surveillance should be implemented.IMPORTANCEInfluenza virus transmission remains a public health concern currently. H3N8 subtype influenza A viruses infect humans and their HAs acquire the ability to bind to both human and avian receptors, posing a threat to human health. We have solved and analyzed the structural basis of dual receptor binding of recently human-infecting H3N8 HA, and we demonstrate that the G228S enhances human receptor binding and adaptation. We also found that HN/4-10 H3N8 HA has distinct antigenic sites, which challenges vaccine efficacy. Taken together, our work is critical to the prevention and control of human H3 influenza virus infection.

Seneca Valley virus (SVV) is an emerging pathogen that causes severe vesicular diseases in swine, posing a significant threat to the global pork industry. DNA and RNA viruses manipulate the host DNA damage response (DDR) to modulate cellular machinery and facilitate their life cycles. However, the interaction between the host DDR and SVV infection remains unexplored. Here, we aimed to comprehensively investigate the DDR and DNA repair signaling pathways during SVV infection. We found that SVV infection causes DNA damage and triggers distinct DDR signaling pathways, including ataxia telangiectasia-mutated (ATM) kinase, ATM-Rad3-related kinase, and DNA-dependent protein kinase. However, it failed to induce the formation of γH2AX and 53BP1 foci, resulting in unrepaired DNA damage. Furthermore, we found that SVV 2B and 2C proteins can activate DDR signaling pathways and impair DNA repair. SVV-induced DDR triggered NF-κB signaling accompanied by upregulation of pro-inflammatory cytokines, as evidenced by the inhibition of ATM kinase, abolished SVV-induced NF-κB activation. Inhibition of the ATM pathway attenuated SVV replication. These findings expand our understanding of host DDR manipulation during viral infection and provide crucial insights into a novel mechanism exploited by SVV to regulate the inflammatory response for efficient replication.IMPORTANCEDDR is a cellular machinery that senses and repairs host DNA lesions to maintain genome integrity. Viruses have evolved diverse strategies to manipulate host DDR for replicative efficiency. SVV is an emerging virus that causes vesicular diseases in pigs and severely threatens the swine industry. However, the interaction between SVV and DDR remains unclear. Here, we found that SVV modulates host DDR pathways to facilitate viral replication. Our results demonstrated that SVV infection causes DNA damage, activates ATM-mediated DNA double-strand break response, and impedes DNA repair. SVV 2B and 2C proteins induced DNA damage and activated the DDR pathway while impairing repair mechanisms. This study revealed a fine-tuned molecular mechanism of SVV-modulated DDR that contributes to viral replication, facilitating deeper insight into SVV replication.

Orthoflaviviruses remodel the endoplasmic reticulum (ER) network to construct replication organelles (ROs) for RNA replication. In this study, we demonstrate that the Japanese encephalitis virus (JEV) NS1 protein concentrates ER membranes in the perinuclear region, which provides a substantial membrane source for viral replication. Subsequently, the virus forms main replication organelles within this membrane-concentrated area to facilitate efficient replication. This process relies on the ER localization signal, glycosylation, dimerization, and membrane-binding sites of the NS1 protein. In conclusion, our study highlights the role of the NS1 protein in the formation of the ROs by JEV, providing new insights into orthoflavivirus replication.IMPORTANCEOrthoflaviviruses use the endoplasmic reticulum (ER) membranes for replication by forming invaginations to assemble the replication organelles. Here, we found that Japanese encephalitis virus (JEV) utilizes the NS1 protein to concentrate a significant number of ER membranes in the perinuclear area, thereby providing a membrane source for viral replication and facilitating the formation of main replication organelles (MROs). This process depends on the ER localization signals of NS1, as well as its glycosylation, dimerization, and membrane-binding sites, but not on the cytoskeleton. In summary, our study highlights how NS1 remodels ER membranes to facilitate the formation of MROs for JEV, thereby accelerating viral replication.

Herpes simplex viruses (HSV) cause many skin diseases, particularly in immunocompromised patients. HSV-1 infection of murine skin recapitulates many aspects of human pathology. However, many protocols rely on mechanical or enzymatic skin disruption to induce lesions, although this can alter skin homeostasis and prime antiviral inflammation before inoculation. To investigate the initial events following HSV-1 primary skin infection before the onset of symptoms, we developed a novel murine ex vivo explant model using gentle depilation without further scarification and infected keratinocytes from the outside with minimal tissue damage. Two-photon microscopy showed that HSV-1 spread exclusively in the epidermis. The infection centers increased in number and size over time and contained hundreds of infected keratinocytes. We investigated the HSV-1 spread at the cellular level, using reporter strains with fluorescently tagged capsid protein VP26, and observed the formation of nuclear capsid assembly sites and nuclear capsid egress and the recruitment of the inner tegument protein pUL37GFP, the outer tegument protein VP11/12GFP, and the envelope protein gDGFP to cytoplasmic capsids. By using electron microscopy, the skin appeared intact, and keratinocytes contained many nuclear capsids, primary virions in the nuclear envelope, cytosolic membrane-associated capsids, and enveloped virions. Our protocol provides a robust and reproducible approach to investigate the very early events of HSV-1 spread in the skin, to characterize the phenotypes of HSV-1 mutants in terminally differentiated skin tissues, and to evaluate potentially antiviral small molecules in a preclinical ex vivo infection model.

Importance: This study describes a novel murine ex vivo skin explant model to investigate early events in HSV-1 infection without causing significant tissue damage. To infect from the outside, via the apical keratinocytes, this method relies on gentle depilation, which maintains skin integrity. HSV-1 spread exclusively within the epidermis, with infection centers increasing over time and involving hundreds of keratinocytes. Using advanced microscopy techniques, we tracked HSV-1 spread at the cellular level and intracellular assembly of all intermediate virus structures. This model offers a valuable tool for studying the initial stages of HSV-1 infection, assessing viral mutant phenotypes, and testing antiviral compounds in a more physiological context to provide critical insights into HSV-1 pathogenesis and therapeutic strategies.

Human immunodeficiency virus (HIV), a retrovirus of the Lentivirus genus, targets CD4⁺ T cells, causing immune dysfunction and AIDS. Approximately 8% of the human genome consists of human endogenous retroviruses (HERVs), ancient retroviral remnants that may interact with HIV. Despite antiretroviral therapy, challenges such as drug resistance, poor immune reconstitution (PIR), and reservoirs remain. This GEM discusses the impact of HIV on HERVs, the potential roles of HERVs in PIR and reservoirs, and provides insights into future research directions.

IFI16 (interferon-γ-inducible protein 16) is an innate-immune DNA sensor that detects viral dsDNA in the nucleus. It also functions as an antiviral restriction factor, playing a crucial role in regulating the latency/lytic balance of several herpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV). We previously demonstrated that IFI16 achieves this by regulating the deposition of H3K9me3 marks on the KSHV genome. Here, we explored whether IFI16 impacts the KSHV latency/lytic balance through additional mechanisms. Our analysis of the IFI16 interactome revealed that IFI16 binds to the class-I HDACs, HDAC1 and HDAC2, and recruits them to the KSHV major latency protein, latency-associated nuclear antigen (LANA). Previous reports have suggested that LANA undergoes lysine acetylation through unknown mechanisms, which results in the loss of its ability to bind to the KSHV transactivator protein (RTA) promoter. However, how the LANA acetylation-deacetylation cycle is orchestrated and what effect this has on KSHV gene expression remains unknown. Here, we demonstrate that LANA, by default, undergoes post-translational acetylation, and during latency, IFI16 interacts with this acetylated LANA and recruits HDAC1/2 to it. This keeps LANA in a deacetylated form, competent in binding and repressing lytic promoters. However, during lytic reactivation, IFI16 is degraded via the proteasomal pathway, leading to the accumulation of acetylated LANA, which cannot bind to the RTA promoter. This results in the de-repression of the RTA and, subsequently, other lytic promoters, driving reactivation. These findings shed new light on the role of IFI16 in KSHV latency and suggest that KSHV utilizes the cellular IFI16-HDAC1/2 interaction to facilitate its latency.

Importance: Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesviruses etiologically associated with several human malignancies, including Kaposi's sarcoma, primary effusion B-cell lymphoma, and multicentric Castleman's disease. Understanding the molecular mechanisms governing the establishment and maintenance of latency in γ-herpesviruses is crucial because latency plays a pivotal role in oncogenesis and disease manifestation post-infection. Here, we have elucidated a new mechanism by which IFI16, a previously discovered antiviral restriction factor, is hijacked by KSHV to recruit class-I HDACs on latency-associated nuclear antigen (LANA), resulting in the latter's deacetylation. The acetylation status of LANA is critical for KSHV latency because it governs LANA's binding to the KSHV replication and transcription activator (RTA) promoter, an immediate-early gene crucial for lytic reactivation. Depletion of IFI16 results in the accumulation of acetylated LANA, which is incapable of maintaining latency. These newly discovered interactions between IFI16 and LANA and between IFI16 and HDAC1/2 enhance our understanding of KSHV latency regulations.