Journal of Virology最新文献

H9N2 avian influenza viruses (AIVs) remain a significant economic burden on poultry production and a persistent zoonotic threat. Hemagglutinin (HA), a surface glycoprotein mediating viral entry and pathogenesis, critically depends on thermostability for its function. Our previous study indicated that recent H9N2 AIVs have experienced a reduction in hemagglutination activity and exhibit low HA thermostability; however, the underlying molecular determinants for this instability remain poorly defined. To address this gap, we employed an in vitro-directed evolution approach to identify HA mutations that enhance thermostability. By subjecting a diverse HA mutant library to iterative heat selection at 48°C, we isolated several HA-stabilizing mutations, including L29I, N133S, N210D, G266R, D387N, A423T, and E509G, and confirmed their effect by site-directed mutagenesis. Further characterization revealed a complex interplay between HA stability, receptor binding specificity, and acid tolerance. Our findings demonstrate that enhancing HA stability can exert pleiotropic effects on key viral properties, highlighting the importance of understanding these relationships for developing effective mitigation strategies against H9N2 AIVs.

Importance: H9N2 avian influenza viruses pose a persistent threat to poultry production and human health, demanding improved control strategies. This study addresses a key knowledge gap by uncovering the molecular determinants that modulate the stability of the hemagglutinin (HA) protein in H9N2 viruses. We identify specific HA mutations that increase thermostability, a property potentially linked to environmental persistence. Furthermore, our findings demonstrate a critical interplay between HA stability and essential viral functions, including receptor binding, hemagglutination activity, replication kinetics, and overall acid tolerance. By disentangling these properties, we provide insights into the mechanisms underlying HA-mediated viral entry and infectivity, which could inform the development of more effective vaccines and therapeutics.

Reverse genetics (RG) systems are essential tools for basic virological studies and applied studies using engineered recombinant viruses in various research fields. While the circular polymerase extension reaction (CPER) has been widely applied to prepare a full-length infectious complementary DNA (cDNA) of positive-sense RNA viruses, its use for negative-sense RNA viruses (mononegaviruses) remains limited. Here, we report the first CPER-based RG system for rabies virus (RABV), a member of mononegaviruses. Infectious RABV was successfully rescued from cells transfected with helper plasmids and the CPER product, the assembled overlapping DNA fragments encoding the full-length viral genome cDNA and regulatory elements. Using this system, we generated wild-type, point-mutant, reporter-expressing, and chimeric RABVs, all of which retained their expected biological properties. Deep sequencing revealed that CPER-derived viruses occasionally harbor low-frequency mutations undetectable by Sanger sequencing, highlighting PCR-related artifacts as a limitation. In addition, CPER products with a pUC19 backbone could be directly applied for Escherichia coli transformation and cloning of RABV full-genome cDNA plasmids, offering a flexible, ligase-free cloning strategy for conventional RG. Our work establishes CPER as a versatile platform for engineering recombinant RABVs, facilitating rapid generation and genetic manipulation of RABV with potential applications for research on other mononegaviruses.IMPORTANCEReverse genetics systems allow researchers to generate recombinant viruses with precise genetic modifications, advancing studies of viral replication, pathogenicity, and vaccine development. However, constructing a full-length viral genome expressing plasmids is often time-consuming and technically demanding. To bypass the cloning process, a simple, cloning-free reverse genetics platform based on the circular polymerase extension reaction (CPER) has been applied for positive-sense RNA viruses. In this study, we applied the CPER-based reverse genetics system for rabies virus (RABV), a mononegavirus, enabling rapid and flexible generation of recombinant RABVs, including mutant, reporter-expressing, and chimeric clones. Our approach greatly facilitates genetic engineering of RABV and provides a versatile framework that can be extended to other mononegaviruses, thereby accelerating both basic and applied virology research.

Reovirus serotype 3 (T3D^PL) serves as a safe, tractable model for studying fundamental virus-host interactions and is also under investigation as an oncolytic therapy. Many viruses, including reoviruses, use sialic acids as attachment factors in addition to protein receptors; however, the relative contributions of these interactions, particularly at physiological temperatures, remain poorly defined. T3D^PL engages sialic acids and JAM-A proteins through the tail and head domains of the σ1 cell attachment protein, respectively. In this study, murine E0771 breast cancer cells were found to resist reovirus infection due to a deficiency in high-affinity receptors. T3D^PL bound to E0771 cells at 4°C but not 37°C, revealing that wild-type sialic acid interactions are thermally unstable at physiological temperatures. Exogenous JAM-A expression restored stable binding and infection at 37°C. Notably, engineered reovirus variants lacking the σ1 head domain but possessing either an RGD motif or specific mutations in the sialic acid-binding domain overcame the restriction and could bind and infect E0771 cells efficiently at 37°C. Through neuraminidase treatment and sialic acid-deficient and dependent cell models, enhanced interactions by sialic acid-binding domain mutations were empirically confirmed to be mediated by sialic acid. Serial passaging of σ1-truncated virus rapidly selected for mutations that strengthened sialic acid-binding at 37°C. Together, these findings demonstrate that high-affinity receptor requirements can be bypassed by enhancing interactions at physiological temperatures with sialic acids or through RGD interactions. This work provides insight into how viruses adapt to receptor scarcity and offers a strategy to overcome receptor heterogeneity in oncolytic virotherapy.

Importance: Reoviruses are promising oncolytic agents, yet clinical efficacy can be hindered by heterogeneous receptor expression in tumors. This study demonstrates that reovirus can bypass high-affinity receptor requirements by optimizing sialic acid interactions or incorporating RGD motifs. Crucially, the data reveal that wild-type reovirus attachment to sialic acids is thermally unstable at physiological temperature (37°C), a restriction masked by traditional 4°C assays. Specific mutations were found to stabilize these interactions at 37°C, providing a mechanistic basis for viral adaptation to receptor-deficient environments. These findings establish new experimental approaches to study attachment at 37°C, which can be applied broadly to discover unanticipated mutational effects on viral entry. Ultimately, evaluating virus-cell attachment under physiological conditions is essential for accurately predicting viral tropism and facilitates the design of next-generation oncolytic therapies better equipped to overcome receptor scarcity and thermal barriers in complex tumor environments.

Ferrets are widely used to model airborne transmission of influenza viruses in humans. Airborne transmission is evaluated by infecting donor ferrets with a high virus dose and monitoring transmission to contact animals sharing the same airspace. Humans can be infected with a broad range of influenza virus doses. Therefore, we evaluated the relationship between inoculation dose and transmission for two pandemic influenza viruses in ferrets. Donor ferrets were inoculated with 10⁰ to 10⁶ tissue culture infectious dose 50 (TCID₅₀) of the 2009 pandemic H1N1 or 1968 pandemic H3N2 virus and were then paired with respiratory contacts. Using the proportion of donors that became infected across virus doses, we calculated the infectious dose 50 (ID₅₀). Subsequently, by comparing the proportion of contacts that became infected, we calculated the transmissible dose 50% (TD₅₀): the donor inoculation dose that resulted in transmission to 50% of contacts. For the 2009 pandemic H1N1 virus, the ID₅₀ and TD₅₀ were equivalent at <1 TCID₅₀. However, for the 1968 pandemic H3N2 virus, the ID₅₀ and TD₅₀ were 10^0.5 and 10^4.08 TCID₅₀ (95% CI: 10^2.34-10^5.82), respectively. The increased TD₅₀ for the H3N2 virus was associated with significant reductions in peak viral titers and viral shedding in donors over decreasing virus inoculation doses. Collectively, these studies define a new measure of transmission that permits comparisons of transmissibility between viral strains and subtypes in ferrets. We show that the 1968 pandemic H3N2 virus has a higher TD₅₀ and reduced transmissibility in ferrets relative to the 2009 pandemic H1N1 virus.

Importance: Ferrets are the gold standard animal model used to assess the transmissibility of influenza viruses. Airborne transmission is evaluated by infecting donor ferrets with a high virus dose and monitoring transmission to contact animals sharing the same airspace. However, the relationship between inoculation dose and transmission has not been evaluated in ferrets. Therefore, we performed studies evaluating airborne transmission of the 2009 pandemic H1N1 and 1968 pandemic H3N2 viruses over log scale reductions in donor inoculation doses. Using the results of these studies, we define a new measure of transmission, the transmissible dose 50%: the donor inoculation dose at which a virus is transmitted to 50% of contacts. Importantly, this metric permits the evaluation of transmissibility over a log scale. We demonstrate that the 1968 pandemic H3N2 virus has reduced transmissibility compared to the 2009 pandemic H1N1 virus in ferrets.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a major viral disease that poses a serious threat to the global swine industry. Although progress has been made in understanding its life cycle, the molecular mechanisms underlying PRRSV entry and replication remain incompletely understood. Multiple RNA viruses hijack the endocytic sorting complex required for transport (ESCRT) machinery to orchestrate various stages during infection. In the current study, we identified ESCRT-II subunit ELL-associated protein 20 (EAP20) as an important host factor involved in PRRSV entry and replication. Mechanistically, EAP20 participated in the transport of internalized PRRSV particles to early endosomes via the clathrin-mediated endocytosis pathway. During replication, EAP20 interacted with PRRSV nonstructural protein (Nsp) 2, Nsp5, and Nsp9. Specifically, EAP20 anchored the core replicase Nsp9 on the perinuclear endoplasmic reticulum (ER) and coordinated with the transmembrane proteins Nsp2/Nsp5 to form ER-derived double-membrane vesicles. Collectively, our findings demonstrate that PRRSV exploits EAP20 for viral entry and replication, highlighting EAP20 as a novel proviral factor and a potential antiviral target.

Importance: PRRSV remains one of the most economically significant pathogens in the global swine industry. Current control strategies are largely hindered because PRRSV pathogenesis has not been fully elucidated. In this study, we identified EAP20, a core subunit of ESCRT-II, as a multifaceted proviral factor that participated in PRRSV entry and replication. These findings provide new insights into the interplay between PRRSV and the host ESCRT machinery, laying a foundation for the development of more effective strategies for PRRS control.

The impact of physiological stress conditions on Kaposi's sarcoma-associated herpesvirus (KSHV) infection remains poorly understood. One such stressor, hypoxia, is regulated by the transcription factor HIF-1α. We recently reported that hypoxia, or HIF-1α expression alone, can promote lytic infection in cells that typically support latent infection under normoxia. Here, we show that hypoxia-induced lytic infection is reversible, leading to an abortive lytic cycle if the hypoxic condition ceases. Additionally, we found that HIF-1α induces lytic de novo infection only if expressed within the first 24 h post-infection (hpi). We show that HIF-1α can bind to viral promoters and induce lytic genes only during this early window of infection, before the KSHV genome undergoes heterochromatinization and establishes latency. In contrast, regardless of the timing of HIF-1α expression during KSHV infection, the induction of HIF-1α host target genes remains unaffected. These results indicate that the heterochromatinized KSHV DNA becomes resistant to HIF-1α-mediated activation after latency is established. These findings may explain why, despite the expression of HIF-1α in Kaposi's sarcoma tumors, KSHV remains in latency, because HIF-1α cannot induce lytic genes once the viral DNA is heterochromatinized. Importantly, we also demonstrate that inhibition of the epigenetic repressor PRC2, which associates with lytic promoters after 24 hpi, restores HIF-1α's ability to bind viral promoters and induce lytic gene expression post-latency. Collectively, our results indicate that not only the presence of HIF-1α, but also the timing and duration of its expression during KSHV infection, are critical determinants of its ability to drive lytic infection.IMPORTANCEThe current view is that the default pathway of KSHV infection is the establishment of latency, however, how this is altered under physiological stress conditions remains largely unknown. We previously showed that hypoxia, or the expression of its transcription factor HIF-1α alone, promotes the establishment of lytic rather than latent KSHV infection. In this study, we show that the duration of hypoxia, as well as the timing and duration of HIF-1α expression, are crucial determinants in facilitating lytic de novo KSHV infection. Notably, we found that PRC2-mediated heterochromatin inhibits the HIF-1α-mediated upregulation of lytic genes as chromatinization of the KSHV genome progresses during infection. Our findings offer a deeper understanding of how epigenetic regulation intersects with host stress responses to influence viral pathogenesis.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes lifelong latency in the host but can reactivate in the lytic cycle under specific pathophysiological conditions. The replication and transcription activator (RTA) serves as the master regulator of this latent-to-lytic switch. Although RTA is well known as a potent viral transcriptional activator and E3 ubiquitin ligase, its role in modulating host gene expression remains incompletely understood. In this study, we identified eukaryotic translation elongation factor 1δ (EEF1D) as a previously unrecognized host inhibitory factor that inhibits KSHV reactivation. Functional analyses revealed that ectopic expression of EEF1D suppressed viral lytic replication, whereas EEF1D depletion enhanced viral reactivation. KSHV RTA counteracts this inhibition through two complementary mechanisms: interaction with EEF1D protein to promote its ubiquitin-proteasome-mediated degradation and, more prominently, repression of EEF1D transcription through promoter silencing. Dual-luciferase reporter assays further revealed that this transcriptional repression is conserved among primate γ-herpesviruses, including KSHV, rhesus rhadinovirus, and Epstein-Barr virus, but is absent in murine γ-herpesvirus 68. Mechanistically, RTA induces DNMT3A-dependent hypermethylation of the EEF1D promoter, a process facilitated by the transcription factor, PATZ1. Collectively, these findings reveal a previously unrecognized repressive function of RTA on the host gene EEF1D, highlighting an additional layer of viral strategy to promote lytic reactivation.

Importance: Kaposi's sarcoma-associated herpesvirus (KSHV) establishes lifelong latency in host cells but can periodically reactivate, a process that is essential for viral spread and disease development. The viral replication and transcription activator (RTA) serves as the master switch of this transition; however, the host factors that inhibit reactivation and the mechanisms by which RTA overcomes them remain incompletely defined. In this study, we identified the host protein eukaryotic translation elongation factor 1δ (EEF1D) as a previously unrecognized inhibitor of KSHV lytic replication. However, RTA inhibited EEF1D expression at both the protein and transcriptional levels. These findings expand the functional repertoire of RTA by revealing its ability to repress host gene transcription, providing new insights into viral persistence and pathogenesis.

Glycoprotein B (gB) serves as the viral fusion protein for herpes simplex virus (HSV), mediating fusion between viral and host membranes resulting in infection. As such, gB represents a critical target for the host immune system with high potential relevance for vaccine design. Here, we investigated the mechanisms of protection for a panel of gB-specific monoclonal antibodies (mAbs) in a mouse model of neonatal HSV (nHSV) infection. Depending on dose, viral neutralization contributed, but Fc effector functions were critical for mAb-mediated protection against nHSV mortality. Moreover, adeno-associated virus-mediated in vivo expression of a gB-specific mAb in mice provided transgenerational protection against HSV-1 and HSV-2 mortality. These findings demonstrate that antibodies targeting gB can serve as potent therapeutics and that they require diverse functional profiles to afford optimal protection, informing vaccine design.IMPORTANCEAntibodies represent promising drugs for the prevention and treatment of viral infections, especially when efficacious vaccines are unavailable. Determining the dominant mechanisms of Ab-mediated protection is a critical step in the design and optimization of potential antibody therapies. In this study of antibody-mediated protection of neonatal mice from herpes simplex virus, efficacy and mechanism of action of antibodies that recognize viral glycoprotein B (gB) were dependent on dose, effector functions, and viral neutralization capacity. Overall, while viral neutralization likely contributes to monoclonal antibody-mediated protection, the ability for gB-specific antibodies to mediate Fc domain-dependent effector functions was unexpectedly crucial.

Many distinct viruses exploit cell surface glycans, particularly heparan sulfates and sialic acids, as initial attachment factors to facilitate entry into host cells. Because these interactions are highly conserved across diverse viral families, they have long been viewed as attractive targets for the development of broad-spectrum antiviral strategies. Over the past decades, numerous approaches have attempted to block these early binding events, including genetic or enzymatic removal of glycans from the cell surface, masking of cell surface glycans, the use of engineered decoy receptors, and the development of multivalent inhibitors. Despite promising in vitro results, no antiviral therapy based on this mechanism has yet advanced to routine clinical use. Here, the biological roles of heparan sulfates and sialic acids in viral entry are examined, and the range of antiviral strategies designed to interfere with these interactions is discussed. The major challenges that have limited clinical translation are highlighted, including insufficient potency, potential off-target effects, the risk of resistance, and challenges related to routes of administration. Finally, recent technological advances that may help overcome these barriers and enable the development of clinically viable viral attachment inhibitors are proposed.