Journal of Virology最新文献

The tubulin code, defined by diverse post-translational modifications of microtubules, fine-tunes microtubule dynamics, regulating downstream cellular signaling. Among these, detyrosination of α-tubulin has long been recognized, yet its upstream regulation and physiological roles in viral infection remain unclear. Here, we show that influenza A virus (IAV) infection induces heterogeneous cell death morphologies in macrophages, including pyroptotic "ghost" cells and apoptotic beaded apoptopodia. Beaded apoptopodia were enriched with detyrosinated α-tubulin, a long-lived, stable microtubule modification generated by enzymatic removal of the C-terminal tyrosine residue. We found that the detyrosination was dependent on caspase-1-mediated inflammasome signaling. Pharmacological inhibition of vasohibin-1 (VASH1) suppressed detyrosination without affecting viral replication, identifying VASH1, but not VASH2, as the predominant carboxypeptidase responsible for this modification upon IAV infection. Overexpression of VASH1 enhanced detyrosination and shifted cell death from pyroptosis toward apoptosis. These findings uncover a cytoskeletal pathway that modulates inflammasome signaling toward immunologically silent apoptosis, thereby limiting inflammatory cell lysis. This mechanism highlights the physiological significance of VASH1-mediated detyrosination in shaping host responses to viral infection.

Importance: Programmed cell death is an essential host response to viral infection, but whether infected macrophages undergo inflammatory or non-inflammatory forms of death has important consequences for disease progression. In this study, we found that influenza A virus infection induces a modification of microtubules known as detyrosination, which stabilizes their structure. This change was driven by the host enzyme vasohibin-1 through activation of the inflammasome, a key signaling complex that normally promotes inflammatory cell death. Remarkably, enhanced detyrosination shifted dying cells away from inflammatory membrane rupture toward apoptosis, an immunologically silent cell death pathway that preserves membrane integrity. Our findings identify microtubule detyrosination as a stress-induced host response during influenza A virus infection, highlighting a novel mechanism by which cytoskeletal modification influences the outcome of infection.

The productive replication of highly pathogenic Nipah virus (NiV) relies on the host cell for the synthesis and correct folding of the viral glycoproteins, which can cause ER stress and activation of the unfolded protein response (UPR). While the UPR can exert proviral functions by restoring ER homeostasis, it can also have antiviral effects. Here, we show that irreversible ER stress induced by thapsigargin resulted in broad expression of UPR target genes and potently inhibited NiV infection. The finding that UPR target gene upregulation was not detectable in NiV-infected cells at 18 h p.i., raised the question of how NiV regulates UPR activation to prevent thapsigargin-like antiviral effects. To address this, we analyzed the effects of NiV glycoprotein expression on UPR activation and found that both NiV glycoproteins F and G, like many other viral glycoproteins, activated the highly conserved IRE1/XBP1 branch of the UPR. Interestingly, upon coexpression of both NiV glycoproteins and thereby induced cell-cell fusion, the activation did not increase. Instead, UPR activation relatively decreased with increasing syncytium formation, an effect that was not observed if cell-cell fusion was blocked. These results support the idea that syncytium formation limits UPR activation despite ongoing viral glycoprotein synthesis. This as yet undescribed mechanism is likely a fusion-dependent countermeasure to prevent an overload of the ER folding capacity by dilution and suggests that NiV-induced syncytium formation not only is an important way to promote NiV spread from cell-to-cell but also regulates ER stress to limit potential UPR-induced antiviral responses.IMPORTANCEThe unfolded protein response (UPR) is a cellular signaling pathway to counteract ER stress. Many enveloped viruses, which force the infected cell to synthesize high amounts of viral surface glycoproteins, induce the UPR but regulate its activation by diverse strategies to prevent UPR-mediated antiviral effects. To date, nothing is known about UPR activation in infections with Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family. Here, we demonstrate that NiV glycoproteins activate the IRE1/XBP1 branch of the UPR. However, this activation is limited by the cell-cell fusion mediated by the glycoproteins, probably due to dilution effects. This study is the first to investigate the interplay between NiV and UPR activation and proposes a novel strategy by which fusogenic viruses may limit the ER stress responses triggered by their glycoproteins.

The phosphoinositide 3-kinase (PI3K)-Akt pathway is a key signaling cascade regulating diverse cellular processes, including proliferation, survival, autophagy, translation, and metabolism. White spot syndrome virus (WSSV), a major pathogen devastating global crustacean aquaculture, has been demonstrated to exploit the PI3K-Akt pathway to facilitate its proliferation. However, the precise mechanism underlying this viral modulation remained unclear. In this study, we demonstrate that WSSV infection induces activation of the PI3K-Akt pathway during the early infection stage in Penaeus vannamei. Mechanistically, we reveal that the WSSV immediate-early protein IE1 interacts with and activates host Src64B kinase via its Y₁₂₉FTS tyrosine motif. This specific interaction promotes recruitment of the PI3K regulatory subunit alpha (PI3Kp85α), thereby triggering the downstream PI3K-Akt signaling. By activating this pathway, WSSV establishes a favorable environment for its proliferation by suppressing host apoptotic and autophagic defenses. Our findings unveil a previously unknown mechanism of WSSV immune evasion through Src-PI3K-Akt signaling hijacking and identify components of this signaling hub as potential therapeutic targets for anti-WSSV strategies.

Importance: Viruses usually hijack host signaling pathways to enhance infectivity and evade immune defenses. Understanding these interactions is critical for elucidating viral pathogenesis and developing effective antiviral strategies. Here, we demonstrate that the WSSV immediate-early protein IE1 binds to and activates host Src64B kinase, which in turn recruits PI3Kp85α and activates the PI3K-Akt signaling cascade. Activation of this pathway suppresses apoptosis and autophagy, thereby facilitating viral proliferation. These findings advance our understanding of WSSV pathogenesis and identify the Src-PI3K-Akt signaling as a promising therapeutic target for anti-WSSV intervention.

With the increasing prevalence of antibiotic-resistant bacteria, phage therapy has garnered significant attention. Holin and lysin play essential roles in the phage-induced lysis of bacteria. This study investigated the functions of the holin protein Hol 46 and the lysozyme protein Lys 17 from the phage PZL-Ah152 and the mechanisms underlying the action of the fusion protein Hol 46_Lys 17. Assays, including membrane protein extraction tests and fluorescence microscopy, verified that the Hol 46 protein localized to the cell membrane and significantly inhibited the growth of Aeromonas hydrophila Ah152. We determined that the Hol 46 C-terminal domain and glutamic acid residue at position 66, along with the lysine residues at positions 63 and 64, were critical for its cell-penetrating activity. Furthermore, the Hol 46_Lys 17 fusion protein was developed to combine the membrane-disrupting capacity of Hol 46 with the lytic action of Lys 17. Hol 46_Lys 17 exhibited not only broader antibacterial effects against Aeromonas (24/38) but also against Escherichia coli (3/12) and Salmonella (5/29). Transcriptomic studies revealed that treatment with Hol 46_Lys 17 led to significant changes in the expression of genes related to flagellar synthesis, bacterial chemotaxis, and TCSs. Finally, animal studies were performed to confirm the safety and effectiveness of Hol 46_Lys 17 in treating intestinal infections caused by A. hydrophila in crucian carp. Our data showed that phage lytic system-related proteins hold great potential for the treatment of infections caused by antibiotic-resistant bacteria.IMPORTANCEAs a zoonotic and fish-pathogenic bacterium, Aeromonas hydrophila causes significant harm worldwide. Owing to the emergence of A. hydrophila strains with multidrug resistance, phage therapy has garnered extensive attention. Holin and lysin, phage-derived antibacterial proteins, play crucial roles in antimicrobial activity. The glutamic acid at position 66 and lysine residues at positions 63 and 64 in the C-terminal domain of the Hol 46 protein from phage PZL-Ah152 were essential for its A. hydrophila cell-penetrating activity. The Hol 46_Lys 17 fusion protein exhibited broad-spectrum antibacterial activity, including effects against Salmonella and Escherichia coli. Transcriptomic assays further revealed the effects of Hol 46_Lys 17 on A. hydrophila Ah152 at the molecular level. In vivo studies confirmed its efficacy and safety for the treatment of intestinal infections in crucian carp.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is one of the most severe viral diseases affecting swine. ASFV employs sophisticated strategies to subvert host immune responses; however, the function of the viral protein g5Rp in viral pathogenesis remains incompletely defined. In this study, we demonstrate that g5Rp plays a critical role in viral replication by impairing host translation and autophagy. Overexpression of g5Rp enhanced viral replication and increased p30 protein levels, whereas siRNA-mediated knockdown of g5Rp suppressed both, underscoring its essential proviral function. Proteomic profiling of infected porcine macrophages (3D4/21 cells) revealed that g5Rp dysregulated 122 host proteins, predominantly involved in translation, autophagy, and apoptosis pathways. Mechanistically, g5Rp directly interacted with eIF5A and RPS15, disrupting their complex formation and thereby inhibiting translation initiation and autophagic flux. Structural analyses identified key residues (SER¹¹⁸, SER²⁰⁶, and ASN⁶¹) critical for this interference. Mutation of these residues abrogated g5Rp activity. Furthermore, virtual screening identified 9″-methyl salvianolate B as a potent g5Rp inhibitor, which restored eIF5A hypusination, promoted autophagy, and suppressed ASFV replication in vitro. Collectively, our findings establish g5Rp as a pivotal regulator of ASFV pathogenesis and a promising target for antiviral drug development.

Importance: ASFV has caused significant economic losses to the global pork industry, and no effective treatment or prevention currently exists. In this study, the interaction of g5Rp with the host proteins eIF5A and RPS15 was identified for the first time, and its crucial role in the viral life cycle was clarified. Resolving the crystal structure of g5Rp revealed its binding site to the host protein, which provides a new target for developing antiviral strategies against g5Rp. Additionally, the screened 9″-methyl salvianolate B, a small-molecule inhibitor, has shown the potential to effectively reduce viral replication and restore host protein synthesis. These findings not only deepen our understanding of the mechanism of ASFV infection but also lay the foundation for developing effective anti-ASFV treatment strategies in the future, which has important scientific implications.

Lonafarnib, an oral antiviral that targets the fusion glycoprotein of respiratory syncytial virus (RSV), has demonstrated efficacy in vitro and in vivo. However, because the RSV has evolved to become resistant to other fusion inhibitors, there is a concern that the same could occur for lonafarnib. Here, we identified resistance to lonafarnib in the RSV A2 strain and a recent clinical isolate, RSV ON1, via in vitro selection at scale. Cell‒cell fusion and recombinant live RSV analysis confirmed that the mutations at K394, K399, and T400 of the cysteine-rich region of the fusion protein mediate high-level resistance. Lonafarnib resistance mutations also confer cross-resistance to other fusion inhibitors of clinical interest. All-atom molecular dynamics simulations revealed that these resistance mutations confer reduced stability to the fusion protein, thereby diminishing its binding affinity with lonafarnib. To address this vulnerability proactively and increase the barrier to resistance development, we designed the first potent proteolysis-targeting chimera (PROTAC) fusion protein degrader, compound 0179841, which uses lonafarnib and cereblon as ligands. This PROTAC effectively inhibited RSV replication. Collectively, our findings indicate that RSV develops resistance to lonafarnib in the cysteine-rich region of the fusion protein. This work sheds light on the mechanisms by which RSV evolves resistance to lonafarnib and provides a foundation for the rational design of antivirals aimed at preventing resistance.IMPORTANCERespiratory syncytial virus (RSV) infection poses a substantial public health challenge. Resistance to several potent fusion inhibitors, which are currently in various stages of clinical development, can readily emerge. Through a drug repurposing screen, we identified lonafarnib as an RSV fusion inhibitor; however, concerns exist regarding the potential development of resistance. Here, large-scale in vitro selection experiments revealed specific mutations within the highly conserved cysteine-rich region of the fusion (F) protein that confer high-level lonafarnib resistance across diverse RSV strains. These resistance mutations also confer cross-resistance to other clinical-stage fusion inhibitors. Mechanistic investigations demonstrated that these mutations reduce F protein stability, thereby diminishing the binding affinity of lonafarnib. As a proof of concept for an alternative antiviral strategy, we rationally designed the first potent proteolysis-targeting chimera (PROTAC) F protein degrader, compound 0179841, by utilizing lonafarnib and cereblon ligands. This novel antiviral agent effectively inhibits RSV infection by inducing degradation of the F protein. This work elucidates the molecular basis of RSV resistance to lonafarnib and establishes a strategy for developing next-generation antivirals aimed at preempting resistance.

The COVID-19 pandemic was marked by successive waves of SARS-CoV-2 variants with distinct properties. The Omicron variant that emerged in late 2021 showed a major antigenic shift and rapidly spread worldwide. Since then, Omicron-derived variants have maintained their global dominance, for reasons that remain incompletely understood. We report that the original Omicron variant BA.1 evolved several traits that converged in facilitating viral spread. First, Omicron displayed an early replicative advantage over previous variants when grown in a reconstructed human nasal epithelium model. The increase in Omicron replication was more marked at the physiologically relevant temperature of 33°C found in human nasal passages. Omicron also caused a decrease in epithelial integrity, as measured by transepithelial electrical resistance and caspase-3 activation. Furthermore, Omicron caused a more marked loss of motile cilia at 33°C than other variants, suggesting a capacity to impair mucociliary clearance. Omicron induced a broad transcriptional downregulation of ciliary genes but only a limited upregulation of host innate defense genes at 33°C. The lower production of type I and type III interferons in epithelia infected by Omicron compared to those infected by the Delta variant, at 33°C as well as 37°C, confirmed the increased capacity of Omicron to evade the innate antiviral response. Thus, Omicron combined replication speed, motile cilia impairment, and limited induction of innate antiviral responses when propagated in nasal epithelia at physiological temperature. Omicron has the capacity to propagate rapidly but stealthily in the upper respiratory tract, which likely contributed to the evolutionary success of this SARS-CoV-2 variant.

Importance: The COVID-19 pandemic was initially characterized by a rapid succession of viral variants that emerged independently of each other, with each of these variants outcompeting the previous one. A major evolutionary shift occurred in late 2021, with the emergence of the highly divergent Omicron BA.1 variant. Since then, all the dominant SARS-CoV-2 variants have been derived from Omicron, for reasons that remain incompletely understood. Here, we compared the replication of SARS-CoV-2 variants in a human nasal epithelium model grown at 37°C and also at 33°C, a temperature that approximates that found in the nasal cavity. In this primary epithelial model, Omicron showed an early replicative advantage that was more marked at 33°C. However, Omicron triggered only a minimal antiviral interferon response at this temperature. Omicron could thus propagate rapidly while partly escaping the innate response at physiological nasal temperature, which helps account for the efficient dissemination of this variant worldwide.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related sarbecoviruses encode a set of accessory proteins (3a, 3b, 6, 7a, 7b, 8, 9b, and 10) that control host responses to infection and promote virus growth. Of these accessory proteins, 7a is set apart by its intracellular localization near CoV budding sites and its incorporation into secreted virions. To investigate 7a functions during CoV infections under biosafety level 2 conditions, we constructed recombinant mouse hepatitis viruses (rMHVs) (rMHV strain A59) that express sarbecovirus 7a genes. Comparative infections revealed that 7a increased viral replication and viral output in immortalized murine cell cultures and in primary bone marrow-derived macrophages (BMDMs). This proviral effect was independent of a previously reported 7a-mediated interferon antagonizing activity. 7a is a type I transmembrane protein with a short cytoplasmic tail that operates in subcellular trafficking and signal transduction. To further elucidate tail functions, we generated a set of rA59 viruses expressing substitutions in the tail di-lysine motifs. Several substitutions reduced 7a proviral activities; notably, the K119A change in rA59-7a-KRATE eliminated 7a support of virus yield. The K119A change also reduced mouse-adapted SARS-CoV-2 virus yields in infected BALB/c mice. 7a expression was proinflammatory in BMDMs, as measured by cytokine arrays. Cytoplasmic tail substitutions tempered these proinflammatory responses, implying connections with proviral activities. SARS-CoV-2-infected macrophages have been implicated in inflammatory COVID-19, and these findings point to 7a cytoplasmic tails as potential contributors to cytokine-mediated disease.

Importance: This study shows that SARS-CoV-2 accessory protein 7a promotes infection of a phylogenetically distinct embecovirus and, in doing so, elicits proinflammatory and potentially disease-relevant host responses. The proviral and proinflammatory activities were traced in part to a short 7a cytoplasmic tail. The findings localize and highlight a specific proviral component in a sarbecovirus accessory protein.

In the post-antibiotic era, bacteriophages have emerged as viable alternatives for combating antibiotic-resistant infections. Analogous to the emergence of antibiotic resistance, bacteria can also develop resistance to phages, a process often accompanied by a reduction in bacterial fitness. In this study, we investigated the mechanisms behind the development of phage resistance in Salmonella enterica serovar Enteritidis strain WJ48 during laboratory coevolution with phage GRNsp8, as well as the associated fitness costs and community dynamics. Three types of phage-resistant mutants, VP81, VP82, and VP84, were isolated. VP81 exhibited a frameshift mutation in the gene encoding glycosyltransferase and displayed partial resistance to GRNsp8. Both VP82 and VP84 carried frameshift mutations in btuB, conferring complete resistance to GRNsp8. Deletion of btuB abolished phage adsorption and conferred complete resistance. Meanwhile, complementation with btuB restored susceptibility. In vitro competition assays showed that the btuB-deletion strain was competitively disadvantaged relative to the wild-type strain within 24 h, exhibiting a relative fitness value of <1. Consistently, in vivo experiments in mice showed a marked attenuation of virulence in the mutant strain. Specifically, the LD₅₀ of the btuB-deficient strain in mice was 6.8 × 10⁸ CFU, 121 times higher than the wild-type strain. During the 9-day coevolution experiment, a single-point mutation in btuB consistently dominated and represented the primary mode of resistance. These findings shed light on the adaptive trade-offs that bacteria undergo to evade phage infection and provide valuable insights for designing more rational and effective phage therapy strategies that exploit these trade-offs.IMPORTANCEAs emerging antibacterial agents in the post-antibiotic era, Bacteriophages also face the challenge of bacterial resistance. However, phage resistance development by bacteria is frequently accompanied by a reduction in bacterial fitness. To elucidate the adaptive trade-offs associated with resistance, in this study, we used Salmonella enterica serovar Enteritidis strain WJ48 and the broad-host-range bacteriophage GRNsp8 as a model system. We found that the acquisition of phage resistance by bacteria was significantly associated with a reduction in virulence. These findings deepen our understanding of bacteria-phage coevolution but also offer key insights into leveraging the resistance-fitness trade-off to inform the strategic design of more effective phage therapies. The results highlight the potential for improving the application of phages in agriculture and animal husbandry, supporting the sustainable development of phage-based antimicrobial strategies.

Annexin A2 (ANXA2) is known to promote the replication of diverse RNA viruses, often through interactions with specific viral proteins. However, whether it also employs a broad-spectrum, virus-independent strategy to facilitate viral replication remains unclear. Here, we identify ANXA2 as a novel negative regulator of the host antiviral response by targeting the RIG-I-like receptor (RLR) signaling pathway. We demonstrate that overexpression of ANXA2 suppresses type I interferon (IFN) production induced by RNA viruses or poly(I:C), whereas ANXA2 deficiency enhances IFN production and restricts viral replication both in vitro and in vivo. Mechanistically, ANXA2 functions as a scaffold protein that concurrently disrupts two critical steps in RLR signaling: it impedes the recruitment of MAVS by MDA5 through interacting with the CARD domain of MDA5, and it inhibits the MAVS-TRAF3 interaction by binding to the linker region of MAVS. Consequently, ANXA2 deficiency leads to enhanced type I IFN production in mice, which effectively restrains replication of encephalomyocarditis virus and vesicular stomatitis virus. Collectively, our study uncovers a novel and broad-spectrum immunomodulatory function of ANXA2, wherein it dampens antiviral innate immunity by sabotaging key protein-protein interactions (MDA5-MAVS or MAVS-TRAF3) within the RLR pathway, thereby presenting a potential target for developing antiviral strategies.

Importance: Subsequent to RNA viral infection, a series of complex cascade reactions are initiated, leading to the production of type I interferons and, consequently, the resistance of the organism to viral infection. This study elucidates the function of Annexin A2 (ANXA2) as a novel key negative regulator in the host antiviral immune response. Mechanistically, ANXA2 achieves its inhibitory effect by disrupting critical signaling steps in the RLR pathway, specifically interfering with key interactions between MDA5 and MAVS, as well as between MAVS and TRAF3. These findings are significant in that they reveal an unknown mechanism by which viruses exploit host proteins to evade immunity, and they position ANXA2 as a potential therapeutic target for developing novel antiviral strategies. The validation of these findings in an ANXA2-deficient mouse model, which exhibits enhanced interferon production and restricted viral replication, serves to further reinforce the physiological relevance of these observations.