Influenza A virus (IAV) remains a major threat to human and animal health, highlighting the need for efficient approaches to identify host factors and antivirals. Reporter viruses are critical tools for these efforts, but incorporating large reporter genes often compromises viral fitness and genetic stability. Here, we developed a replication competent reporter IAV by fusing an 11-amino acid HiBiT tag into the nonstructural protein 1 (NS1) of the A/WSN/1933 (H1N1) backbone. The modified virus retained parental virion morphology, comparable replication kinetics, and in vivo tissue tropism, while enabling the highly sensitive, rapid, and quantitative detection of viral replication. Using this reporter virus, we established a robust high-throughput screening (HTS) platform with excellent assay quality and validated its capability to identify host regulators of IAV infection. Application of this platform to host-encoded micropeptides (miPEPs) led to the identification of cytokine-inhibitory micropeptide 53 (CIM53) as a novel regulator of IAV replication. Mechanistically, CIM53 promoted IAV replication by intrinsically suppressing antiviral innate immune responses. Furthermore, although CIM53 enhanced viral replication, its immunomodulatory activity contributed to reduced lung injury and improved survival when combined with Oseltamivir (OSV) treatment in infected mice. Our study establishes a highly practical screening platform for virological research and highlights CIM53 as a potential host-directed adjunct therapy for severe IAV Infection.
Prototype foamy viruses (PFVs) are complex retroviruses that establish long-term latent infections in hosts without causing disease, positioning them as potential safe gene transfer vectors. Understanding the host proteins involved in PFV replication and their interaction mechanisms may enhance gene transfer efficiency. However, only a few cellular proteins are known to influence PFV replication. Based on the transcriptomic analysis of PFV-infected HT1080 cells, we observed a potential significance of RING finger protein 152 (RNF152) in modulating PFV replication. Overexpression of RNF152 significantly inhibits PFV replication, whereas RNF152 knockdown enhances viral replication. Mechanistically, RNF152 interacts with the Gag protein to promote its polyubiquitination at lysine 396 (K396), thereby facilitating its degradation via the ubiquitin-proteasome system. Furthermore, RNF152 reduces the size and number of PFV virus-like particles (VLPs) by inhibiting the multimerization of Gag. Collectively, our findings reveal a previously unrecognized mechanism that influences PFV infection. Additionally, we elucidate the role of RNF152 in affecting virus replication for the first time, providing valuable insights into the mechanisms of virus replication and demonstrating the importance of ubiquitination modification in affecting viral replication.
Gastric cancer (GC) is a prevalent malignancy worldwide, yet effective early diagnostic tools remain lacking, and the role of the virome, a key component of the tumor microenvironment, in GC progression is largely unknown. This study aimed to characterize the virome landscapes in peripheral blood and feces of GC patients versus healthy controls, and to identify viral signatures associated with GC onset and metastasis. We performed viral metagenomic sequencing on pooled libraries from 100 GC patients (45 non-metastatic, 55 metastatic) and 50 healthy controls, followed by taxonomic annotation, diversity assessment, LEfSe differential abundance testing, and co-occurrence network analysis. In blood, the GC virome shifted from a bacteriophage-dominated profile in controls to one overwhelmingly dominated by Anelloviridae (> 80%), with significantly decreased alpha diversity. In contrast, the gut virome of GC patients showed increased alpha diversity and coexistence of diverse bacteriophages. LEfSe identified betatorquevirus in blood as a key discriminatory taxon for GC. Network analysis revealed negative correlations between Anelloviridae and multiple bacteriophage families, suggesting niche competition. We also discovered 67 provisional novel anellovirus species and one novel gemykibivirus in GC patient blood. Collectively, our findings indicate that GC is associated with compartment-specific virome remodeling in blood and gut, and that expansion of blood anelloviruses holds promise as a non-invasive biomarker. This study provides a foundational resource for understanding the virome's role in GC.
Porcine circovirus type 2 (PCV2), a major causative agent of PCV2-associated diseases, poses a serious threat to the global swine industry. The stability of PCV2 capsid (Cap) protein is critical for viral replication; however, the underlying mechanisms regulating Cap stability during PCV2 infection remain poorly understood. In this study, co-immunoprecipitation assays were used to demonstrate that PCV2 Cap protein can maintain its own stability by binding to the cellular Y-box-binding protein 1 (YBX1) during viral infection. Furthermore, binding domain mapping experiments revealed two specific regions essential for this interaction: the N-terminal arginine-rich motif (ARM) of PCV2 Cap (amino acid sequence: 1MTYPRRRYRRRRHRPRSHLG20) and the amino acid residues 189RRRR192 within the C-terminal domain (CTD) of YBX1. Virus rescue experiments further confirmed that substituting all arginine residues with alanine in the N-terminal ARM of Cap completely abolished its binding to YBX1. Analysis of viral replication capacity showed that YBX1 promotes PCV2 replication by stabilizing the viral Cap protein. Additionally, knockdown of YBX1 significantly reduced the protein levels of PCV2 Cap, and this effect was reversed either by treatment with the proteasome inhibitor MG132 or by restoring YBX1 expression. Collectively, these findings demonstrate that YBX1 promotes PCV2 replication by directly interacting with the ARM of PCV2 Cap, thereby inhibiting the proteasomal degradation of Cap during viral infection.
The Chinese goral (Naemorhedus griseus) is identified as a vulnerable species on the Red List of China's Biodiversity and listed as a national second-class key protected wild animal in China. Despite its widespread distribution in China, there has been scant research into the pathogens harbored by this species, and our understanding of the virus diversity remains limited. In the present study, we performed the whole virome profiling of a rescue-failed Naemorhedus griseus, which identified a diverse viral community across multiple organ tissues of the animal. Specifically, abundant bacteriophages, several plant-derived viruses, and multiple mammalian viruses, i.e., two distinct parvoviruses, novel picobirnaviruses, and a novel phenuivirus. Additionally, virus-carrier determination analysis that Naemorhedus griseus-borne viruses are related to plants, mammals, and arthropods. These data provide the first insight into the genetic diversity of Naemorhedus griseus-borne viruses, and their transmission dynamics across humans, domestic animals, arthropods, and wildlife. This study not only expands the existing repertoire of viral genomic information and delineates the host range of these pathogens but also underscores the imperative of implementing a One Health strategy for the surveillance and control of wildlife-borne viral pathogens.
Enteroviruses, including Coxsackievirus B3 (CVB3), are significant human pathogens that cause severe diseases, such as viral myocarditis, pancreatitis, and encephalitis. ABL proto-oncogene 2 (ABL2), a non-receptor tyrosine-protein kinase, regulates diverse physiological processes and participates in virus infection; however, its role in enterovirus infection remains uncharacterized. Here, we demonstrate a novel host-virus interaction: enteroviruses degrade ABL2 via the ubiquitin-proteasome system through their non-structural protein 2B. Furthermore, ABL2 functions as an antiviral restriction factor during enterovirus infection, specifically inhibiting the early stages of viral replication. Mechanistically, ABL2 directly interacts with RAC1, a Rho family GTPase, and downregulates RAC1 protein levels, thereby suppressing RAC1-dependent activation of the PI3K/AKT signaling pathway. In summary, our study reveals a post-translational mechanism by which enteroviruses evade host antiviral defenses, providing a rationale for therapeutic development against enteroviral diseases.
Repeated influenza exposures generate complex antibody landscapes, yet how pre-existing antibodies are associated with subsequent vaccine responses remains unclear. We longitudinally tracked 21 adults stratified by pre-vaccination neutralizing titers to examine B-cell dynamics using flow cytometry, B cell receptor (BCR) sequencing, and monoclonal antibody characterization. After vaccination, participants with high baseline titers showed a longitudinal increase in the frequency of HA+CD27-IgD+ naive-phenotype B cells. BCRs recovered from hemagglutinin (HA)-binding (HA+) B cells in the high-titer group displayed lower somatic hypermutation and shorter CDR3s, whereas those recovered from the lower-titer groups showed higher mutation levels and more extensive class switching. Correspondingly, monoclonal antibodies from the high-titer group generally showed narrow reactivity, while those from lower-titer groups showed broader neutralizing activity against historical strains. Clonal tracing further suggested that mAbs from lower-titer groups were more often linked to pre-existing sequences. Together, these findings suggest that pre-existing antibody levels are associated with differences in B-cell repertoire maturation and antibody breadth after influenza vaccination.
The co-circulation of multiple viruses can lead to distinct pathological outcomes, yet how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection influences other viral infections remains poorly understood, despite its documented high frequency during the pandemic. In this study, we investigated how the proteolytic activity of SARS-CoV-2 3C-like protease (3CLpro) influences the replication of influenza A virus. In silico analysis identified candidate 3CLpro cleavage sites across numerous viral proteins, and biochemical assays confirmed that 3CLpro catalyzes the degradation of influenza virus nucleoprotein (NP) and polymerase acidic protein (PA) in a manner requiring its protease activity. This degradation of NP and PA, which are essential for viral genome packaging and transcription, disrupted the influenza replicative cycle and suppressed viral replication, both upon ectopic 3CLpro expression and during SARS-CoV-2 infection. Our data uncover a direct, enzyme-based mechanism by which SARS-CoV-2 can suppress influenza virus replication during coinfection. We provide a molecular explanation for the sharp, global decline in influenza activity observed during the COVID-19 pandemic and illustrate how enzymatic weapons of one virus can be repurposed to restrain a competing pathogen.
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus for which no specific antiviral therapy is currently available. During the large outbreak in Foshan, Guangdong Province, China, in July 2025, CHIKV rapidly spread to neighboring regions and caused more than 16,000 confirmed cases. In this study, the predominant outbreak strain of CHIKV was selected as the reference sequence to establish a panel of complementary biosafe tools for antiviral compound screening and mechanistic investigation. A virus replicon particle (VRP) system for CHIKV was first constructed and applied to compound library screening, resulting in the identification of three candidate antiviral compounds: MDL-12330A, bazedoxifene acetate, and anidulafungin. To further validate their antiviral activities and investigate their potential mechanisms, CHIKV functional evaluation systems were subsequently established, including vesicular stomatitis virus (VSV)- and murine leukemia virus (MLV)-based pseudovirus systems for viral entry, a replicon RNA system for post-entry replication-associated processes, a replication-defective nsP4 mutant replicon RNA system for primary translation, and a virus-like particle (VLP) system for viral particle assembly and budding assessment. Using these complementary systems, we systematically evaluated the antiviral profiles of the three candidate compounds across multiple stages of the CHIKV life cycle. This analysis revealed distinct stage-specific inhibitory patterns and provided insights into their potential antiviral mechanisms, which warrant validation using authentic CHIKV infection to assess their translational potential.
Human metapneumovirus (hMPV) is a major cause of pediatric acute lower respiratory tract infections (ALRTIs), yet vaccine development has been hindered by the intrinsic metastability of the prefusion F glycoprotein (pre-F). Here, we used a structure-based design strategy to stabilize hMPV pre-F while preserving neutralization-sensitive epitopes. By applying combined stabilizing elements, including disulfide bonds, a designed salt bridge, and a trimer-interface sequence swap, we generated VM-874, a pre-F-stabilized trimer that was expressed at high levels in 293F cells and exhibited improved thermal stability and stress-resistant antigenicity. VM-874 also retained binding to multiple conformation-sensitive monoclonal antibodies following thermal and storage stress. VM-874 elicited high serum neutralizing titers against both hMPV A2 and B1 strains and conferred protection in BALB/c mice and cotton rats, as evidenced by reduced pulmonary viral burden and attenuated lung pathology after challenge. Although VM-874 elicited antibodies that cross-bound respiratory syncytial virus (RSV) F protein, no RSV-neutralizing activity was detected, underscoring the need to distinguish cross-reactivity from heterologous protection in combined RSV/hMPV vaccine strategies.
Viral proteases are key targets for the development of broad-spectrum antiviral drugs development. However, screening platform capable of accurately assessing inhibitor activity within physiologically relevant cellular environments remain urgently needed. Traditional methods, such as fluorescent protein assays and Förster resonance energy transfer (FRET), suffer from significant limitations, including susceptibility to non-specific conformational interference by test compounds and an inability to faithfully reflect intracellular inhibitory effects. To address these challenges, we constructed two modular biosensors (TS3AR and C3SIR) based on engineered ascorbate peroxidase (APEX). Their detection mechanism relies on specific cleavage of the substrate recognition sequence by the target protease, which triggers the reassembly of split APEX fragments, restores enzymatic activity, and generates fluorescent signals generated via cascade amplification reaction. Validation using the coronavirus main protease (Mpro) as a model showed that the TS3AR sensor achieved the signal-to-noise ratio up to 1500-fold for enzyme activity detection, while the C3SIR sensor effectively avoided the false positives caused by conformational interference seen in traditional methods and accurately identified high-potency Mpro inhibitors, including Enstrelvir, PF-00835231, and Nirmatrelvir. Moreover, by replacing the protease recognition sequence, these modular biosensors can be flexibly adapted for activity analysis and drug evaluation of Mpro from various coronaviruses (e.g., SARS-CoV-2, MERS-CoV) as well as other viral proteases (e.g., Enterovirus 71, Epstein-Barr virus and Hepatitis A virus). Overall, this platform provides a reliable, highly specific intracellular screening tool to accelerate the development of broad-spectrum therapeutics against both emerging and existing viral threats.
Mammalian embryonic stem cells (ESCs), despite lacking functional interferon (IFN) signaling, are remarkably resistant to viral infection. However, the mechanistic basis of their antiviral defenses remains incompletely characterized. In this study, we investigated the antiviral functional role and molecular mechanism of tripartite motif-containing protein 25 (TRIM25), a well-known key driver of IFN-dependent innate immunity, in IFN-deficient mouse ESCs (mESCs). TRIM25 exhibited a broad spectrum of antiviral activity in mESCs. RNAi depletion or CRISPR-mediated knockout of TRIM25 markedly enhanced the replication and propagation of both encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV). Mechanistically, the antiviral activity of TRIM25 in mESCs is independent of both IFN production and constitutive ISG expression. In virus-infected mESCs, TRIM25 translocated from the nucleus to the cytoplasm and directly bound viral RNA, where it formed cytoplasmic condensates with GTPase-activating protein-binding protein 1 (G3BP1) that colocalized with dsRNA foci. Genetic perturbation of G3BP1 similarly compromised the antiviral defenses of mESCs, revealing an essential synergism between TRIM25 and stress granule components in restricting viral replication. Our study identifies TRIM25 as a pivotal RNA-sensing effector in ESCs, delineating a previously unrecognized IFN-independent axis of intrinsic immunity that bridges viral RNA surveillance with stress granule-mediated suppression, thereby expanding the paradigm of pluripotent cell-autonomous antiviral strategies.
Tick-borne encephalitis virus (TBEV) poses a severe threat to public health, causing neurological disorders with high morbidity and mortality in endemic regions. Therefore, developing safe and effective strategies for TBEV vaccines has long been a focus of attention. Herein, leveraging our well-established NS1 trans-complementation platform, we successfully constructed a high-titer replication-defective TBEV (TBEV-ΔNS1) using the BHK-21 cell line stably expressing Omsk hemorrhagic fever virus (OHFV) NS1 (designated BHKNS1). Retention of the NS1 deletion in TBEV-ΔNS1 was confirmed by continuous passaging in BHKNS1 cells, as no replicative virus was detected in naive BHK-21 cells. The safety profile of TBEV-ΔNS1 was further validated, as administration of a high dose of TBEV-ΔNS1 to ICR mice did not induce any clinical symptoms. Notably, TBEV-ΔNS1 conferred complete protection against lethal wild-type (WT) TBEV challenge in ICR mice, whereas non-immunized control mice exhibited 100% mortality, significant weight loss, and viremia. This protective efficacy is tightly correlated with robust humoral and cellular immune responses, as demonstrated by the induction of high titers of TBEV-specific IgG, neutralizing antibodies, and IFN-γ-secreting CD8+ T cells following a single immunization. Collectively, our findings provide a proof-of-concept for the NS1 trans-complementation platform in TBEV vaccine development.
The ubiquitin-proteasome system (UPS) plays a central role in antiviral defense but is also frequently hijacked by viruses to facilitate their replication. Here, we demonstrate that the host deubiquitinase OTUB2 stabilizes the viral replication factor NSP8 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through a dual-track mechanism. OTUB2 directly removes polyubiquitin chains from NSP8 to prevent its degradation. In parallel, OTUB2 stabilizes the viral papain-like protease (PLpro), which further promotes NSP8 stability through deubiquitination. Together, these effects preserve the functional integrity of the viral replication-transcription complex. Mechanistically, OTUB2-mediated stabilization of NSP8 potentiates NSP8-dependent suppression of type I interferon signaling, thereby promoting viral replication and immune evasion. Importantly, inhibition of OTUB2 disrupts OTUB2-mediated stabilization of NSP8 and PLpro, resulting in a marked reduction in viral replication and disease severity in cell culture systems and a hamster infection model. Collectively, our findings reveal a previously unrecognized mechanism by which SARS-CoV-2 utilizes the host deubiquitination system to stabilize its replication machinery and identify OTUB2 as a potential target for host-directed antiviral intervention.
Monkeypox virus (MPXV), a pathogenic orthopoxvirus, has caused major outbreaks and emerged as a global public health threat. Although antivirals approved for smallpox are used therapeutically against monkeypox, their clinical utility is limited by drug availability and emerging resistance. The conserved strategy by which viruses remodel host nucleotide metabolism to secure biosynthetic precursors for replication and spread has emerged as a pivotal target for the development of broad-spectrum antiviral therapeutics. In this study, leveraging the high genetic and biological similarity between vaccinia virus (VACV) and MPXV, we employed VACV as a surrogate model to screen 10 FDA-approved inhibitors targeting nucleotide metabolism enzymes, aiming to identify potential novel inhibitors against MPXV. Mycophenolate mofetil (MMF), an inosine 5'-monophosphate dehydrogenase type II (IMPDH2) inhibitor, displayed potent inhibition effects against both VACV and MPXV. Subsequent downstream time-course studies revealed that MMF targets a post-entry stage of the viral replication cycle. Mechanistic studies suggest that MMF inhibits IMPDH2 activity by suppressing ubiquitin-specific protease 5 (USP5)-mediated deubiquitination of IMPDH2 and inducing rod-and-ring (R&R) assembly, leading to reducing dNTP pools and enhancing antiviral effects. In conclusion, our findings demonstrate that MMF is an effective antiviral drug against VACV and MPXV infection and establish a host-directed therapeutic strategy to combat future orthopoxvirus outbreaks.
Human immunodeficiency virus type 1 (HIV-1) Tat is essential for efficient viral transcription and replication, and its stability is tightly controlled by host factors. In our previous study, we showed that ZNF598 stabilizes Tat and promotes HIV-1 replication through an E3 ligase activity-independent mechanism. Here, we identify FAT10 as a critical mediator of this effect. FAT10 knockout in HEK293T cells or FAT10 knockdown in Jurkat T-cell infection models markedly impaired the ability of ZNF598 to suppress Tat K48-linked ubiquitination, increase Tat abundance, and enhance HIV-1 replication. Mechanistically, FAT10 reduced Tat K48-linked ubiquitination, stabilized Tat, and promoted HIV-1 transcription and replication. Direct GST pull-down assays further demonstrated that FAT10 directly binds Tat in vitro, and this interaction was retained by a FAT10 mutant lacking the C-terminal diglycine motif, supporting a predominantly non-covalent mechanism rather than canonical FAT10ylation. Consistently, ZNF598 enhanced the FAT10-Tat interaction, whereas ZNF598 knockdown attenuated FAT10-mediated Tat stabilization and proviral effects. In addition, the N-terminal 1-32 amino acids of Tat were required for responsiveness to both ZNF598 and FAT10. Finally, FAT10 expression was increased in HIV-1-infected cells and in CD4+ T cells from HIV-infected individuals, where it was associated with clinical markers of disease activity and viral load. Together, these findings define a ZNF598-FAT10-Tat regulatory axis that promotes HIV-1 transcription and replication.
