ABSTRACT Identifying the host factors that mediate avian influenza virus adaptation in mammals is important for monitoring zoonotic potential. Although viral polymerase adaptations are known to influence cross-species transmission, the engagement of specific host factors with divergent viral polymerases remains to be explored. We examined human DEAD-box RNA helicases (DDXs) as potential regulators of influenza polymerase activity. A screen of 16 DDXs identified DDX10 as a factor that selectively enhanced the polymerase activity and replication of human-adapted H1N1 viruses, including the 2009 pandemic strain, but not avian-origin H9N2 viruses. This differential activity was associated with DDX10 showing stronger interaction with human-origin PB1 (Cal04/H1N1) than with avian-origin PB1 (BJ16/H9N2). Strain specificity was mediated by two residues (336 and 364) within the PB1 catalytic domain: introducing H1N1-type residues (I336 and I364) into H9N2-PB1 conferred DDX10 responsiveness, while reciprocal mutations in H1N1-PB1 abolished it. This work defines DDX10 as a potential host factor that differentially supports influenza polymerase activity, revealing a specific molecular interface that contributes to the replication efficiency of distinct viral subtypes in human cells and providing insight into host-adaptive mechanisms. IMPORTANCE This study identifies DDX10 as a strain-specific host factor that differentially regulates influenza A virus replication. By demonstrating that DDX10 selectively enhances human-adapted H1N1 viruses over avian-origin H9N2 viruses through direct interaction with the viral PB1 protein, and by mapping two critical residues (positions 336 and 364) in PB1 that govern this selectivity, our work provides a mechanistic framework for understanding how a single host factor can modulate viral fitness and host adaptation. These findings have important implications for predicting cross-species transmission potential and may guide the development of host-targeted antiviral strategies. This study identifies DDX10 as a strain-specific host factor that differentially regulates influenza A virus replication. By demonstrating that DDX10 selectively enhances human-adapted H1N1 viruses over avian-origin H9N2 viruses through direct interaction with the viral PB1 protein, and by mapping two critical residues (positions 336 and 364) in PB1 that govern this selectivity, our work provides a mechanistic framework for understanding how a single host factor can modulate viral fitness and host adaptation. These findings have important implications for predicting cross-species transmission potential and may guide the development of host-targeted antiviral strategies.
R. Ullah, Wei Chen, Ling-Kai Zhang et al.· Journal of Virology· 0 citations
H9N2 avian influenza virus (AIV) remains a global threat to poultry health and has zoonotic potential. Antigenic drift in the hemagglutinin (HA) protein complicates vaccine efficacy and diagnostic accuracy, highlighting the need for precise epitope characterization. In this study, the HA protein of H9N2 AIV was expressed in a eukaryotic system, and two monoclonal antibodies (mAbs), 9C12 and 9F4, were generated. Both mAbs specifically bound HA, as shown by ELISA, Western blot, and immunofluorescence, but lacked hemagglutination inhibition activity. Epitope mapping revealed two minimal linear epitopes: 123FSSSRSYQ130 within the vestigial esterase domain and 201NLYTRTDTT209 within the receptor-binding domain. Alanine scanning revealed key residues required for antibody binding, whereas structural modeling confirmed that both epitopes are surface exposed. Sequence analysis demonstrated strong conservation across H9N2 strains, with the 9F4 epitope showing near-complete invariance, whereas both epitopes exhibited low conservation among other influenza A virus subtypes. These findings define two novel, nonneutralizing epitopes on H9N2 HA that expand the antigenic map and represent promising targets for subtype-specific diagnostic assays.
Porcine epidemic diarrhea virus (PEDV) causes devastating enteric disease in piglets, yet the mechanistic basis of antibody-mediated neutralization remains poorly understood. Here, we determined the structure of PEDV HNXX-strain spike domain B (S1B) simultaneously bound by C62, a neutralizing porcine monoclonal antibody against PEDV G2 strains, and N34, a non-neutralizing porcine PEDV antibody. The structure reveals that C62 targets a conserved, cryptic epitope that is accessible only when S1B adopts an “up” conformation. Functionally, we showed that C62 has substantially stronger activity than N34 in triggering S-trimer disassembly and inducing the formation of proteinase K-resistant, post-fusion-like S2 structures. Despite the weaker triggering activity of N34, both C62 and N34 can function as artificial receptors. Notably, although the C62 epitope is conserved across both G1 and G2 strains, C62 exhibits G2-strain-biased neutralizing activity. We further showed that differences in cell-surface membrane fusion activity among PEDV spikes correlate with distinct viral entry pathways and are jointly determined by the S1A and S1B sequences. Together, our findings identify a strain-specific vulnerable site on the PEDV S-trimer and provide insight into how cell-surface membrane fusion activity may influence viral entry pathway selection and antibody neutralization efficacy.
Jian-Bo Liu, Sheng Wang, Zimu Li et al.· bioRxiv· 0 citations
The Eurasian avian□like (EA) H1N1 swine influenza virus (SIV), derived from avian influenza viruses (AIV), poses a serious threat to public health due to its capacity for cross□species transmission and pandemic emergence. The molecular determinants underlying its replication advantage over AIV remain poorly defined. Here, we identify RNA□binding motif protein 6 (RBM6) as a novel host factor that differentially regulates the replication of EA H1N1 SIV and AIV. Mechanistically, RBM6 binds to the critical M901 site of the viral M segment, thereby modulating RNA splicing. Substitution of M901C with M901T markedly reduced RBM6 binding, impaired M segment splicing, and attenuated viral replication both in vitro and in vivo. Conservation analysis revealed that M901T is common in avian strains, whereas M901C is predominantly maintained in swine strains, underscoring M901C as a determinant of swine adaptation. Complementation experiments further demonstrated that swine RBM6, but not avian RBM6, restored EA H1N1 SIV replication. Taken together, our findings uncover a previously unrecognized role of RBM6 in shaping influenza virus replication and highlight the RBM6-M901C axis may serve as potential targets for controlling influenza virus adaptation and interspecies transmission.
Jiahui Zou, Shaoyu Tu, Huimin Sun et al.· bioRxiv· 0 citations
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