The Mechanism Of Avian Metapneumovirus Fusion Protein-mediated Membrane Fusion And Inhibition Of Interferon-β

Avian metapneumovirus(aMPV) is member of the genus Metapneumovirus in the subfamily Pneumovirinae of Paramyxoviridae. aMPV causes actue rhinotracheitis(TRT) in turkeys and swollen head syndrome(SHS) in chickens, which has been considered major threats to poultry industry. The entry of aMPV into host cells primarily requires the fusion of viral and cell membranes, which can be mediated by fusion(F) protein alone. However, little is known about mechanism of aMPV F protein inducing membrane fusion. Furthermore, aMPV is along with secondary bacterial and/or viral infections, but the immunosuppressive effect of aMPV is unclear. This study aimed to study the mechanism of membrane fusion mediated by F protein and immunopathogenesis of aMPV.Both aMPV and human metapneumovirus(hMPV) belong to the members of the genus Metapneumovirus. For hMPV F protein, trypsin- and/or low pH-induced membrane fusion is a strain-dependent phenomenon. Here, we revealed that three subtypes of aMPV(aMPV/A, aMPV/B, and aMPV/C) F proteins mediated cell-cell fusion without adding trypsin. Indeed, in the presence of trypsin, only the fusogenicity of aMPV/C F protein was increased. Mutagenesis of the amino acids at position 100 and/or 101, located at a putative cleavage motif of a MPV F proteins, revealed that trypsin-regulated fusogenic activity of aMPV F proteins was determinated by the residues at positions 100 and 101. Furthermore, we revealed that aMPV/A and aMPV/B F proteins induced cell-cell fusion independent of low pH, whereas the aMPV/C F protein did not. Mutagenesis of the amino acid at position 294 of the aMPV/A, aMPV/B, and aMPV/C F proteins showed that residue 294 G played a determinant role in F protein-mediated cell-cell fusion at low pH environment. Dissection of the basic requirements for cell-cell fusion mediating by aMPV F proteins would contribute to better understanding of the mechanism of aMPV entry.The cleavage of aMPV/B F protein is independent of exogenous trypsin, indicating that aMPV/B F protein might be cleaved by endogenous proteases, but the proteases have still not been identified. Here, we provided the first evidence that the transmembrane serine proteases 12(TMPRSS12) facilitated the cleavage of aMPV/B F protein. We revealed that the overexpression of TMPRSS12 enhanced the cleavage extent of aMPV/B F protein, fusogenicity of aMPV/B F protein, and viral replication. Subsequently, the knockdown of TMPRSS12 with specific small interfering RNAs(siRNAs) reduced the cleavage extent aMPV/B F protein, F protein fusogenicity, and viral replication. We also demonstrated that 100 and 101 amino acid sites of aMPV/B F protein were recognized by TMPRSS12. Mutation analysis revealed that the histidine, aspartic acid, and serine residue(HDS) triad of TMPRSS12 was essential for the proteolysis of aMPV/B F protein. Importantly, we observed TMPRSS12 mRNA expression in target chicken organs of aMPV/B. Overall, our results indicated that TMPRSS12 was critical for proteolysis of aMPV/B F protein and highlight that TMPRSS12 served as a potential target to develop novel therapeutics and prophylaxis for aMPV.aMPV F protein-mediated membrane fusion of virus-cell is primarily triggered by F protein binding to its receptor(s) on host cell surface. Nevertheless, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV(aMPV/B) F proteins contain a conserved Arg-Asp-Asp(RDD) motif, suggesting that aMPV/B F protein may mediate membrane fusion and virus infection via RDD binding to integrin. To test the hypothesis, we firstly demonstrated integrin-specific peptides significantly reduced fusogenicity of aMPV/B F protein and viral replication. Specifically we identified integrin αv or/and β1 as the cell surface molecules mediated F protein fusogenicity and viral replication by using antibody blocking, siRNAs knockdown, and overexpression. Secondly, we found overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. Finally, when RDD motif of aMPV/B F protein was mutated to Arg-Ala-Glu(RAE) impaired binding and fusogenic activity of aMPV/B F protein. Overall, these results suggested that integrin αvβ1 served as a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which would provide new insights into fusogenic mechanism and pathogenesis of aMPV.Clinical observations indicate that aMPV infection cause immunosuppression for chickens, whereas, the immunosuppressive effect and immunopathogenesis of aMPV in chickens is still unclear. Type I interferons(IFN-I) like IFN-β, critical component of the host defence, play significant roles in host response against viral and bacterial infections. Here, we discovered that the response of IFN-β was downregulated in aMPV/B-infection both chickens and DF-1 cells. Meanwhile, we demonstrated that aMPV/B F protein displayed inhibitory effect for the production of IFN-β in DF-1 cells. Moreover, mutagenesis of the amino acids at 100, 101 and 93 of aMPV/B F protein revealed that the cleavage level of F protein determined the extent of aMPV/B F protein inhibiting IFN-β production. Furthermore, siRNAs knockdown and peptide treatment demonstrated that aMPV/B F protein downregulating IFN-β by TLR3 and TLR4 pathways. siRNAs knockdown and flow cytometry revealed that cluster of differentiation 14(CD14) was involved in aMPV/B F protein downregulating IFN-β production. Importantly, aMPV/B F protein impaired the immunological effect of lipopolysaccharides(LPS). Collectively, these results indicated that immunosuppressive effect of aMPV/B for chicken was likely caused by F protein counteracting the production of IFN-β via targeting CD14. Our findings would shed light on the immunopathogenesis of aMPV in chickens.aMPV F proteins undergo cleavage and attach receptor(s) to take place conformational changes, which promotes virus-cell membrane fusion. Dissection of the basic process for membrane fusion mediating by aMPV F proteins will benefit for understanding of aMPV entry and infection. In this study, we demonstrated trypsin- and low pH-mediated fusogenicity of aMPV F proteins was determined by residues at positions 100, 101 and 294. Meanwhile, we revealed that TMPRSS12 was an activating protease for cleavage of aMPV/B F protein. Moreover, we showed that integrin αvβ1 serveed as functional receptor for aMPV/B F protein-mediated membrane fusion and viral replication. Furthermore, we demonstrated that the cleavage level of F protein determined the extent of aMPV/B F protein inhibiting IFN-β production, and CD14 was involved in aMPV/B F protein downregulating IFN-β production. Our study might contribute substantially to a better understanding pathogenesis of aMPV. Further research on this aspect would shed light on the mechanism of membrane fusion mediated by F protein and the immunosuppresive effect of aMPV on chicken.