The NS1Protein Phosphorylation Research Of Influenza A Viruses

Influenza A viruses are enveloped viruses within the family Orthomyxoviridae. They contain a single-stranded, negative sense, segmented RNA genome consisting of eight segments of viral RNA (vRNA), which encode11known proteins. NS1protein is encoded by the segment8th, containing230-270amino acid. NS1do not exist in virus particles but in the infected cells. In the early time of virus infection, NS1majority accumulate in the nucleus, then export into the cytoplasm in the later time. There are two major domains on NS1protein:RNA-binding domain at N-terminal and effector domain in C-terminal. NS1has multiple function during viral infection, such as its inhibition of host immune response and synthesis of host protein. These functions are to the benefit of efficient replication and infection of virus.Interplay of host immune system and virus determine the efficient of viral infection. First, infected cells trigger the antivirus immune machine to defend viral replication and the protein synthesis of virus; for another, viruses have evolved multiple mechanisms to circumvent these defenses. NS1is a multi-functional protein that antagonize host immune responses. It also performs a plethora of activities, which may additionally contribute towards efficient virus replication and virulence during infection:Interaction with nucleoprotein (NP) promote the replication of viral genome; recruitment of translation initiation factor onto the viral mRNA.Phosphorylation sites on NS1of influenza virus A/WSN/1933(H1N1) were identified by mass spectra. The residues were mutated to Ala and phospho-mimic Glu/Asp to analysis their function on virus and IFN production. All mutated viruses were rescued by using of reverse genetic system and tittered for the growth curve. Several mutants (T80E and S83D) showed the lower growth curve compared with wild type (WT), indicating that these sites have an effect on virus replication. Intracellular localization of these NS1mutants were also observed with indirect immunofluorescence assay, however, none of them show different location with NS1WT. by using Real-Time PCR, we found that the IFN-antagonism ability of NS1was reduced by mutation T80E and S83D. The RNA-binding ability of NS1mutant (T80E) was reduced by detected by immunoprecipitation with poly(U)-agarose. We hypothesis that reduction of RNA binding ability of T80E may cause the enhancement of RIG and viral RNA, which may lead to more production of IFN and lower virus replication. By the study of phosphorylation on NS1protein, we further understand the interaction of virus and host, and battle of viral virulence factor with host immune system.