Effect Of Influenza A Virus NS1 Protein On NS Segment Splicing And VRNPs Nucleocytoplasmic Shuttling

Avian influenza is an avian infectious disease syndrome caused by influenza A virus of orthomyxovirus.Virus Ribonucleoproteins are the basic units of viral replication.In the early stage of IAV infection,the v RNPs were transported into cellular nucleus by importin ?/? complex to participate in replication and transcription of viral genes.The newly synthesized proteins assemble with v RNAs in the nucleus and the formed v RNPs are exported to the cytoplasm to be matured.The Nuclear export protein,Matrix protein 1 of avian influenza virus,the cellular chromosomal maintenance 1 and v RNPs form a exporting complex.NS segment is the shortest segment of IAV.NS1 protein,as the the non-structure protein of virus,ecoded by NS segment has a high level expression during infection.NS1 is the multifunctional viral protein,such as repressing the host immune response,regulating gene expression by affecting splicing and transcription of both the host and virus m RNA.We first observed that NS1 protein enhances the transcription and splicing of NEP m RNA,and the RNA binding domain plays a key role in transcription while the effector domain is important for splicing of NS segment.Then the experiment of immunofluorescence and fused with green fluorescent protein tag display that NS1 protein changes the subcellular localization of NEP protein.Absent of NS1 protein,the NEP located in nucleus.While NS1 protein coexpression with NEP protein,made the nucleus exportion more apparently.No matter if the NS1 protein presented,NEP protein is colocalization with cellar CRM1 protein.The physiological meaning of NEP exportion is believed to assist the exportion of v RNP.So we use the method of reverse genetics to recombine a NS1 mutant virus.The localization of v RNPs are observed through the immunofluorescence.The result showedthat 96 % NP protein located in cytoplasmic after infecting the wild type in 4 hours.Only 0.5 % NP protein located in cytoplasmic after infecting the NS1 1-38 residues truncated virus 4 hours while 28 % NP protein located in cytoplasmic after infect in 11 hours and 30 % NP protein located in cytoplasmic after infect NS1 1-80 residues truncated virus in 4 hour.Meanwhile,we detected the titer of wild type virus and the NS1 truncated virus.The titer of wild type virus is 10000 times more than NS1 protein deficient virus.But titer of NS1 1-80 residues truncated virus is only 10 times less than the wild type.Both suggested that if the NS1 protein completely missing,the influenza virus can barely replicate,while retaining the NS1 RNA binding domain,replication competent virus particles is slightly lower than the wild type virus.These indicated that the RNA binding domain of NS1 protein plays a key role of virus replication.In summary,our research shows that NS1 protein affects the splicing and transcription of NS m RNA,in order to regulate the exportion of v RNPs complex and participate in the influenza a virus life cycle.