Regulation Mechanism Of Influenza A Virus Replication By Phosphorylation And Dephosphorylation Of Nucleoprotein

Influenza A viruses are widespread human pathogens that are responsible for seasonal flu epidemics and occasional severe pandemics of respiratory disease,with significant morbidity and mortality.Influenza A virus is a member of the Orthomyxoviridae family of enveloped viruses.The genome of influenza A virus contains eight negative-sense single-stranded RNA segments that encode viral proteins.Nucleoprotein(NP),three polymerase subunits(PB1,PB2 and PA),and viral RNA(vRNA)form the major components of the viral ribonucleoprotein(vRNP)complex,which is responsible for viral transcription and replication.NP is encoded by the segment 5 of vRNA and is an abundant protein in the virus particle.The intracellular trafficking and oligomerization of NP,and the interaction of NP with PB1,PB2 and vRNA in the vRNP complex are important for regulating the viral polymerase activity and the virus replication cycle.NP molecules self-associate into oligomeric structures by the insertion of the tail-loop of one NP molecule into the groove of a neighboring NP molecule,which promotes the formation of vRNP complex.NP interacts with PB1 and PB2 but not PA.In addition,NP binds RNA with high-affinity and with no specificity.Besides,NP promotes the nuclear import and export of vRNP.Nuclear export signals(NESs)and nuclear localization signals(NLSs)play important roles in the nuclear-cytoplasmic shuttling of NP during viral infection.Our previous study demonstrates that NP contains two chromosome maintenance protein 1(CRM1)-independent NESs(NES1 and NES2)and a CRM1-dependent NES(NES3).The phosphorylation and dephosphorylation can occur on serine,threonine and tyrosine.The phosphorylation and dephosphorylation of viral proteins may positively or negatively regulate their functions,which plays important roles during the influenza virus infection.Several phosphorylated residues in the proteins of influenza A virus have been reported.In the present study,we explored the regulation mechanism of influenza A virus replication by phosphorylation and dephosphorylation of NP.Threonine 188(T188)was identified as a novel phosphorylated residue in the NP of A/WSN/1933(H1N1)by using mass spectrometry.T188 is located within NES2 which is CRM 1-independent.Sequence homology alignments demonstrated that the T188 residue and this threonine linker regions are highly conserved among all the analyzed influenza A and B viruses.T188 and its hydroxyl group within NES2 are exposed on the surface of the oligomeric NP and accessible by other proteins,indicating that it could be a functional phosphothreonine.We introduced a glutamate(E)substitution at T188 to mimic the constitutively phosphorylated residues and an alanine(A)substitution at T188 to mimic the constitutively dephosphorylated residues.We examined the cellular localization of NP at various times and found that the phosphorylation of T188 inhibited NES2-dependent NP nuclear export by IFAs and cell counting.In addition,the phosphorylation of NP T188 affected the polymerase activity of the vRNP complex.WT and T188-mutant WSN viruses were rescued using the 12-plasmid reverse genetic system.The protein and mRNA expression levels of T188-mutant viruses were decreased compared to those of WT virus by using western blotting and real-time PCR,respectively.A549 cells were infected with viruses to obtain multiple-cycle growth curves.Recombinant virus carrying the dephosphor-mimetic T188 mutant caused a reduction in virulence compared to WT virus in cells.However,the T188E mutation could not be rescued,suggesting that the phosphorylation of T188 is vital for viral growth.We further determined whether the T188 mutation affects virus replication and pathogenicity in mice,which were intranasally infected the WT and mutant viruses at diffferent titers.Light weight loss,lethality of mice,lung dissection,lesions in the lungs,lung indices and virus titers in the lungs were examined.The T188A mutation caused a decrease in both replication and pathogenicity compared to WT in mice.All these results suggest that phosphorylation and dephosphorylation of T188 alternately occurs during the virus replication cycle and jointly regulate the replication of influenza virus.Besides,we identified serine 69(S69)as a novel phosphorylated residue in the NP of influenza A virus by using mass spectrometry.Sequence homology alignments demonstrated that the S69 residue is highly conserved among all the analyzed influenza A viruses.S69 is exposed on the surface of the oligomeric NP and accessible by other proteins.We constructed the plasmids with E/A substitution at S69 to mimic the constitutively phosphorylated residues or the constitutively dephosphorylated residues,repectively.WT and mutant viruses were rescued using the 12-plasmid reverse genetic system.We observed that the phosphorylation and dephosphorylation of residue S69 regulated viral replication in cells and mice and decreased the viral proteins expression,and found that the phosphorylation of S69 affected the viral polymerase activity and the transcription and replication of viral RNAs.We further detected the interaction of NP and polymerase basic proteins,NP-RNA,NP-NP and found that the S69E affected the interaction of NP and PB2.Moreover,we observed the cellular localization of NP at various times and found that the phosphorylation of T188 inhibited the nuclear import of NP,which indicating the phosphorylation and dephosphorylation of residue S69 might regulated the polymerase activity the vRNP complex through controlling the NP-PB2 binding and nuclear import of NP.In summary,we found two novel phosphorylation sites of NP,T188 and S69.The phosphorylation and dephosphorylation of T188 regulated viral replication by affecting the nuclear export of NP and the polymerase activity of the vRNP complex.And we observed that the phosphorylation and dephosphorylation of residue S69 regulated viral replication by controlling NP-PB2 binding,NP nuclear import and the viral polymerase activity.Our findings provide further insights for understanding the replication of influenza A virus and a new target for antiviral drug design and development.