Effect Of Site-Directed Mutagenesis Of Amino Acids In The NLS3 Region Of Nucleoprotein On Replication And Proliferation Of H1N1 Subtype Influenza Virus

As a multi-functional protein of influenza A virus(IAV),the functional domains of nucleoprotein are indispensable structural basis for its functions.Among them,the nuclear localization signals(NLSs)play a key role in mediating NP into the cellular nucleus and initiating the assembly of vRNP.At present,there are many reports about the functions of NSL1 and NSL2 of NP,but it is not clear for NSL3 in the role of replication and proliferation of IAV.The sequence alignment of 1,578 H1N1 influenza viruses in GenBank revealed that there were significant differences at the 350,353,371,373,377,and 400 amino acid sites in the NLS3 region of NPs between human pandemic and seasonal strains.In this study,T,V,M,T,S,and R at these six positions were respectively substituted for K,I,V,A,N,and K by the overlappingping PCR used A/WSN/1933(H1N1)strain as the donor of NP gene or mother virus.Then the effects of these site-directed mutagenesis on the biological activities were studied in vitro such as the efficiency of nuclear localization of NP,polymerase activity,and viral replication and proliferation.The analysis of confocal laser scanning microscopy confirmed that T373 A mutation significantly reduced the efficiency of nuclear localization of NP(p<0.001),while the other five site mutations had no significant effect(p>0.05).In addition,it was showed by the dual-luciferase reporter system assay that the activity of polymerase was increased significantly after the substitution of T350 K,V353I or S377N(p<0.05),but the activity was decreased significantly after T373 A mutation(p<0.05),while the mutation of R400 K or M371 V had no significant effect on the activity of polymerase(p>0.05).In order to further explore the effect of these mutations on the replication and proliferation of the four site-mutated rescued viruses in MDCK cells,the levels of cRNA,vRNA and RNA together with viral titers were detected by the real-time fluorescence quantitative PCR and the viral growth kinetics curve,respectively.The results showed that the substitution of V353 I or S377 N can significantly up-regulate the total RNA level in infected cells(p<0.05)with the significant increase in virus proliferation in MDCK cells(p<0.05),while the M371 V or R400 K mutation had no significant effect on them(p>0.05).All these results showed that the amino acids 353 and 377 in the NLS3 region of nucleoprotein are the key sites associated with the efficiency of nuclear localization of NP,polymerase activity,replication and transcription of vRNA,and virus proliferation,while the amino acid 371 or 400 has no significant effect on these indicators.This study preliminarily explored the effect of site-direct mutagenesis of some amino acids in NLS3 region of nucleoprotein on viral replication and proliferation in vitro,which can not only help us deeply understand the functions of NP and the mechanism of infection and pathogenesis of IAV,but also provide us experimental basis for exploring new and potential targets of antiviral drug.