Mechanism For Inhibition Of Influenza Virus Replication By NP Mutant And The Key Proteins In The Formation Of Influenza Virus VLPs

Influenza viruses can not only infect human beings and many kinds of animals, but also kill then frequently, and accordingly, cause serious social and economic problems. To develop new and powerful medicines and vaccines to prevent and control influenza, the characteristics and interactions between viral proteins of influenza viruses must be studied in details.Nucleoprotein is the major structure protein of influenza virus. It is highly conserved in all the subtype of influenza virus and plays a multiple role in virus life. So NP is a favorite target for medicine design.In this study, using mini-replicon system (NP/PB1/PB2/PA) to transcribe the virus like RNA which contain the open reading frame (ORF) of firefly luciferase gene, we tested the function of transcription and inhibition to wild-type NP with a NP mutant (NPA387R) which was proved to be a inhibitor of wild-type NP, and several mutants based on NPA387R. We found that all the mutants lost the transcription function in mini-replicon system. The mutant deleting the unconventional nucleic localization signal (NLS1) on NPA387R was able to inhibit the wild-type NP in mini-replicon system, while the mutant deleting both NLS1 and bipartite NLS (NLS2) was not. To clarify the relationship between the nucleic localization signals and the inhibition function, we mutated three amino acids in the NLS2 on the NLS1 deletion NPA387R mutant. In the mini-replicon system, and the mutant showed to inhibit wild-type NP, which indicated that the nucleic localization signal was not important in the inhibition.To prove this point in detail, we established two stable cell lines, one of which expressed A387R, and the other expressed the mutant with three-amino acid mutation in the NLS2 and the NLS1 deletion A387R mutant. We found that the mutant A387R localized in the nucleus while the other one localized in cytoplasm under the immuno-confocal microscopy. Both stable cell lines could not support the replication of PR8 influenza virus, while their parental MDCK cells could. The result suggested that the mutants could inhibit the wild-type NP even they were in cytoplasm.To determine the mechanism that wild-type NP was inhibited by the mutants, we used Co-immunoprecipitation (Co-IP) method to test the interaction of the proteins. We found that the homology or nonhomology interaction existed between the mutants and wild-type NP. In addition, we adopted sucrose density gradient centrifugation to analysis the interaction between the mutants and wild-type NP. The result showed that wild-type NP mainly lay in the substratum of the sucrose gradient and the mutant NP mainly in the superstratum where BSA appeared. However, after the mutants and wild-type NP co-expressed in the cells, wild-type NP moved upward to the upper layers and the mutant NP moved downward to the lower layers. Our findings suggested that the mutants interfered the oligomerization of wild-type NP, and accordingly affected the formation of functional RNPs which is necessary in the replication of influenza viruses. Our finding also offered a notion that the novel anti-flu medicines should designed targeting at NP protein in cytoplasm.In order to determine the key proteins in the formation and release of influenza virus like particles (VLPs), we used eukaryotic expression system which could express three genes in one plasmid. We constructed the plasmid expressing HA, NA and M1, the plasmid expressing HA and NA, and plasmid expressing HA only. We transfected these plasmids into 293T cell and detected the hemagglutination (HA) of the supernatant of the transfected cell culture. The hemagglutination was detected when three proteins (HA, NA and M1) or two proteins (HA and NA) were expressed, but not when only HA protein were expressed. After the supernatants were super-centrifugated, VLPs were found under transmission electron microscope when three proteins (HA, NA and M1) or two proteins (HA and NA) were expressed, but not when only HA protein were expressed. Our findings indicated that HA and NA proteins are necessary in the VLPs'formation and release, which is useful in the development of novel flu vaccines.In summery, we clarify the mechanism of mutant NP inhibit the function of wild type NP and virus replication, and we confirm that HA and NA are the major proteins for influenza virus VLP formation and release. We hope to make contribution to the anti-influenza medicine research and vaccine development.