Studies On The Host Factors Involved In Influenza Virus Replication Based On The CRISPR/Cas9 System

The complicated interactions between pathogens and hosts determine the host range and the severity of the disease.After influenza virus infection,on the one hand,changes of specific amino acids in viral proteins are thought to be related to the severity of the disease or the host adaptation of avian influenza viruses,for example,when the D701N mutation in the polymerase basic protein 2(PB2)occurs,the viral polymerase activity,the viral replication and the pathogenicity to animals of avian influenza viruses are enhanced,thus leading to adaptation to mammalian hosts,which may be mediated by the increased binding to importin-?7 and thus the enhanced nuclear import of viral ribonucleoproteins(vRNPs);On the other hand,host genes could also affect influenza virus replication,such as Interferon-induced Transmembrane Protein 3(IFITM3),which inhibits the replication of influenza viruses by interfering with virus-endosome membrane fusion,thus blocking the release of vRNPs into the cytosol.Therefore,the study of interactions between influenza viruses and hosts is of great significance for revealing inter-species transmission and pathogenicity of influenza viruses.The Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated 9(CRISPR/Cas9)system has been widely used for host gene modifications to study gene functions.In this study,we firstly deleted IFITM3 gene in Hela cells or importin-a7 gene in A549 cells based on the CRISPR/Cas9 system,successfully constructing the gene knockout cell lines which were used for effect confirmation of host genes in influenza virus replication.The growth properties of influenza A viruses(including the seasonal H1N1 and H3N2 influenza viruses,avian influenza viruses,and swine influenza viruses)and influenza B viruses were systematically compared in the importin-a7 knockout cells.It was found that,similar to previous studies,importin-a7 affected the replication of influenza A viruses such as human influenza viruses and avian influenza viruses,in addition,our study was the first to find that importin-a7 affected the replication of influenza B viruses and Eurasian avian-like H1N1 swine influenza viruses(EA H1N1 SIVs).To further study the effects of importin-a7 on the pathogenicity of the PB2 D701N mutation in EA H1N1 SIVs,the D701N mutant viruses based on A/Hunan/42443/2015(H1N1)were rescued by the reverse genetics system.Growth curves and confocal microscopy results in A549-importin-a7-KO cells showed that the viral replication and the nuclear import of vRNPs of both viruses depended on importin-?7,and importin-a7 was required for the increased viral replication and nuclear import of vRNPs induced by the D701N mutation.To further investigate the phenotypic effects of the D701N mutation on the pathogenicity of EA H1N1 SIVs,in vitro experiments demonstrated that the D701N mutation increased the polymerase activity and the viral replication of EA H1N1 SIVs in mammalian cells,and in vivo experiments showed that the D701N mutation enhanced the pathogenicity of EA H1N1 SIVs in mice.In summary,the PB2 D701N mutation in EA H1N1 SIVs enhanced the replication and pathogenicity of the viruses by the increased nuclear import of vRNPs mediated by importin-a7.In addition to the above-mentioned known host factors,there are still a large number of new candidate genes involved in influenza virus replication to be discovered,we performed a genome-wide CRISPR/Cas9-based knockout screening to identify host factors crucial for A/Hunan/42443/2015(H1N1)virus replication.Here we identified 258 candidate genes,some of which are known essential genes for the replication of influenza viruses,such as TMPRSS2,ST6GAL1,UBR4 and SLC35A2,there are also many novel host genes whose functions need to be further investigated.In summary,our study successfully constructed the stable cell lines with specific knockout of host genes involved in influenza virus replication based on the CRISPR/Cas9 system.Using this technology platform,we found,for the first time,that importin-a7 had effects on the replication of influenza B viruses and EA H1N1 SIVs,and the PB2 D701N mutation could increase the replication and pathogenicity of EA H1N1 SIVs by the increased nuclear import of vRNPs mediated by importin-a7.Further CRISPR/Cas9-based knockout library screened out a series of host factors that may affect the replication of influenza viruses.