| Influenza A virus(IAV)is a membrane-bound RNA virus.It can be divided into 18 hemagglutinin(HA)subtypes and 11 neuraminidase(NA)subtypes based on the different HA and NA proteins.IAV continuously evolves in nature,posing a serious threat to human and animal health and causing significant economic impact globally.The genome of IAV is composed of 8 segmented genes with a genome size of approximately 13 kb and limited coding capacity,encoding only a dozen viral proteins.Therefore,at each stage of its replication cycle,it requires host genes to complete the virus’ s proliferation.The lifecycle of influenza virus includes several steps,such as attachment,endocytosis,uncoating,transcription and translation of the genome,assembly of virus particles,and budding.However,the critical host factors and related molecular mechanisms required for its infection and replication in host cells are not yet fully understood.To investigate the critical host factors and molecular mechanisms involved in IAV infection and replication in host cells,this study constructed an A549 whole-genome CRISPR knockout library cell line(A549-GeCKO library cells).High-throughput sequencing was used to confirm that the coverage of the library cells met the experimental requirements.Multiple rounds of lethal infection with influenza virus were performed on the library cells,with cell survival used as a screening phenotype.Surviving cells were obtained through positive selection after infection,and the sg RNA carried by the surviving cells and their corresponding host genes were identified using high-throughput sequencing.Combined with bioinformatics and statistical analysis,a subset of candidate target genes was selected and single-gene knockout cell lines were constructed.Preliminary verification showed that genes such as GNE,UNC50,ODF3L2,FMO5,and GABPB2 play important roles in the process of influenza virus replication.This study focused on GNE and UNC50,revealing their roles and molecular mechanisms in influenza virus replication.To reveal the role of GNE in influenza virus infection,two GNE knockout monoclonal cell lines,GNE-KO-G62 and GNE-KO-G64,were constructed.It was verified that GNE knockout could inhibit the infection of different subtypes of influenza virus,and the expression of h GNE1 isoform in GNE knockout cells could restore viral infection.On the other hand,overexpression of GNE could promote influenza virus replication,demonstrating that GNE is an important host gene in the process of influenza virus infection.Further research indicated that GNE mainly participates in the adsorption and endocytosis stages of the influenza virus replication cycle.As a rate-limiting enzyme in the biosynthesis of sialic acid,GNE can regulate the synthesis of Neu5 Ac,the precursor of sialic acid,thereby affecting the synthesis of α-2,3 and α-2,6 linked sialic acid receptors of influenza virus,and thus affecting the binding of influenza virus to host cells and subsequent endocytosis.Additionally,GNE may also regulate innate immune responses through its sialylation of host proteins.This study also found that the UNC50 protein is crucial for influenza virus replication.To reveal the molecular mechanism of UNC50 protein in influenza virus replication,two UNC50 gene knockout monoclonal cell lines,UNC50-KO-U9 and UNC50-KO-U20,were constructed.It was confirmed that UNC50 knockout can inhibit the replication of various subtypes of influenza virus and does not affect the adsorption,internalization,and v RNP formation of influenza virus.This suggests that UNC50 may be involved in the late stage of influenza virus replication,i.e.,the synthesis and expression of viral proteins and the assembly and release of virus particles.Ultra-thin sectioning of virus-infected UNC50-deficient cells and A549 wild-type cells revealed a significant reduction in virus budding on the outer side of the cell membrane after UNC50 deficiency.Immunoprecipitation experiments revealed that UNC50 interacts with the HA and NA proteins of influenza virus,and UNC50 affects the targeting of HA and NA to the cell membrane.After UNC50 knockout,HA and NA are retained inside the cell and cannot be transported to the cell membrane,and VLP experiments also indicate that UNC50 affects the budding ability of HA and NA,confirming that UNC50 affects the assembly of virus particles by affecting the transport of HA and NA to the cell membrane.Further analysis showed that UNC50 may affect the vesicular transport pathway related to the Golgi apparatus by affecting the expression of the GBF1 protein,resulting in the inhibition of the vesicular transport pathway of HA and NA.In summary,this study identified important host genes involved in influenza virus infection through genome-wide CRISPR screening,and revealed the stage and molecular mechanism of GNE and UNC50 in influenza virus replication,providing a theoretical basis for the prevention and treatment of influenza virus. |
