Construction Of Cullin-2 Knock-out Cell Line And Effect On Replication Of Influenza Virus

The infection of Influenza A virus(IAV)can induce the host to produce excessive inflammatory responses,especially the production of "cytokine storm" in the lung,and specific mechanism of it is still unknown.Previous studies of our lab showed that there was interaction between the influenza virus and Cullin-1-related E3 ligase complex(CRL1),and it was closely related to the NF-?B pathway,which can produce inflammatory cytokines.This study focused on other CRLs,especially the CRL2 complex,to research the relationship between CRL2 and the influenza virus replication,and to explore the molecular mechanism of CRL2 affecting the influenza virus infection in the host,aiming to provide new ideas for the research of influenza pathogenesis.The CRL complex,as the largest family of E3 ligases,is responsible for recognition and ubiquitination of substrate proteins.As a cell parasitic organism with a simple structure,viruses often use the CRL complexes of the host to activate,hijack or disturb the degradation of their substrates during the process of invading and infecting host cells,creating an environment beneficial for replication.In this study,by analyzing the transcriptomics data of A549 cells infected with influenza A virus Ca09(H1N1)6 hours after infection,it was found that the transcription level of cullin2,cullin5 and CRL activation inhibitor CAND1 gene was significantly down-regulated after 6 hours of influenza virus infection,indicating that Various CRL-related genes may play an important role in the early stage of influenza virus infection.In order to study the effect of CRL complex on influenza virus replication and the expression of host cytokines,we designed sgRNA according to genes of 8 Cullin proteins and the NAE1 enzyme required for CRL activation,constructed them into LentiCRISPRv2 vector,which was transfected into 293 T cells,harvested packaged lentivirus,infected A549 cells,and screened with puromycin.As a result,NAE1,cullin2 and cullin3 gene knockout cells were obtained,and then the Cullin-2 protein stable knockout cell line was obtained by limiting dilution method,named CUL2-KO.After identification by Western blot,PCR and sequencing,the cell line was successfully constructed.On the basis of obtaining the CUL2-KO knockout cell line,this study further explored the effect of cullin2 gene knockout on influenza virus replication.The influenza virus WSN(H1N1)was used to infect the cells of cul2-ko cell line and the control group,and we collected cell samples and cell supernatant at different time points(6h,12 h,24h,36h).Through virus titration and Western blot detection,it was found that cullin2 gene knockout had no significant effect on influenza virus replication.However,using qRT-PCR detection,it was found that cullin2 gene knockout can enhance the up-regulation of IFN-?,IL-6 and other cytokines caused by IAV infection.Further research found that the Cullin-2 protein interacts with the NF-?B pathway activation inhibitor I?B? protein.Cullin-2 protein may affect the expression of cytokines by regulating the degradation of I?B?.The specific molecular regulatory mechanism remains to be further studied.In order to explore the possible mechanism that cullin2 gene knockout enhanced the transcriptional upregulation of cytokines caused by IAV infection,We conducted a proteomic analysis of CUL2-KO cells and analyzed the impact of cullin2 gene knockout on the expression of other proteins,and found that the expression of proteins related to cytokine expression has changed,such as the up-regulation of IFN-? receptor IFNGR1,interleukin-1 receptor-related kinase IRAK2 and other proteins,which provided more ideas for clarifying the relationship between CRL2 and influenza pathogenicity.