Study On The Mechanism Of Host Factor TOR1AIP2 Regulating Influenza Virus Replication

Influenza virus,a zoonotic pathogen,often causes pandemics and poses a huge threat to human health and the development of livestock and poultry breeding.Influenza viruses are prone to mutate,constantly escape the immune response of host cells,and develop resistance to existing anti-influenza drugs.Therefore,it is urgent to find new ways against influenza.After the influenza virus invades the host cells,it will use the host resources to self-replicate,completing the processes of adsorption,endocytosis,membrane fusion,v RNP complex nuclear import,transcription,translation,packaging and release.The host factors play vital roles in the entire replication process.Therefore,searching for host factors involved in the replication process of influenza viruses and exploring the molecular mechanisms by which host factors regulate influenza virus replication will help to find potential anti-influenza drug targets and lay a theoretical foundation for the prevention and control of influenza virus.Our laboratory previously used a genome-wide siRNA library to screen many host genes involved in the replication of the H5N1 NA-Venus report virus.Among them,we selected TOR1AIP2 to explore its impact on influenza virus replication.TOR1AIP2 can encode two proteins,IFRG15 and TOR1AIP2.We explored the molecular mechanisms of the two proteins on influenza virus replication in this study.By using RNAi interference technology,siRNAs targeting IFRG15 were transfected into A549 cells,and the effect of IFRG15 on influenza virus replication was detected.It was found that down-regulation of IFRG15 not only inhibited the replication of H5N1 NA-Venus reporter virus,but also inhibited wild-type A/Anhui/2/2005(AH05)(H5N1)and A/WSN/33(H1N1)influenza virus replication.Meanwhile,down-regulating expression of IFRG15 had no effect on VSV-EGFP virus replication.In order to explore the molecular mechanism of IFRG15 promoting influenza virus replication,Western blotting detection found that IFRG15 promoted the synthesis of influenza virus M2 protein.The result of dual luciferase reporter system found IFRG15 did not affect influenza virus polymerase activity.To explore whether IFRG15 interacts with virous influenza virus proteins,the Co-IP test was used to detect that IFRG15 did not interact with all viral proteins.Since IFRG15 is an interferon responsive protein,an interferon stimulation test was used to detect whether it affects the activation of the interferon pathway.The results showed that IFRG15 did not affect the activation of the interferon ? and ? pathways.The TOR1AIP2 knockout cell lines were constructed by CRISPR/Cas9 gene knockout technology to detect the effect of TOR1AIP2 knockout on influenza virus replication was tested.By plaque assay,TOR1AIP2 positively regulated influenza virus replication.Western blotting results indicated that the expression levels of influenza virus proteins were significantly decreased after TOR1AIP2 was knocked out.The q RT-PCR experiment found that the m RNA synthesis of NP protein was significantly inhibited after TOR1AIP2 was knocked out.In order to reveal the phase in which TOR1AIP2 promotes influenza virus replication,flow cytometry and Western blotting results showed that TOR1AIP2 did not affect the adsorption and endocytosis of influenza virus.The results of laser confocal experiments showed that TOR1AIP2 did not affect the fusion of influenza virus into the endosomal membrane,and the v RNP complex could be released into the cytoplasm through membrane fusion.Through immunofluorescence staining of NP protein and detection of NP protein content in the cytoplasm and nucleus after virus infection,it was found that TOR1AIP2 knockout significantly inhibited NP protein from entering the nucleus,indicating that TOR1AIP2 knockout inhibited v RNP complex from entering the nucleus.The dual luciferase reporter system was used to detect the polymerase activity of influenza virus,and it was found that TOR1AIP2 knockout did not affect the polymerase activity.In order to elucidate the mechanism by which TOR1AIP2 inhibited the nuclear import of the v RNP complex,Co-IP experiments were performed to test whether there was an interaction between TOR1AIP2 and each viral protein,and we found no interaction between TOR1AIP2 and all viral proteins.In addition,TOR1AIP2 had no interaction with nuclear import proteins involved in influenza virus replication.This study found that both IFRG15 and TOR1AIP2 encoded by TOR1AIP2 can promote influenza virus replication,and TOR1AIP2 affects the nucleation of the v RNP complex of influenza viruses.However,the molecular mechanisms of IFRG15 and TOR1AIP2 promoting influenza virus replication remain to be further studied.This study confirmed that IFRG15 and TOR1AIP2 positively regulate influenza virus replication,enriching the host factor network involved in influenza virus replication and providing a basis for subsequent studies on the molecular mechanisms of IFRG15 and TOR1AIP2.