Study On The Mechanism Of Host Factor TMUB1 Participating In The Replication Regulation Of Influenza Virus

Influenza virus belongs to the orthomyxoviridae family,influenza virus genus,an important zoonotic pathogen,can cause human and animal infection and disease.The high variability of influenza virus can easily cause influenza pandemic and inter-species transmission,which poses a great threat to human health.Therefore,the development of effective anti-influenza drugs is of great significance.High-throughput screening of host factors involved in influenza virus replication can be realized through genome-wide siRNA library and reported influenza virus,providing a favorable target for the development of anti-influenza virus drugs.According to the previous genome-wide siRNA library screening result,host factor Transmembrane and ubiquitin-like domain-containing 1(TMUB1)was identified.In this study,the plaque assay confirmed that both TMUB1 knockdown by siRNA interference and TMUB1 knockout by CRISPR/Cas9 technology significantly inhibited influenza virus replication.To explore the specific stage of TMUB1 involved in influenza virus life cycle,Western blot was used to detect the effect of TMUB1 on the influenza virus protein expression at different time points after virus infection,it was found that TMUB1 knockout could significantly inhibit the expression of various viral proteins at 4 h after virus infection,and confirmed that TMUB1 affected the early stage of virus infection.The effect of TMUB1 on influenza virus entry stage was further carried out by using a variety of methods.Flow cytometry assay proved that TMUB1 had no effect on influenza virus adsorption stage.The low PBS-HCl(pH 1.3)washing assay verified that TMUB1 had no effect on the endocytosis stage of influenza virus.By Lysotracker red DND-99 and anti-M1 antibody staining,we found that TMUB1 had no effect on PH of acid organelle of the cell and the membrane fusion stage of influenza virus.Finally,it was found that TMUB1 knockout could significantly inhibit the nuclear accumulation of influenza virus by using NP antibody immunofluorescence staining.Furthermore,dual-Luciferase reporter assay and co-immunoprecipitation experiment were performed and proved that TMUB1 had no significant effect on the polymerase activity of influenza virus.It has been reported that TMUB1 is a negative cell cycle regulatory protein.To explore whether TMUB1 promotes influenza virus replication through the cell cycle regulation,flow cytometry assay was used and verified that TMUB1 knockout was beneficial to the cell cycle progression,and TMUB1 knockout eliminated influenza virus infection induced G0/G1 phase arrest.To further explore the molecular mechanism of TMUB1 affecting influenza virus replication through regulating cell cycle,qRT-PCR was used to detect the effect of TMUB1 knockout on the mRNA transcription level of associated proteins in G1/S phase of cell cycle.The result showed that the m RNA transcription level of cylin E was significantly increased after TMUB1 knockout.TMUB1 knockout relieved influenza virus infection induced inhibition of cylinE expression by qRT-PCR and Western blot detection.This study confirmed that TMUB1 is a host factor promoting influenza virus replication and mainly affecting the nuclear accumulation of influenza virus replication cycle.Influenza virus mayinhibit the expression of cylinE through TMUB1,block the cell cycle in the G0/G1 phase,and thus facilitate the replication of influenza virus.In this study,we illuminated the molecular mechanism of host factor TMUB1 promoting influenza virus replication by targeting cell cycle,which provided a favorable target for the development of antiviral drugs.