The Mechanism Of NUP93 Protein Promoting Influenza Virus Replication Through Interaction With PB1.

Avian influenza virus(AIV)is an important zoonotic pathogen.Avian influenza has caused serious economic losses to the poultry industry and threatened human health.The replication of avian influenza virus is strictly dependent on the host cell.The interaction between virus and host protein exists in every process of virus replication,and it is one of the important mechanisms for viruses to complete the process of infection,replication,pathogenesis and cross-species infection.The avian influenza virus PB1 protein is the center of the viral polymerase and contains an RNA-dependent RNA polymerase(RdRp)activity domain with polymerase activity,which can bind to vRNNA and cRNA.The Nterminus of PB1 interacts with PA,and the C-terminus of PB1 interacts with PB2.Therefore,the screening of host proteins that interact with PB1 is of great significance for revealing the biological function of avian influenza virus.In this study,the host factors interacting with avian influenza virus PB1 were initially screened by mass spectrometry,and it was found that avian NUP93 protein interacted with influenza virus PB1 to promote the replication of influenza virus on DF-1 cells.Overexpression of NUP93 can enhance the activity of influenza virus polymerase,and at the same time promote the entry of influenza virus NP into the nucleus.The main research contents are as follows:1.Screening of avian host factors interacting with influenza virus PB1DF-1 cells were transfected with HA-PB1 plasmid,and the interacting proteins were fished using anti-HA immunomagnetic beads.Fishing samples were analyzed by SDSPAGE electrophoresis and silver staining,and the eluates and differential bands of the samples were analyzed by mass spectrometry.62 proteins were initially screened to interact with PB1.Based on the mass spectrometry results,we selected five proteins with higher scores for validation,namely NUP93,NPEPPS,TPM3,VIM and NPM1.Three si RNAs were designed for each gene,and the above genes were silenced respectively to detect their effect on the replication of influenza virus.The experimental results show that silencing NUP93 has a significant inhibitory effect on influenza virus.2.NUP93 interacts with PB1 and promotes influenza virus replicationThe results of Co-IP showed that when the NUP93-Flag and HA-PB1 plasmids were co-transfected,the anti-HA and anti-FLAG immunomagnetic beads could fish each other for NUP93 or PB1 protein.After digestion with RNase A,the interaction of NUP93 with PB1 remained.Furthermore,under co-transfection conditions,NUP93 and PB1 were found to co-localize intracellularly.In this study,NUP93 was overexpressed to detect its effect on influenza virus replication.Flow cytometry analysis showed that NUP93 could enhance the replication of GFP fluorescently labeled influenza virus in DF-1 cells;Western blotting and virus titer results showed that NUP93 could increase the NP protein expression of JX/H5N6 and DW/H5N1 viruses in cells,and the virus titer also significantly improved.Therefore,NUP93 is a positive regulator of IAV proliferation on cells.3.NUP93 enhances influenza virus polymerase activity and promotes NP entry into the nucleusThe effect of NUP93 on the polymerase activity was investigated by influenza virus polymerase activity detection system established in our laboratory.The results showed that overexpression of NUP93 could enhance the activity of influenza virus polymerase.To further explore the mechanism by which NUP93 promotes viral proliferation,we investigated the effect of NUP93 on vRNNP entry and exit.The results showed that overexpression of NUP93 could promote the nuclear entry of vRNNP after 2 h of infection with JX virus.The similar results were obtained when protein levels were measured in the same treated cells.