Study On The Interaction Of Host Proteins DFFA And TRIM6 With Influenza Virus Proteins

Influenza A virus(IAV)is an important zoonotic pathogen that causes frequent epidemics and occasional pandemics in humans.Since the H1N1 subtype influenza pandemic in 1918,there have been four human influenza pandemics,which pose a serious threat to human public health security.Based on the antigenicity of the surface glycoproteins hemagglutinin(HA)and neuraminidase(NA),IAVs are classified into 18 different HA subtypes and 11 different NA subtypes.Anti-influenza virus drugs are available for the prevention and control of influenza virus infections and epidemics.At present,the most widely used anti-influenza drugs are diamondoids and zanamivir,which target the matrix protein(M2)and neuraminidase protein(NA)of influenza virus,respectively.Now,drug-resistant strains have emerged in clinical application.Therefore,it is inportant to explore new drug targets and develop new anti-influenza drugs for the prevention and control of influenza virus.The nucleoprotein(NP),a major component of the viral ribonucleoprotein(vRNP)complex,has a significant function in virus life cycle.NP peotein is an excellent anti-influenza drug targets due to highly conserved in various subtypes of influenza viruses.To screen for host interacting partners of NP protein,we applied a yeast two-hybrid strategy by using NP as bait.DFFA was identified and its interaction with NP was further demonstrated by co-immunoprecipitation(Co-IP)in both transfected and infected mammalian cells.Furthermore,we found the knockdown or knockout of expression DFFA gene suppressed influenza virus replication,whereas the overexpression of DFFA also suppressed virus replication.We further found that over-expression or knockdown of expression DFFA gene affect apoptosis.The results showed that apoptosis was not the only condition for DFFA to regulate influenza virus replication.Co-immunoprecipitation assay showed that DFF35 was modified after transfection in 293T cells,and this modification was weakened after co-transfection with NP protein.Mass spectrometry analysis showed that there was phosphorylation modification of DFF35,so the regulation of DFF35 protein for influenza virus may be related to its phosphorylation level.Based on previous laboratory screening,TRIM6 protein from the TRIM family was found to interact with NP protein and PB1 protein of influenza virus through immunoprecipitation experiment.Down-regulation of TRIM6 expression by siRNA interference can promote influenza virus replication about 12-fold.On the contrary,the overexpression of TRIM6 reducted about 2.6-fold.The result show that influenza virus replication on 293T cells can be negatively regulated by TRIM6.Studies on the mechanism of TRIM6 regulating influenza virus replication found that TRIM6,as a member of TRIM family,had no effect on the expression of NP and PB1 proteins.But the expression of NP protein inhibited the expression of TRIM6,and TRIM6 inhibited the level of SUMOylation of NP protein.In conclusion,our study identified some host factors that interact with influenza viruses,verified their interactions,and further explored the mechanisms which DFFA and TRIM6 affect influenza virus replication.Our findings enriched the understanding on the regulation of influenza virus replication network by host factors.