Research Of The Interaction Between Chicken CCT5 Protein And Avian Influenza Virus NP And PB2 Protein To Promote Virus Replication

Avian influenza virus(Avian influenza virus,AIV)are the part of the Orthomyxoviridae family and placed in the the genus of AIV.AIV occurs naturally among wild aquatic birds worldwide.While,AIV some subtypes of AIV also infect humans.Some H5 subtype influenza viruses prevalent in poultry are highly pathogenic AIV(HIPV),which is a major threat to public health and poultry breeding industry.Therefore,understanding the pathogenic mechanism of AIV has theoretical significance for influenza virus prevention and control.The interaction between AIV and host protein is the key part of virus replication and pathogenicity.In the past 20 years,lots of research have demonstrated that human host factors play a key role in the life cycle of influenza virus.However,little is known about the role of avian host factors in influenza virus replication.In this study,immunoprecipitation-mass spectrometry was used to detect the avian host factors that interact with influenza virus.It was found that chicken CCT5 protein(a cytoplasmic chaperone protein containing TCP1 subunit 5)directly interacted with AIV NP and PB2.Further studies showed that virus infection significantly promoted the expression of CCT5 in DF-1(chicken embryo fibroblast DF1 cells),and the up-regulated CCT5 in turn promoted the nuclear output of NP protein,promoting the proliferation of AIV.The results show that avian CCT5 plays an important role in supporting AIV replication.The main research contents of this topic are as follows:1.Screening of interacting avian host factors by immunoprecipitation-mass spectrometry.In this study,p CAGGS-HA-NP plasmid was constructed and transfected into DF1 cells.After expressing NP protein,DF1 cells were infected with influenza virus H5N6,and specific NP antibody fishing interaction proteins were detected by immunoprecipitation;.SDS-PAGE electrophoresis and silver staining were used to find differential proteins.72 potential NP protein interaction host factors were identified by protein spectrum analysis.Compared with the NP interacting proteins published in the literature,it was found that CCT5,FARSA,MTHFD1 L,NOP56 and RUVBL1 might interact with NP proteins.Then,si RNA was used to silence these five candidate host factors respectively to detect their roles on influenza virus NP protein and m RNA levels.The results showed that CCT5,FARSA,MTHFD1 L silencing significantly inhibited the expression of influenza virus NP protein,among which CCT5 gene silencing had the most obvious inhibitory effect on NP/PA/PB2 expression.2.Construct CCT5 expression plasmid and verify the interaction between CCT5 and viral structural protein.In the experiment,the avian CCT5 gene was cloned and ligated to the p3 Xflag vector,and co-transfected with the HA-NP/PB2 expression plasmid into DF1 cells,the HA,Flag antibody were used to fish each other and the interaction was tested by immunoprecipitation technique.The endogenous antibodies CCT5 and NP/PB2 were used to fish each other after the virus was infected with CCT5-Flag protein alone.Meanwhile,this result was verified by immunofluorescence technique.CCT5 interacted with NP and PB2 proteins either overexpressed or infected with influenza virus,so there is a guess that CCT5 may be the ligand of NP and PB2 proteins and participate in the replication process of influenza virus.3.To verify the relationship between CCT5 and influenza virus replication.Considering the fluorescence quantitative results showed that si-CCT5 could significantly inhibit the expression of NP,PA and PB2 proteins of influenza virus.After using flow analysis,it was found that DF1 cells silenced by CCT5 could inhibit the replication of H5N6 influenza virus with GFP fluorescence labeling.The subsequent results showed that the expression of CCT5 proliferated with the increase of infection time and infection gradient of H5N6 and H1N1(HM/DW)influenza virus.This indicates that influenza virus promotes the expression of CCT5.When studying the effect of CCT5 on influenza virus RNA production,it is found that CCT5 silencing can inhibit influenza virus RNA synthesis.On the contrary,CCT5 expression can promote influenza virus RNA synthesis.Furthermore,indirect immunofluorescence assay confirmed the co-localization of CCT5 and NP in cells,and promoted the nucleation of NP protein.