Study On The Mechanism Of Host Protein DENND1B Affecting The Replication Of Influenza A Virus

Influenza virus,a member of the the family Orthomyxoviridae,is the infectious agent causing diseases and deaths in mammals and poultry.According to the antigenic differences of the viral NP and M proteins,influenza viruses are classified into four types,A,B,C and D.Except for infecting humans,influenza A virus(IAV)can infect many other animals,such as poultry,horses,pigs,and seals.Influenza B and C viruses mainly infect humans,and influenza D virus only causes some bovine infections.The natural host of IAV is wild waterfowl.The circulation of IAV in humans and other animals poses a huge threat to global economy and public health security.Vaccine immunization is still the most effective measure to prevent and control IAV infection.However,due to the rapid mutation of the viral HA protein of IAV,it is necessary to update the vaccine strain every one to three years,which brings great obstacles to the development and application of vaccines.Therefore,it is of great significance to study the replication cycle of IAV in order to identify new conserved anti-influenza targets and develop new antiviral drugs.DENND1B,a potential host factor for the replication of IAV,is obtained by genome-wide siRNA library screening.On this basis,the supportive role of DENND1 B in IAV replication was validated by siRNA mediated knockdown of DENND1 B expression,CRISPR/Cas9 system-based knockout of DENND1 B,and retroviral-or lentiviral-mediated overexpression of DENND1 B.Further studies showed that the N-terminal domain of DENND1 B is the key domain for regulating viral replication.We also found that DENND1 B interacted with the PB1 and PA protein of IAV,and the C-terminus of DENND1 B is the key domain in mediating the interaction with PB1.Notably,we found that influenza viral infection induce AP2B1 subcellular localization change,and host factor DENND1 B can regulate the expression and cellular localization of AP2B1 protein.In addition,up-regulation of host factor DENND1 B was accompanied by the aggregation of DENND1 B,AP2B1 and viral NP protein into the cellular membrane and co-location,which suggested that DENND1 B mainly affects the replication of IAV by affecting the endocytosis.This study preliminarily revealed the molecular mechanism of host factor DENND1 B regulating the endocytosis of IAV,enriched the interaction network between host factors and viral protein,and further deepened the understanding of IAV replication cycle.