Membrane Transport Is Involved In The Study Of The Mechanism Of Hepatitis C Virus Infection

Hepatitis C virus(HCV)has infected over 71 million people worldwide.Infection with HCV is associated with chronic inflammation,fibrosis and a high risk of developing hepatocellular carcinoma(HCC).As an obligate intracellular virus,HCV depends on a large number of host factors to complete its lifecycle.Finding the new host factor involved in HCV helps us better understand the life cycle of HCV.Claudin-1 is an HCV coreceptor required for viral entry.Although extensive studies have focused on Claudin-1 as an anti-HCV target,little is known about how the level of Claudin-1 at the cell surface is regulated by host vesicular transport.Here,we identify an interaction between Claudin-1 and Sec24C,a cargo-sorting component of the coat protein complex II(COPII)vesicular transport system.Claudin-1 partially colocalizes with Sec24C.In vitro COPII vesicle reconstitution assay shows Claudin-1 is sorted into COPII-coated vesicles and is a COPII cargo molecule.Blocking COPII transport inhibits HCV entry by reducing the level of Claudin-1 at the cell surface.Further experiments demonstrate that the Claudin-1 YV motif is responsible for the interaction between Claudin-1 and Sec24C.A Sec24C mutant with the cargo binding site mutated no longer binds to the Claudin-1.Sequence analysis of the C-terminal cytoplasmic tails from Claudins family proteins reveals that there is a conserved YV motif within the carboxy terminus of several Claudins.Claudins with a C-terminal YV or V motif can associate with the COPII component Sec24C,suggesting that the COPII machinery plays key roles in the proper localization and functions of Claudins.Together,we find Claudin-1 is transported from the endoplasmic reticulum(ER)by interacting with Sec24C through its C-terminal YV and is eventually targeted to the cell surface.These findings provide mechanistic insight into the role of COPII vesicular transport in HCV entry.HCV exploits lipid metabolism in various fashions to facilitate their lifecycle.In infected hepatocytes,HCV increases the local concentration of PI4P to form membranous web-like structures and recruits PI4P effector proteins to build replication complexes.Retromer mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network(TGN)or to plasma membrane.The Retromer comprises two multisubunit complexes:a cargo recognition complex composed of Vps26,Vps29,and Vps35 and a heterodimer of sorting nexins.Recently,PI4P has been shown to facilitate the dissociation of Retromer cargoes at the TGN.Whether Retromer,a PI4P-related host transport machinery,unloads its cargo at HCV replication sites remains inconclusive.We sought to characterize the role of Retromer in HCV replication.Here,we demonstrate the interaction between Retromer subunit Vps35 and HCV NS5A protein by immunoprecipitation and GST pulldown.Vps35 colocalizes with NS5A and PI4P in cells harboring OR6 replicon or infected by JFH1 virus.HCV replication is inhibited upon silencing Retromer subunits.CIMPR,a typical Retromer cargo,participates in HCV replication.Our data suggest that Retromer component Vps35 is recruited by NS5A to viral replication sites where PI4P unloads CIMPR.These findings demonstrate the dependence of HCV replication on Retromer and identify Retromer as a potential therapeutic target against HCV.