Fusion Expression Of HCV Co-receptor OCLN Extracellular Loops And Functional Study

Hepatitis C virus is a small enveloped RNA virus belonging to the Flaviviridae family,genus Hepacivirus,and that causes chronic liver disease in humans,including liver fibrosis,cirrhosis,and Hepatocellular carainoma.However,the HCV genotypes are highly variable,and lack of effective small animal models,HCV vaccine remains unavailable to date.Although current antiviral therapy comprising direct-acting antivirals(DAAs)can achieve a quite satisfying sustained virological response(SVR)rate,it is still limited by viral resistance,long treatment duration,combined adverse reactions,and high costs.DAAs are inhibitors which target the step of replication during the HCV life.HCV receptor analogue may be a novel candidate as an efficient inhibitor of HCV entry which can induce a synergy in the efficacy of the antiviral effect.Human occludin(OCLN)is an essential HCV cell entry factor which are human-specific co-receptors,therefore restricting the natural host of HCV.Previous studies identified an α-helical domain within gp41 composed of a trimer of two interacting peptides.The crystal structure of this complex,composed of the peptides N36 and C34,is a six-helical bundle.To interrogate determinants of OCLN-mediated HCV infection and to study the functions of OCLN extracellular loops,we designed model proteins that consist of the HIV-1 gp41 NHR and CHR segments(six-helical bundle,6HV1)linked by short protein linkers or OCLN extracellular loops.The proteins were successfully cloned and effectively expressed in prokaryotic expression system.The protein,named D1ECL1S+D2ECL2,was found to have the strongest effort to inhibited HCV infection at non-cytotoxic concentration.Circular dichroism studies indicate that D1ECL1S+D2ECL2 adopt stable a-helical folds consistent with design.Thermal denaturation indicated that D1ECL1S+D2ECL2 is highly stable at 80℃(melting temperature,Tm,of 89.09 ± 2.0℃)and comparable in stability to 6HV1.Moreover,the time-of-addition experiment revealed that D1ECL1S+D2ECL2 predominantly function at the early steps during HCV entry.HCV inhibits HCV entry by acting directly on the viral particle which is consistent with our hypothesis.We also demonstrate that D1ECL1S+D2ECL2 inhibits HCVcc as well as HCVpp entry,regardless of the genotype.Taken together,our findings provide a novel strategy for developing potent and broad-spectrum HCV entry inhibitors,which may complement the current direct-acting antiviral medications for chronic hepatitis C.We also provide a novel avenue for recombinant OCLN protein expression in vitro,which may shed light on structural studies for OCLN.