Hepatitis C virus envelope glycoproteins 2(E2) containing 18 conserved cysteine residues plays an im portant role inmediating viral cell entry and eliciting neutralizing antibodies.Although studies had shown that cysteine residues contributed to structure and function of E2 protein,significance of each cysteine in E2 function and immunogenicity among different HCV genotypes remains unclear.Here we performed a comprehensive cysteine-alanine mutagenesis scan of all 18 cysteine residues in full-length E1E2 of H77 and Con-1 strain.Results showed that all the individual cys-ala mutations in E2 protein of both strain did not affect E2 expression and membrane transport, however, abrogated Huh7.5 cells entry of the corresponding pseudoparticles.Cys494, Cys508,Cys552, Cys564, Cys607 and Cys644 in H77 strain were indispensable to conformation-dependent recognition to both H48 and H53 mAbs,while Cys429 and Cys503 were critical only to H48 recognition and Cys597 were only to H53.Results also showed that disulfide 1,3,4,and 5 were strictly indispensable,while 7 and 8 were not so strictly indispensable, and 2,6 and 9 were dispensable for H48 and H53 recognition.Further studies showed that E2 antibodies in chronic HCV patient were conformation-dependent.Individual Cys-Ala mutants in Con-1 and H77 affected CD81 binding in similar patterns.Finally,none of these mutations had an increase in E2 immunogenicity.This research contributes to the current knowledge in HCV E2 immunogenicity and HCV vaccine design.