Hepatitis C virus (HCV) is the main cause of chronic hepatitis worldwide. Current treatment of HCV is expensive with unpleasant side effects and no vaccine available until now. HCV has been classified into at least 7 genotypes and over sixty subtypes. The HCV glycoprotein E2 is associated with E1 to make heterodimer and mediate entry into target cells. There are three variable regions within E2 glycoprotein designated hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region. It has been proposed that the variable regions are surface exposed. Deletion of HVR1 leads to attenuation of cell culture derived HCV (HCVcc) that can be restored by adaptive mutations. However, deletion of HVR2 or the igVR is lethal to HCVcc infectivity. In this study, we used phylogenetic analyses to select nine closely and distantly related strains of HCV to construct full length chimeric genomes harbouring these alternative HVR2 and igVR sequences. We used in-vitro transcribed RNA to transfect Huh 7.5 cells. All chimeras, except MD5.HVR2, had relatively similar capacity to replicate RNA in Huh 7.5 cells. Secreted chimeric HCVcc viruses were used to infect naive Huh7.5 cells. We observed that the infectivity titre of MD5.HVR2 and TN9.igVR are 1 Log and ½ Log lower than the parental WT, respectively. The chimeric viruses with HVR2 and the igVR sequences of G.1a H77c isolates were completely non-infectious and infectivity could not restored by passaging cell associated and/or cell free virus. These studies suggest that the E1E2 glycoprotein complex within infectious HCV produced in cell culture is highly sensitive to sequence change in HVR2 and igVR. The mechanism by which HCV is attenuated by sequence change in HVR2 and igVR will be investigated.