Hepatitis C virus (HCV) cell entry is a complex, multistep process requiring numerous host cell factors, including the tight junction protein claudin-1 (CLDN1). It is not known whether CLDN1 and the HCV glycoproteins physically interact. Therefore, the focus of this work was to study genetic interactions between CLDN1 and HCV. We used CRISPR technology to generate CLDN1 knockout (KO) Huh-7.5 cells, which could not be infected by genotype 2a Jc1 HCV unless CLDN1 expression was restored. Passage of Jc1-transfected CLDN1 KO cells resulted in the selection of a virus that could infect these cells. This virus encoded a single mutation, H316N (numbered relative to the HCV polyprotein), in the E1 glycoprotein. Whereas Jc1 H316N efficiently infected cells lacking CLDN1, such infection was blocked by an antibody targeting CLDN6, another member of the claudin family that is expressed in these cells. Furthermore, HuH6 cells, which express CLDN6, but not CLDN1, were infectable only with the mutant virus. Thus, this mutant virus adapted to the loss of CLDN1 by developing the capacity to utilize other CLDNs. Indeed, CLDN1/CLDN6 double-KO Huh-7.5 cells supported infection by the mutant virus only when CLDN1, CLDN6, or CLDN9 was expressed. Finally, this phenotype was not genotype dependent, given that the H316N mutation rendered a Japanese fulminant hepatitis 1 chimeric HCV genome encoding the genotype 5a glycoproteins able to utilize CLDN6 for host cell entry. Conclusion: These data demonstrate plasticity of HCV virus-host interactions, where a previously CLDN1-dependent virus was capable of evolving to use CLDN6. They also reveal a role for E1 in determining entry factor usage and imply a direct, physical interaction between E1 and CLDNs.