Cellular protein α-actinin has been shown to involve in HCV replication in Huh7 HCV replicon cells . However , it is still elusive howα-actininregulates HCV RNAsynthesis . In this study , we attempted to study the mechanismhowα-actinin facilitates HCVreplication. α- Actinin was transfectedinto JFH1-infected Huh 7.5 cells . The results showed that overexpression of α-actinin significantly increased HCV nonstructural protein expression as well as intracellular and supernatant HCV RNAlevels . More inf ective virus particles were also secreted into media. Membrane flotation analysis indicated that the vast maj ority of both fulllength actinin and NS5A were colocalized in the detergent-resistant membrane ( DRM) fractions . Knockdown of α-actinin altered the membrane association pattern of NS5A. Immunofluorescence microscopy analysis showedthat NS5A was unable to display atypical pattern of cytoplasmic distribution and colocalize with calnexin on endoplasmic reticulum. Taken together , these results indicate that α-actinin regulates HCVreplicationthrough facilitation of NS5A’s associati on with endoplasmic reticulummembrane , and may provide newinsights into fundamental cellular processes and identify novel targets f or antiviral intervention.