Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein

Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic t rans-activation activity. To investigate its potential role in viral replic ation, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identi fied a newly discovered soluble N-erhylmaleimide-sensitive factor attachmen t protein receptor-like protein termed human vesicle-associated membrane pr otein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP- 33 and NS5A. interestingly hVAP-33 was also shown to interact with NS5B, th e Viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domai ns of hVAP-53: NS5A binds to the C-terminus, whereas NS5B binds to the N-te rminus of hVAP-33. Immunofluorescent staining showed a significant colocali zation of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coil ed-coil domain followed by a membrane-spanning domain at its C-terminus. Ce ll fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These intera ctions provide a mechanism for membrane association of the HCV RNA replicat ion complex and further suggest that NS5A is a part of the viral RNA replic ation complex. (C) 1999 Academic Press.