title: Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein.
abstract: Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-ethylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-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, the viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-33: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell 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 interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex.
authors.affiliation: Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, California 90033, USA.
authors.forename: H
authors.initials: H
authors.lastname: Tu
authors.forename: L
authors.initials: L
authors.lastname: Gao
authors.forename: S T
authors.initials: ST
authors.lastname: Shi
authors.forename: D R
authors.initials: DR
authors.lastname: Taylor
authors.forename: T
authors.initials: T
authors.lastname: Yang
authors.forename: A K
authors.initials: AK
authors.lastname: Mircheff
authors.forename: Y
authors.initials: Y
authors.lastname: Wen
authors.forename: A E
authors.initials: AE
authors.lastname: Gorbalenya
authors.forename: S B
authors.initials: SB
authors.lastname: Hwang
authors.forename: M M
authors.initials: MM
authors.lastname: Lai
mesh_terms: D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D016174:Hepacivirus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013347:Subcellular Fractions; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; C114051:VAPA protein, human; C491919:Vapa protein, rat; D033921:Vesicular Transport Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase