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 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.
Tu
H
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Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, California 90033, USA.
Gao
L
L
Shi
S T
ST
Taylor
D R
DR
Yang
T
T
Mircheff
A K
AK
Wen
Y
Y
Gorbalenya
A E
AE
Hwang
S B
SB
Lai
M M
MM
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
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