JVI Accepts, published online ahead of print on 14 February 2007
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J. Virol. doi:10.1128/JVI.01366-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Participation of Rab5, an early endosome protein, in hepatitis C virus RNA replication machinery

Michelle Stone, Shuaizheng Jia, Won Do Heo, Tobias Meyer, and Kouacou V. Konan*

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park PA, Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA

* To whom correspondence should be addressed. Email: kvk10{at}psu.edu.


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Abstract

Like most positive-stranded RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits ER-to-Golgi protein traffic (Konan et al., J. Virol. 77, 7843-7855). However, both NS4AB and NS4B can induce "membranous web" formation, first reported by Egger et al. (J. Virol. 76, 5974-5984), which is also observed in HCV-infected cells (Rouille et al., J. Virol. 80, 2832-2841) and cells that bear subgenomic NS5A-GFP replicon (Moradpour et al., J. Virol. 78, 7400-7409). To determine the intracellular origin of the web, we have examined NS4B co-localization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein, NS4B. Furthermore, an immuno-isolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild type and constitutively active Rab5 were associated with NS4B. Interestingly, expression of dominant negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 siRNAs into NS5A-GFP replicon cells resulted in significant decrease in GFP fluorescence. Finally, Rab5 protein was found to co-immunoprecipitate with HCV NS4B. These studies suggest that EE proteins including Rab5 may play a role in HCV genome replication or web formation.




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