Research Article: Rab18 Binds to Hepatitis C Virus NS5A and Promotes Interaction between Sites of Viral Replication and Lipid Droplets

Date Published: August 1, 2013

Publisher: Public Library of Science

Author(s): Shadi Salloum, Hongliang Wang, Charles Ferguson, Robert G. Parton, Andrew W. Tai, Guangxiang Luo.


Hepatitis C virus (HCV) is a single-stranded RNA virus that replicates on endoplasmic reticulum-derived membranes. HCV particle assembly is dependent on the association of core protein with cellular lipid droplets (LDs). However, it remains uncertain whether HCV assembly occurs at the LD membrane itself or at closely associated ER membranes. Furthermore, it is not known how the HCV replication complex and progeny genomes physically associate with the presumed sites of virion assembly at or near LDs. Using an unbiased proteomic strategy, we have found that Rab18 interacts with the HCV nonstructural protein NS5A. Rab18 associates with LDs and is believed to promote physical interaction between LDs and ER membranes. Active (GTP-bound) forms of Rab18 bind more strongly to NS5A than a constitutively GDP-bound mutant. NS5A colocalizes with Rab18-positive LDs in HCV-infected cells, and Rab18 appears to promote the physical association of NS5A and other replicase components with LDs. Modulation of Rab18 affects genome replication and possibly also the production of infectious virions. Our results support a model in which specific interactions between viral and cellular proteins may promote the physical interaction between membranous HCV replication foci and lipid droplets.

Partial Text

Hepatitis C virus (HCV) is a positive-sense RNA virus in the family Flaviviridae that is estimated to chronically infect up to 170 million people worldwide. The 9.6 kb genome encodes three structural and seven nonstructural proteins. One of these nonstructural proteins, NS5A, is an RNA-binding phosphoprotein essential for both viral replication and viral particle assembly [1]. It is composed of a N-terminal amphipathic helix that mediates membrane association [2]–[4] followed by three domains separated by two low-complexity sequences [5]. Domain I is responsible for NS5A dimerization [6] and has been proposed to contribute to RNA binding [7], [8]. A role for this domain in HCV RNA replication has been supported by the finding that many adaptive mutations that enhance HCV replication in cell culture map to Domain I [9], [10], In contrast, the majority of Domain II and the entirely of Domain III are dispensable for RNA replication, while deletion of Domain III virtually abolishes viral particle assembly [1].

In this study, a strategy combining affinity purification with mass spectrometry to identify novel host factors that associate with NS5A in cells infected with a cell-culture infectious HCV strain revealed that Rab18, a lipid droplet-specific Rab GTPase, interacts with NS5A. A similar strategy using Strep-tag affinity purification of NS5A has been previously used to identify oxysterol-binding protein (OSBP) as an NS5A-interacting protein [44]. That study used a subgenomic replicon, as opposed to our use of a fully infectious HCV construct, which may have enhanced our ability to detect host proteins involved in virion production. In addition, we used SILAC to reduce the identification of false-positive protein interactions.




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