Proteomic Study Of HCV Gene Replication Complex Associated With Lipid Raft

Formation of a detergent resistant membrane-associated replication complex composed of viral proteins, replicating RNA and modified cellular membranes is the general strategy for most of the plus-strand RNA viruses. HCV gene replication is proposed to take similar strategy, residing in Golgi apparatus and endoplasmic reticulum membrane, and inducing a membranous web, where all HCV proteins were found to be associated with lipid raft, forming a membrane-associated multiprotein complex. This kind of membranous web functions as a place for synthesis of the viral RNA and protects viral RNA from accessing to nuclease and protease by possible cellular lipid membranes. Recently it is reported that HCV replication complexes assemble on lipid rafts to form gene replication complex through interactions between HCV viral nonstructural proteins and host protein hVAP33. However, the components of HCV gene replication complex are still not very clear.To identify potential cellular proteins related to HCV replication complexes (RC), we purified detergent resistant membranes from HCV subgenomic replicon cells by discontinuous sucrose gradient. HCV NS proteins and RNAs were mainly located in the detergent resistant membrane fractions and sensitive to detergent resistant membrane depletion reagent. The proteins of detergent resistant membrane fractionswere separated by two-dimensional gel electrophoresis (2DE) and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. All together, 325 proteins spots were identified which could be categorized as signaling proteins, intracellular transport proteins, translation machine components, mitochondria proteins, signaling molecules, intracellular transport actors, cytoskeleton and cytoskeleton associated proteins. Lipid raft-derived cellular proteins, such as actin, eIF3-subunit2, eIF3-P44, UNR protein, HnRNP K, HnRNP G, HnRNP L, and UNR-interacting proteins were reported to interact with HCV 5' IRES by proteomics approach. Cellular proteins reported to interact with HCV proteins, such as Histone, 14-3-3 protein, FYN and Apo E, cyclophilin B were also found.To identify the proteins associated with HCV RC, bioinformatics analysis were first undertaken. Protein-protein interaction network analysis among the identified proteins revealed that the cellular proteins associated with HCV gene and viral proteins are mainly cytoskeleton proteins, signaling molecules and HnRNPs. Secondly, comparing proteome strategy was used to address the altered detergent resistant membrane proteins in replicon cells compared with its parental Huh7 cells, sixty proteins were up regulated which included proteins connected with HCV in the protein interaction networks, such as HSC71, T-complex protein and eIF3. Transient expression and confocal microscope analysis showed that cellular proteins, such as ubiquitin fusion degradation protein 1 homolog (UFD1L), SNAP-γ and sytaxin17, PA28β Ras-GTPase-activating protein binding protein 1 (G3BP1) and nucleophosmin (NPM) were colocalized with HCV NS5A. Further functional analysis showed that, while blockage of SNARE-mediated membrane traffic impaired HCV replication complex and inhibited HCV replication in replicon cells, knockdown of G3BP1 by siRNA in replicon cells also significantly reduced HCV replication, which implicate the importance of G3BP1 and SNARE family proteins in HCV replication.The above results suggest that detergent resistant membrane derived cellularproteins, including G3BP1, may be the components of HCV RC, which may help to understand the mechanism of HCV replication and develop novel antiviral against HCV infection.