Abstract
Hepatitis C virus (HCV) replication complex resides in detergent-insoluble subcellular domains or lipid rafts. We used two proteomics approaches to characterize the protein content of lipid rafts isolated from Huh7 cells and its modification upon HCV replication. Using two-dimensional electrophoresis and mass spectrometry, we identified approximately 100 protein spots in the isolated lipid rafts; among them, 39 were reproducibly modified in HCV replicon cell lines as compared with control cell lines. We also used stable isotope labeling by amino acids in cell culture (SILAC) combined with one-dimensional electrophoresis separation and mass spectrometry. Using this approach, we identified 1036 individual proteins based on peptides selected with at least 95% confidence; among them, 413 proteins were identified with at least two peptides. Quantification analysis identified 150 proteins modified by at least 2.5-fold (110 up-regulated and 40 down-regulated) in HCV-replicating cells compared with controls. Protein identifications and quantifications obtained by both proteomics approaches were largely concordant. Modulated proteins included a majority of proteins involved in vesicular and protein trafficking and in cell signaling. Remarkably for a large number of proteins, their up-regulation in lipid rafts of HCV replicon cells was due to their relocalization. By using small interfering RNAs directed to the modulated small GTPases Cdc42 and RhoA, we observed an increase in HCV replication, whereas reduction of syntaxin 7 expression resulted in decreased replication of HCV. Our findings indicate that protein subcellular relocalization occurs in HCV-containing cells that can directly affect HCV replication.
Highlights
Hepatitis C virus (HCV) replication complex resides in detergent-insoluble subcellular domains or lipid rafts
We recently reported the upregulation of N-Ras in detergent-insoluble membrane domains and subsequent activation of the phosphatidylinositol 3-kinase-mTOR pathway in Huh7 cell lines expressing a fulllength HCV replicon, leading to protection against apoptosis and reduced HCV replication [10]
To further identify protein modifications in lipid rafts resulting from HCV replication, we utilized the standard proteomics approach of two-dimensional (2-D) PAGE followed by mass spectrometry analysis of individual protein spots
Summary
Cell Culture and Isolation of Detergent-insoluble Fractions—Human hepatoma Huh cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Cellgro/ Mediatech) and 1% penicillin/streptomycin (Invitrogen). Stable Huh clones SFL-2 and SFL-3 expressing the selectable full-length HCV-1b genome and T1 clone expressing the neomycin resistance gene were described previously [10]. These cells were grown in complete DMEM with 400 g/ml G418 (Invitrogen). Detergent-insoluble fractions were isolated as described previously [10].
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
