Modification of host lipid raft proteome upon hepatitis C virus replication.
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.
Mannová
Petra
P
Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Fang
Ruihua
R
Wang
Hong
H
Deng
Bin
B
McIntosh
Martin W
MW
Hanash
Samir M
SM
Beretta
Laura
L
D002457:Cell Extracts / Q000737:chemistry; D002458:Cell Fractionation / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D016174:Hepacivirus / Q000502:physiology*; D006790:Host-Parasite Interactions; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism / Q000821:virology; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D002457:Cell Extracts; D020543:Proteome