Proteomic analysis of HuH-7 cells harboring in vitro-transcribed full-length hepatitis C virus 1b RNA
Abstract
Aim: The present study examined the differential expression of proteins in HuH-7 cells and HuH-7 cells harboring in vitro-transcribed full-length hepatitis C virus 1b RNA (HuH-7-HCV), and elucidated the cellular responses to HCV replication.
Methods: The protein profiles of matched pairs of HuH-7-HCV cells and HuH-7 mock cells were analyzed by 2-D electrophoresis (2DE). Solubilized proteins were separated in the first dimension by isoelectric focusing, and by 12.5% SDS-PAGE in the second dimension. The differential protein expression was analyzed by use of image analysis software to identify candidates for HCV infection-associated proteins.
Results: In total, 29 protein spots showed increases and 25 protein spots showed decreases in signal in HuH-7-HCV cell 2DE profiles as compared with HuH-7 mock cells. In the next step, the 10 spots showing the greatest increase and the 10 spots showing the greatest decrease were excised from gels and the proteins present were identified by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer (MALDI-TOF MS) or MALDI-TOF/TOF MS. In total, 13 proteins were identified successfully. The potential significance of the differential expression due to HCV replication was discussed.
Conclusion: Our study identifies changes in the proteome of HuH-7 cells in the presence of HCV replication and yields information of the mechanism of HCV pathogenesis. These results will be useful for the identification of HCV infection-associated proteins that could be molecular targets for treatment.
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Methods: The protein profiles of matched pairs of HuH-7-HCV cells and HuH-7 mock cells were analyzed by 2-D electrophoresis (2DE). Solubilized proteins were separated in the first dimension by isoelectric focusing, and by 12.5% SDS-PAGE in the second dimension. The differential protein expression was analyzed by use of image analysis software to identify candidates for HCV infection-associated proteins.
Results: In total, 29 protein spots showed increases and 25 protein spots showed decreases in signal in HuH-7-HCV cell 2DE profiles as compared with HuH-7 mock cells. In the next step, the 10 spots showing the greatest increase and the 10 spots showing the greatest decrease were excised from gels and the proteins present were identified by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer (MALDI-TOF MS) or MALDI-TOF/TOF MS. In total, 13 proteins were identified successfully. The potential significance of the differential expression due to HCV replication was discussed.
Conclusion: Our study identifies changes in the proteome of HuH-7 cells in the presence of HCV replication and yields information of the mechanism of HCV pathogenesis. These results will be useful for the identification of HCV infection-associated proteins that could be molecular targets for treatment.