Identification of the 6 differential proteins in Huh7.5.1 wt and HepG2 cells detected by analysis of label-free, iTRAQ, qRT-PCR and western blot assays (Relative differential proteins: HepG2/Huh7.5.1 wt).

<p>Identification of the 6 differential proteins in Huh7.5.1 wt and HepG2 cells detected by analysis of label-free, iTRAQ, qRT-PCR and western blot assays (Relative differential proteins: HepG2/Huh7.5.1 wt).</p

Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines

<div><p>Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.</p></div

The overall workflow.

<p>Huh7.5.1 wt and HepG2 whole-cell proteins were harvested and analyzed using the Label-free and iTRAQ proteomic approaches, and the differential proteins were verified by qRT-PCR and western blot. The functions of selected differential proteins in HCV replication were analyzed further.</p

Quantitative analysis of proteins abundance changes detected using the label-free and iTRAQ approaches.

<p>(A) The volcano plot shows t-test p-values plotted against log2 values of protein fold changes. Data points in the lower center area of the plot have a log2 value close to 0 and a <i>p</i> value approaching 1 and indicate no significant change, whereas points in the upper left and upper right quadrants indicate significant negative and positive changes in protein abundance, respectively. (B) The iTRAQ ratio was transformed by log2 analysis. The proteins with abundance ratio within the range from -1 to +1 indicate no significant changes, and values larger than +1 or lower than -1 indicate significant positive and negative changes in protein abundance, respectively.</p

The effects of six key differential proteins on HCV RNA levels.

<p>(A) qRT-PCR analysis of HCV RNA levels by transfection of siRNA or over-expression of plasmids. (B) Luciferase assay of Jc1-Luc HCVcc in Huh7.5.1 cells transfected with siRNA or over-expression plasmids. (C) QRT-PCR analysis of HCV RNA levels in knockdown of CTSB or over-expression of vimentin in HepG2 cells after transfection with HCV replicon RNA. Western blot results showed the protein level of knockdown and over-expression differential proteins, and β-actin as protein loading control. (D) Huh7.5.1 cells were transfected with siRNA or over-expression plasmids of CTSB, then 24 h later, cells were infected with HCVcc (multiplicity of infection 0.1). At 48 h post infection, immunoblotting was performed with an anti-HCV core antibody. Each bar represents the average of triplicate data points with the standard deviation represented as the error bar. *P<0.05, ** P<0.01 and ***P<0.001 versus negative control</p

The qualitative and quantitative results from the label-free and 4-plex iTRAQ methods.

<p>The qualitative and quantitative results from the label-free and 4-plex iTRAQ methods.</p

The key differentially expressed proteins in IPA networks analysis were validated by qRT-PCR and western blot.

<p>(A) The mRNA levels of six differential proteins in hepatic cell lines. (B) The protein levels of six key differential proteins in hepatic cell lines. NS3 was used as control for HCV-replicon expression for all the analyses, while GAPDH as control. And β-actin was used for protein loading control. *P<0.05, ** P<0.01 and ***P<0.001 versus negative control</p

The interaction networks of differential proteins in Huh7.5.1 wt and HepG2 cell lines by IPA analysis.

<p>The networks of differentially expressed proteins related to Cancer, Gastrointestinal and Hepatic System Disease, Cell Death and Survival, Cellular Development, Growth and Proliferation. Proteins high in Huh7.5.1 wt cells and HepG2 cells are denoted in red and green, respectively. Any known direct connections between these proteins are indicated by solid lines, indirect interactions are shown with dashed lines.</p