Proteome Analysis of Liver Cells Expressing a Full-Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-Infected Patients

Abstract

The development of a reproducible model system for the study of hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large-scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full-length HCV replicon. We detected >4,200 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry. Consistent with the literature, a comparison of HCV repliconpositive and -negative Huh-7.5 cells identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where a total of >1,500 proteins were detected from only 2 g of liver biopsy protein digest using the Huh-7.5 protein database and the accurate mass and time tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting in the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.Hepatitis C virus (HCV) is the most common blood-borne infection in the United States, infecting approximately 2% of the population. Approximately 85% of cases progress to chronic infection, which often results in liver disease, including variable degrees of hepatic inflammation and fibrosis, cirrhosis, and hepatocellular carcinoma. Since the discovery and sequencing of the virus genome in 1989 (7), studies have been performed to elucidate the host/virus interactions pertinent to persistent infection of the liver. To date, global characterization of the host cellular response to infection has centered on the use of expression microarray profiling to identify potential gene markers of HCV-associated liver disease (11,44,45,48). In contrast, only limited studies describing the proteomic analysis of human liver proteins have been reported (21,55,56). Proteomic studies of HCV infection have been particularly limited for several reasons, including the lack of a good cell culture model and the need for large amounts of protein for conventional proteomic analysis.Recent advances in both proteomic methodologies (43,44,49,50) and cell culture models of HCV infection (5, 26) now make it possible to perform global characterization of the host cell protein response within the context of the complete set of HCV genes in vitro. Refined multidimensional liquid chromatographic (LC) separations coupled with mass spectrometry (MS) for proteome analysis has allowed global experiments to be p...

HepatoProteomics: Applying proteomic technologies to the study of liver function and disease

“…A consistently emerging theme from the analysis of tumor tissue from patients with a variety of different disease etiologies including HCV infection, is the apparent decline in abundance of fatty acid oxidation enzymes. [33][34][35][36]41 Similar findings have been observed in two independent proteomic studies of HCV replication in vitro 38,42 and more recently in liver biopsy specimens obtained from HCVinfected individuals who have progressed to various stages of fibrosis (Diamond et al, manuscript in preparation). While the wide-spread nature of fatty acid oxidation down-regulation suggests it may not be beneficial for etiological diagnosis, it remains to be determined whether the molecular basis underlying these perturbations are conserved among the various causative agents leading to chronic liver damage and hepatocellular carcinoma.…”

“…Applying the AMT tag approach, over 1,500 proteins have been identified from only 2 g of a protein digest obtained from a liver biopsy sample. 38 This represents a significant advancement in clinical proteomics now making it possible to track physiologically relevant protein abundance changes in vivo using small patient samples.…”

Genome-Wide Analysis of Host mRNA Translation during Hepatitis C Virus Infection

“…A breakthrough in HCV research was the discovery of an HCV strain, JFH1, capable of replicating efficiently and of producing viral particles in cultured cells (4)(5)(6). In this in vitro model of HCV infection, transcriptomic analyses revealed regulation of the expression of genes involved in apoptosis and cell cycle arrest (7)(8)(9)(10), while proteomic studies identified numerous perturbations in the metabolism of phospholipids and sphingomyelins that are predicted to play important roles in viral replication, assembly, and secretion (11). This suggests that important differences exist between the HCV-regulated transcriptome and proteome that may rely on the differential stability of proteins and/or on gene expression regulation at the mRNA translation level, the process by which proteins are synthesized from existing mRNAs.…”

Quantitative Analysis of Human Immunodeficiency Virus Type 1-Infected CD4+Cell Proteome: Dysregulated Cell Cycle Progression and Nuclear Transport Coincide with Robust Virus Production

“…Given the effects of viral infections on host protein synthesis and processing mechanisms over the course of viral infections, it is not surprising that protein abundance changes have been reported for viral infections. For instance, in hepatitis C virus infection models, abundance changes of proteins, particularly in relation to lipid metabolism, have been reported through the use of mass spectrometry (MS) (45) and in conjunction with stable isotope labeling (54). As described below, we have adopted both approaches for protein expression profiling in our HIV infection system to maximize the number of proteins surveyed.…”