Cellular Response to Hepatitis C Virus Infection: Global Quantitative Proteome AMT Tag Measurements of Cellular Protein Expression
EMSL Project ID
4393
Abstract
Approximately 170 million individuals worldwide are infected with HCV and in the United States, there are approximately 4 million infected individuals and over 10,000 HCV associated deaths annually. The most remarkable aspect of HCV is its striking ability to persist in the infected host, which often leads to progressive liver disease including fibrosis, cirrhosis, and increased risk of hepatocellular carcinoma. Unfortunately, HCV research has been hampered by the lack of an adequate tissue culture system, or an effective animal model for HCV infection. As a result, the mechanisms of HCV replication and pathogenesis remain poorly understood, and little is known about the underlying mechanisms leading to the liver injury that often results from HCV infection.To gain a better understanding of HCV replication and pathogenesis, we propose to use the AMT tag approach to study changes in the liver proteome during HCV infection and HCV-associated liver disease using in vitro and in vivo systems. Although not without its limitations, the HCV replicon system has become the “gold standard” for in vitro analysis of HCV replication. In this system, full-length HCV genome RNAs in the human hepatoma cell line Huh-7 allows for an abundant source of material for total proteome analysis and the establishment of a hepatocyte- specific AMT tag database. Tryptic digests of Huh-7 subcellular fractions would be provided for subsequent high resolution LC tryptic peptide separations and highly sensitive FTICR MS analysis in order to generate highly confident identifications across the total proteome. Our long-term goal is to characterize the changes in cellular protein expression that occur in multiple points along the continuum from infection to liver disease, including cirrhosis and hepatocellular carcinoma. In this regard, the established AMT database could be used to canvass changes in protein expression using tryptic digests from prospective serial liver biopsies. Biopsy material from liver transplant recipients with recurrent HCV after liver transplantation offers the major advantage that one can prospectively examine early, critical intracellular events that may determine the progression to severe liver disease in patients with chronic HCV. At present, this model offers the only opportunity to study this aspect of chronic HCV since there are no animal models of HCV in which progressive liver injury occurs. The limiting amount of protein obtained from liver biopsy samples necessitates the use of proteomic methods that provide increased sensitivity and proteome coverage, an issue that would be addressed by the use of AMT tags.
The overall goals of this EMSL User proposal are three-fold: (1) to initiate the proteomic characterization of the Huh-7 cell line and the creation of an AMT tag database, (2) to obtain proteomic coverage of the HCV replicon expressed in Huh-7 cells, and (3) to utilize these preliminary results for the preparation of an R21 proposal.
The final results of these studies should increase our understanding of the mechanisms of HCV replication and pathogenesis and may provide critical markers for both diagnostic and prognostic applications as well as suggest novel targets for therapeutic and prophylactic intervention.
Project Details
Project type
Exploratory Research
Start Date
2003-10-13
End Date
2005-07-11
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Baas T, CR Baskin, DL Diamond, A Garcia-Sastre, H Bielefeldt-Ohmann, TM Tumpey, MJ Thomas, VS Carter, TH Teal, N Van Hoven, S Proll, JM Jacobs, Z Caldwell, MA Gritsenko, R Hukkanen, DG Camp, RD Smith, and MG Katze. 2006. "Integrated Molecular Signature of Disease: Analysis of Influenza Virus-Infected Macaques through Functional Genomics and Proteomics." Journal of Virology 80(21):10813 (16 pages).
Diamond DL, S Proll, JM Jacobs, EY Chan, DG Camp, RD Smith, and MG Katze. 2006. "HepatoProteomics: Applying Proteomic Technologies to the Study of Liver Function and Disease." Hepatology 44(2):299-308.
Proteome analysis of liver cells expressing a full-length hepatitis C virus (HCV) replicon and biopsy specimens of posttransplantation liver from HCV-infected patients.
Jacobs JM, Diamond DL, Chan EY, Gritsenko MA, Qian W, Stastna M, Baas T, Camp DG 2nd, Carithers RL Jr, Smith RD, Katze MG.
J Virol. 2005 Jun;79(12):7558-69.