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 replicon-positive 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 mug 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.

FIG. 1.

Schematic representation of sample preparation, separation and analysis of the Huh-7.5 replicon model system. Global lysates or subcellular fractions (cytosolic, microsomal, and nuclear) were prepared from Huh-7.5 cells (negative and positive for the HCV full-length replicon) and digested with trypsin. The resulting peptides were initially separated by SCX, followed by reversed-phase capillary-to-liquid chromatography (RP-LC) coupled with mass spectrometry. MS/MS spectra were collected and analyzed by SEQUEST with stringent filtering criteria to provide confident protein identifications.

FIG. 2.

Western blot analysis of Huh-7.5 cells negative and positive for the HCV replicon. Protein lysates from Huh-7.5 cells negative and positive for the HCV replicon were resolved by 14% SDS-PAGE gels. Subsequent Western blot analysis using antibodies to the following HCV proteins: NS5A (predicted molecular weight [MW], 56,000 to 58,000) (A), NS5B (predicted MW, 68,000) (B), NS5B followed by core protein (predicted MW, 21,000) (C) revealed the specific detection of these proteins only in cells containing the HCV replicon. We were not able to detect glycoprotein E1 using a commercially available antibody generated against a glutathione S-transferase fusion protein containing the first 77 amino acids of HCV-1b E1 (data not shown). This may reflect an inability of the antibody to recognize with appreciable affinity the native (full-length, glycosylated) untagged protein. Although we cannot rule out the possibility that E1 is not expressed, this seems unlikely given that both of its flanking partners in the polyprotein precursor (core and E2) are detected.

FIG. 3.

Graphic representation of the distribution of detected proteins from the global Huh-7.5 HCV replicon-positive sample by various cellular classifications. (A) Categorization was based upon GO identification numbers corresponding to cellular component, with approximately 56% of the proteins categorized. (B) Categorization was based upon GO identification numbers corresponding to cellular process, with approximately 55% of the proteins categorized. Values in parenthesis represent the percentage of all IPI Human Database GO identifications for comparison. Results were similar for the Huh-7.5 HCV replicon-negative cell samples.

FIG. 4.

Confirmation of protein abundance changes by western blot analysis. Protein lysates from Huh-7.5 cells negative and positive for the HCV replicon were resolved by 4 to 20% SDS-PAGE gel. Western blot analysis was then performed using antibodies to the following proteins: actin (predicted MW, 41,800) (A), HDGF (predicted MW, 26,800) or thioredoxin (predicted MW, 11,700) (B), and catalase (predicted MW, 59,600) (C). These data indicate that the patterns observed by Western blot analysis are consistent with the data obtained by the peptide hit approach. Both methods detected no change in the expression of actin while catalase was down-regulated and both HDGF and thioredoxin were up-regulated in the presence of the HCV replicon. It is noted that while the predicted MW of HDGF was 26,800, our findings were consistent with the results of previous Western analyses identifying an ∼40-kDa band, presumably associated with cotranslational/posttranslational modification (31).

FIG. 5.

Example of a liver biopsy peptide identified using the AMT tag approach. (A) Representative MS survey scan taken during reversed phase LC-MS/MS of Huh-7.5 samples previously fractionated by strong cation exchange. The peak shown by an asterisk was selected for further identification via collision-induced dissociation (MS/MS). (B) MS/MS scan identifying the peptide SVTELNGDIITNTMTLGDIVFKR with its accompanying theoretical mass and elution time information for placement into a mass tag database. This was one of seven peptides detected that identified protein P07148, fatty acid-binding protein in the Huh-7.5 sample. Shown are the total ion count (C) and specific LC-FTICR-MS scan (no. 1136) (D) of the liver biopsy sample detecting an accurate mass isotopic distribution and elution time (shown by the asterisk), which matches with the peptide mass tag found in the database, identifying this peptide in the biopsy sample. Such a technique removes the need for MS/MS analysis of every sample, increasing throughput and allowing protein identification of small samples.