Hepatitis C virus induced changes to the host cell phosphoproteome

Hepatitis C virus (HCV) is a leading global pathogen affecting over 3% of the world's population and at least 250 000 individuals in Canada alone. Infection with the (+) polarity RNA virus is often characterized by a chronic disease state that can ultimately culminate in liver cirrhosis, steatosis and hepatocellular carcinoma. HCV is the leading indicator for liver transplants in North America. In addition to the high incidence and persistent nature of HCV, current treatment regimens have limited efficacy and there is no vaccine available for prophylactic measures.;Viruses are obligate intracellular pathogens, ergo, they have to rely heavily on host cells in order to propagate. HCV is known to remodulate the cellular environment to create an intracellular milieu conducive to virus replication and propagation. Of the many cellular control mechanisms that viruses can take advantage of, protein phosphorylation is a key factor. Phosphorylation plays a key role in numerous cellular processes including antiviral signalling, protein expression, cell proliferation and cytoskeleton remodelling. Therefore, in an effort to better understand the pathogenic mechanisms of HCV infections; alterations to the host cell phosphoproteome were analyzed in Huh7.5 human hepatocytes containing a subgenomic replicon or in Huh7.5 cells infected with high titre HCVcc (cell cultured virus). Phosphoproteins were enriched from liver cell lines using phospho-serine, threonine and tyrosine antibody immunoaffinity. Phosphoprotein enriched fractions were analysed by high resolution, quantitative, 2D differential-in-gel electrophoresis (2D-DIGE) and phosphoproteins that exhibited statistically significant alterations between control and experimental cell lysates were deemed of interest and identified by mass spectrometry.;Subtle changes to the phosphoproteome were observed in subgenomic replicon Huh7.5 cell lines and included elevations in Hsp90 and amphiphysin II and decreases in tropomyosin 4 and calponin. More dramatic changes to the phosphoproteome were observed in Huh7.5 cells infected with live HCV. Increases in the poly-rC binding protein I and multiple tubulin and tropomyosin isoforms were observed. Very few changes were decreased in relation to mock infected cells but included two isoforms of calponin.;Despite the genotypic and genome coding differences between the subgenomic replicon and infectious HCV, similar protein families were altered when compared to control cell lines. These included cellular proteins associated with; cytoskeletal rearrangement, endoplasmic reticulum stress and protein translation. A number of proteins found to be altered using 2D-DIGE methodologies were confirmed by 2D immunoblotting. Of critical importance, the findings observed by 2D-DIGE were specific to both the phosphorylation status of the proteins as well as the specific isoform of each protein. In certain cases, total proteomic analysis by standard one dimensional separation methods would not have identified those changes discerned here.