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Published on: October 1, 2015
Ultra-structural localisation of hepatocellular PKR protein using immuno-gold labelling in chronic hepatitis C virus
Gerry C MacQuillan1, Paul Caterina, Bastiaan de Boer
1Department of Gastroenterology and Hepatology, Sir Charles Gairdner Hospital, Nedlands, WA, Australia. Gerry.MacQuillan@health.wa.gov.au
Abstract:
The greater resistance of HCV genotype 1 infection to IFN therapy has been partially attributed to functional inhibition of the type 1 interferon induced anti-viral protein PKR in vitro. Whether PKR has antiviral activity against HCV in vivo is unknown. Whilst the ultra-structural localisation of PKR is known in vitro, it is not defined in chronic hepatitis C disease. Using a novel immuno-gold technique we characterised the expression of intrahepatic PKR protein at the ultra-structural level in four patients with chronic HCV disease compared to normal human PBMCs, HepG2 cells and a normal human liver biopsy. All four HCV patients labelled for PKR protein, localising to the nucleus, nucleolus and cytoplasm. Nuclear labelling was confined mainly to the nucleolus and euchromatin. Cytoplasmic labelling was evident within smooth vesicles. Strong immunogold labelling was also evident within the cisternae of the rough endoplasmic reticulum. A similar pattern of ultra-structural nuclear and cytoplasmic PKR protein labelling was seen in PBMCs from healthy donors, HepG2 cells and a normal liver biopsy. The mean nuclear and cytoplasmic count for PKR protein in the HCV group was 21 +/- 4 and 18 +/- 3 gold particles/microm(2), respectively. This represented an increase, though not statistically significant, in nuclear and cytoplasmic labelling for PKR protein in HCV biopsies relative to normal liver tissue.
Insights
Hepatitis C virus (HCV) infection resistance to interferon therapy may involve PKR protein. This study investigated PKR
Area of Science:
- Virology
- Immunology
- Hepatology
Background:
- Hepatitis C virus (HCV) genotype 1 exhibits resistance to interferon (IFN) therapy.
- This resistance is partly linked to the inhibition of the type 1 IFN-induced antiviral protein, protein kinase R (PKR), in vitro.
- The in vivo antiviral activity of PKR against HCV and its ultrastructural localization in chronic hepatitis C remain undefined.
Purpose of the Study:
- To investigate the in vivo antiviral activity of PKR against HCV.
- To define the ultrastructural localization of intrahepatic PKR protein in chronic hepatitis C disease.
- To compare PKR expression in HCV patients with controls.
Main Methods:
- Utilized a novel immuno-gold technique for ultrastructural analysis.
- Characterized intrahepatic PKR protein expression in four chronic HCV patients.
- Compared PKR localization and expression with normal human peripheral blood mononuclear cells (PBMCs), HepG2 cells, and a normal liver biopsy.
Main Results:
- PKR protein was detected in the nucleus (nucleolus and euchromatin), cytoplasm (smooth vesicles), and rough endoplasmic reticulum cisternae in all four HCV patients.
- A similar ultrastructural localization pattern was observed in healthy donor PBMCs, HepG2 cells, and normal liver tissue.
- HCV biopsies showed a non-statistically significant increase in nuclear and cytoplasmic PKR protein labeling compared to normal liver tissue.
Conclusions:
- PKR protein is expressed in the nucleus and cytoplasm of hepatocytes in chronic hepatitis C.
- The ultrastructural localization of PKR in HCV disease mirrors that in normal tissues.
- While not statistically significant, increased PKR labeling in HCV biopsies warrants further investigation into its role in HCV pathogenesis and therapy resistance.
