Role of alternate reading frame potein (ARFP) in the pathogens of chronic Hepatits C viral infection
Date of Award
Doctor of Philosophy in Health Science (PhD)
Biological and Biomedical Sciences
Chronic infection by hepatitis C virus (HCV) eventually leads to liver failure and is responsible for reducing the quality of life for over 170 million people worldwide. HCV encodes ten classic proteins as well as a newly discovered alternative reading frame protein (ARFP) whose synthesis originates from the core region by a +1 frame shift. ARFP is encoded by all HCV genotypes, but its function remains unknown. Although, immunogenicity of genotype 1 and 2 derived ARFP in infected hosts has been reported, limited information is available for genotype 3 ARFP. In view of this, the aim of this study was to understand the basic molecular biology of genotype 3 encoded ARFP as well as to investigate its role in HCV associated disease progression. HCV genotype 3 core/ARFP region was PCR amplified, cloned, and sequenced which revealed that HCV genotype 3 strain (PKI-ICV3) encoded a 160 as ARFP, harboring a C-terminal extension of 36 aa. Recombinant ARFP and peptides were employed in ELISAs with serum samples from chronically infected individuals. It was observed that sera from 84% patients contained ARFP reactive antibodies. Peptide ELISAs showed that all regions of rARFP were immunogenic, with peptide F7 (DSLSPRRAGAKAGPGLSPGT) being the most immunodominant. Core peptides used as controls revealed peptide C3 (NVKFPGGGQIVGGVYVLPRR) to be relatively more immunogenic. When F7 and C3 peptides were incubated with peripheral blood mononucleocytes (PBMCs) from HCV infected individuals, F7 stimulated production of more TNFa and IL-10 cytokines as determined by FACS array. Although anti-ARFP antibodies are found in sera of both acute and chronically infected individuals demonstrating that the protein is expressed in infected individuals, ARFP has never been directly detected in vivo. On the basis of immunochemical studies, we report that genotype-3 encoded ARFP is indeed detectable in paraffin embedded liver biopsy tissues from HCV infected patients and exhibits a nuclear expression which increases with disease severity. ARFP was also found in nuclei of transfected HEK293 cells. Additionally, by employing a set of truncated ARFPs we have identified a putative NLS in ARFP that appears to be bipartite. Since ARFP is a nuclear protein, we hypothesized that it could modulate genomic DNA methylation patterns in cells expressing the protein. By performing methylation specific PCRs with DNA from HCV+ HCC samples, it was found that HCV infection promotes hypermethylation of Rassfl A, a tumor suppressor gene, suggesting that silencing of Rassfl A may be the key event that promotes hepatocellular carcinoma. Moreover, to determine impact of ARFP expression on whole-genome DNA methylation patterns, DNA methylation arrays were employed and used to interrogate the methylome of ARFP expressing HEK293 and llepG2 cells. This exercise revealed a large set of gene promoters whose methylation status is impacted by ARFP expression. Further studies are required to determine the precise biochemical function of ARFP as well as its role in disease progression, especially as it transitions from chronic stage to hepatocellular carcinoma, and to explore if ARFP is a reliable biomarker for monitoring different stages of infection and/or in assessing therapeutic response.
Qureshi, H. H. (2011). Role of alternate reading frame potein (ARFP) in the pathogens of chronic Hepatits C viral infection (Unpublished doctoral thesis). Aga Khan University, Karachi, Pakistan.