The hepatitis C virus (HCV) is a positive sense single stranded RNA virus occupying its own genus within the family Flaviviridae, and is an important human pathogen infecting approximately 2% of the global population. More than 70% of infected individuals develop a persistent infection, which frequently leads to chronic liver disease, cirrhosis, and hepatocellular carcinoma. The virus can not be reliably cultured in cells, and there is no small animal model. The cause of persistent infection is unknown.
The research in our lab seeks to identify points of regulation within the HCV life cycle that may be exploited in HCV specific antiviral drug design. Because of the existing expertise in our lab, we have focussed primarily of RNA structure/function relationships.
The genome has a single open reading frame (ORF) that encodes a polyprotein of about 3000 amino acid residues, which is co and post-translationally processed into the mature viral proteins by both host and viral proteinases. The ORF is flanked by 5’ and 3’ untranslated regions (UTR’s) that have been implicated in the initiation of translation and replication, respectively. The 5’ UTR, 341 nucleotides, is the most conserved region of the HCV genome and constitutes >90% of the HCV internal ribosome entry site (IRES). The IRES is a complex RNA structure responsible for the initiation of viral translation and extends about 30 nucleotides into the 5’ region of the ORF. The current research in my laboratory explores the relationship between the RNA structure within the HCV IRES and the efficiency of cap-independent translation, ultimately relating these parameters to HCV replication, infection, and persistence. We have shown that cobalamins (i.e., vitamin B12 and B12 analogues) can down regulate HCV IRES dependent translation in vitro. B12 dependent inhibition is not observed for cap-dependent reporter genes or reporter constructs incorporating the IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV). We have shown that this inhibition arises from a direct interaction with the HCV IRES resulting in stabilization of structure flanking the start codon. This mechanism is thus far unique in eukaryotic translation and is similar to prokaryotic modes of regulation. We are using cobalamins as a probe to investigate the mechanisms of eukaryotic translation and translational control. We have shown in preliminary human trials that a single injection of vitamin B12 into chronic HCV carriers results in an increase in HCV serum viral load, suggesting that cobalamins may be natural regulators affecting the HCV replication cycle. These results have implications for the so-called positive-sense RNA virus dilemma. The current effort in my laboratory is to develop cell culture systems appropriate to this study and to conduct mutational analysis on the HCV IRES to better characterize this phenomenon. We have initiated experiments to modify the cobalamin structure to produce potential HCV-specific antiviral agents and utilize the natural cobalamin transport system for drug delivery.