Molecular studies of the hepatitis C virus : the role of IRES activity for therapy response, and the impact of the non-structural protein

Sammanfattning: Chronic hepatitis C can lead to life threatening conditions like cirrhosis, liver failure and hepatocellular carcinoma. There are an estimated 170 million chronic carriers of the hepatitis C virus (HCV) world wide, making it a serious global health problem. 70-90 % of the newly infected individuals will fail to clear the virus and develop chronic hepatitis. There is no vaccine, and the only treatment available is a long-term combination therapy of pegylated interferon-á (peg-IFN-á) and Ribavirine (RBV). Unfortunately, this regiment cannot be given to all patients, and of the treated individuals only 50-80 % will succeed in clearing. the virus. Therefore, more efficient treatment approaches are needed. To achieve them, a deeper understanding of the virus and its interaction with the host is necessary. This thesis will focus on two aspects of the HCV life cycle; the translation and replication of the virus genome. In the first section, viral translation mediated by the internal ribosome entry site (IRES) of the HCV genotype 3 and its relation to treatment response was studied. A Pakistani cohort of approximately 144 chronic HCV patients was treated with IFN-á and RBV. Follow up one year post treatment showed 70% sustained responders (SR) and 30% nonresponders (NR) (depicting if the virus was cleared or not). Substantial differences in the IRES nucleic acid sequence between the two groups were observed. This prompted analyses of translation efficiencies of IRES regions derived from 15 SR and 7 NR randomly selected patients. The SR derived IRES displayed significantly lower translation efficiencies than those of the NR group (29.7 ± 13 vs. 69.4 ± 22; P < 0.01). This difference may be a consequence of more disrupted IRES tertiary structure in the SR group compared to the NR group. The results indicate that a correlation between the IRES sequence and the therapeutic outcome may serve as a prediction model of treatment outcome for HCV genotype 3. However, further studies are needed to establish such a model, and to understand if the correlation applies to other genotypes. In the second section, studies were undertaken to elucidate the role of the non-structural protein 4B (NS4B) in the replication of HCV. NS4B is an integral membrane protein of little known function except that it has membrane rearranging properties. Analyses of the NS4B topology when processed from the polyprotein inside cells showed that the NS4B N-terminus translocated into the ER-lumen, extending previous observations from in vitro experiments. The N-terminus translocation was exhibited by all the major six genotypes, manifesting a conserved function important in the viral life cycle. By immunoprecipitation and immunofluorescence experiments using a collection of NS4B mutants, we showed that NS4B homo-oligomerized and that the N-terminal translocation into the lumen was crucial for induction of the membrane associate foci by NS4B. This provided an important insight into the events that create the very environment in which the viral RNA replication takes place. Finally, 14 point mutations located in different regions of NS4B in a HCV subgenomic replicon all affected the viral replication. Even mutations of non-conserved amino acid residues and substitutions on the lumenal side of the ER membrane displayed considerable negative effects; only one mutant exhibited increased replication. These results implied that NS4B may have a direct involvement in viral RNA replication beyond MAF induction. Together, the results emphasize a critical role for NS4B in the viral life cycle. Accordingly, elucidating interacting partners of the NS4B and intrinsic functions of the protein may offer new important insights, and possibly novel treatment strategies.