Cellular proteins binding to the HCV 5' and 3' UTRs

Abstract: Hepatitis C virus (HCV) is a worldwide health problem with more than 170 million people infected by the virus. About 80% become chronic carriers, which may result in hepatocellular carcinoma. The lack of an in vitro cell culture system that allows propagation of the virus has hampered the development of a vaccine or anti-viral drugs against HCV. It is not known why HCV grows poorly in vitro, but we speculate that liver-specific cellular factors that interact with the HCV untranslated regions and which are crucial for the replication and translation of HCV RNA may be absent under cell culture conditions or be present only at suboptimal levels. It is also possible that factors which actively inhibit replication and translation are present in cell culture. This thesis focuses on cellular factors that bind to the 5' and 3' untranslated regions (UTR) of HCV. Cell extracts from liver and HeLa cells were fractionated and RNA-binding proteins were identified by UV cross-linking and RNA gel shifts. We found that poly(rC) binding proteins (PCBP)-1 and PCBP-2 bind to the 5' UTR and that HuR, lupus antigen (La) and heterogenous nuclear ribonuclear protein C (hnRNP C) bind to the 3' UTR. Interestingly, both HuR and hnRNP C also interact with the 3' end of HCV RNA of negative polarity. We further established an in vitro stability assay and found that La-binding to HCV 3' UTR RNA protects the RNA from degradation.In order to study translation of HCV mRNA in human cells, we generated miniviruses consisting of the 5' UTR, capsid-CAT fusion protein and the 3' UTR. mRNAs were synthesised in vitro and electroporated into HeLa cells. We show that the HCV RNA is inefficiently translated compared to capped and polyadenylated mRNAs. The cap and poly(A) structures synergistically load ribosomes onto the HCV internal ribosomal entry site and stimulate translation.