To identify cellular factors that interact with hepatitis C virus RNA, cellular extracts were subjected to UV cross-linking to radiolabeled RNAs corresponding to the hepatitis C virus 5' and 3' untranslated regions of positive and negative polarities. Our results demonstrate that the U-rich region of the hepatitis C virus 3' untranslated region of the positive RNA strand is a hot spot for cellular RNA binding proteins. Two of these proteins were identified as the ELAV-like HuR protein and hnRNP C. Interestingly, HuR and hnRNP C also interacted with the 3' end of the RNA representing the negative strand of the HCV genome. The binding of HuR and hnRNP C to the 3' ends of the HCV RNAs of both negative and positive polarities suggests that HuR and hnRNP C may be involved in the transcription of the HCV RNA genome. Alternatively, they act by protecting the HCV RNAs from premature degradation by binding to their 3' ends. However, we were unable to demonstrate an effect on HCV RNA stability by the HuR protein. These interactions may be necessary for the establishment of chronic active infections that may develop into cirrhosis or hepatocellular carcinoma.

We have analysed hepatitis C virus (HCV) RNAs in an in vitro RNA degradation assay. We found that the 3h end of positive polarity HCV RNA is sensitive to cytosolic RNases whereas the 3h end of negative polarity HCV RNA is relatively stable. Interaction of the HCV 3h untranslated region with the cellular La protein prevented premature degradation of the HCV RNA. One may speculate that HCV RNAs interact with La protein in infected cells to prevent premature degradation of the viral RNAs.

We have investigated whether poly(C)-binding protein (PCBP)-1 and PCBP-2 interact with the hepatitis C virus (HCV) 5h untranslated region. Our results demonstrate that glutathione Stransferase (GST)-PCBP-1 and GST-PCBP-2 fusion proteins bind specifically to the HCV 5h untranslated region. An antiserum raised against PCBP-2 induced a supershift after incubation with RNA-protein complexes formed between proteins in a HeLa cell cytoplasmic extract and the HCV 5h untranslated region, indicating that this interaction occurs intracellularly. The complete internal ribosome entry site was necessary for efficient binding, suggesting that maintenance of the secondary structure was necessary for recognition of the binding site by the PCBPs.

Vaccines based on recombinant viruses represent a promising strategy for the development of a prophylactic vaccine against HIV-1. However, despite a proven capacity to stimulate potent HIV-1-specific immune responses, viral systems have limited utility in homologous prime-boost regimens due to the generation of anti-vector immune responses. It is therefore important to develop a diverse set of vaccine candidates that can be combined in different heterologous prime-boost regimens and/or to identify a vaccine candidate that is less sensitive to anti-vector mediated immunity. In this report, we describe the design and pre-clinical immunogenicity of a Semliki Forest virus-based vaccine, VREP-C, encoding Indian origin HIV-1 clade C antigens. We show that a single immunization with VREP-C stimulates HIV-1-specific IFNgamma ELISPOT responses, which were efficiently boosted by a second and a third homologous VREP-C immunization resulting in highly potent cytotoxic T cell responses. These results suggest that VREP-C may be a valuable component of a future prophylactic vaccine against HIV-1.