FUNCTIONAL-ANALYSIS OF THE INTERACTION BETWEEN HCV 5'UTR AND PUTATIVESUBUNITS OF EUKARYOTIC TRANSLATION INITIATION-FACTOR ELF3

Translation initiation in Hepatitis C Virus is controlled by the prese nce of an internal ribosome entry site element (IRES) principally loca ted in its 5' untranslated region (UTR). Mutation/deletion analyses ha ve shown that the integrity of this structure is essential for initiat ion of cap-independent protein synthesis. We have developed a strategy to swap the position of the two-major domains (II and III) on the 5'U TR sequence. The aim was to further characterize this mechanism by pre serving domain-specific interactions but possibly losing contacts that require any interdomain geometry. The expression of dicistronic mRNAs containing these different UTRs showed that the positioning of the di fferent domains on the 5'UTR is essential for efficient IRES functioni ng. We then used these mutants to identify cellular factors implicated in IRES activity. Using UV crosslinking assays we found that domain I II makes direct contact with two proteins (p170/p120) which can be ass ociated with efficient IRES activity. In particular, we have mapped th e binding sites of these proteins and shown that p120 binds to the api cal loop segment of domain III, whilst p170 binds in the stem portion, independently of domain III position or context. Finally, we provide evidence showing that p170 and p120 represent two subunits of eukaryot ic initiation factor eIF3: p170 and p116/p110.