Modulation of hepatitis C virus internal ribosome entry site-mediated translation by RNA sequence encoding the viral core protein

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) directs the translation initiation of the viral polyprotein in a cap-independent manner. Located within the 5′ nontranslated region of the genome, the IRES is comprised of highly complex RNA secondary and tertiary structures that fold into four defined domains and are crucial for its function. The internal initiation of translation on the viral RNA involves a direct interaction between the 40S ribosome subunit and the HCV RNA at the site of initiator AUG codon, which is located in domain IV. The stability of domain IV stem-loop influences the efficiency of IRES-directed translation, such that mutations which stabilize the structure adversely affect IRES activity. It has been proposed that a viral translation product may interact with this part of the HCV IRES, serving as a feedback mechanism to down-regulate translation and thus promoting viral persistence. Despite a number of earlier reports that suggest that the core protein modulates IRES-mediated translation, it is not clear whether this protein plays any role in translation of HCV. In this study, I examined the influence of the HCV core protein on the translation initiated by the IRES. The expression of core protein from dicistronic reporter plasmids, pRC22F and pRC173F, containing the HCV IRES and RNA encoding 22 as or 173 as of the core protein respectively, did not alter IRES efficiency in comparison to related frameshift products of the core protein sequence. The expression of the core protein provided in trans by recombinant baculovirus did not alter IRES efficiency in vivo, nor did the addition of purified recombinant core protein in an in vitro, cell-free translation system with IRES-containing reporter transcripts. However, at the RNA level, the additional core-coding sequence in pRC173F transcripts is capable of suppressing IRES activity in vitro and in vivo. Using deletion analysis, we found that the suppression of translation was alleviated when RNA sequence encoding the core protein, from nt 386 to 510, which possess two putative stem-loop structures, V and VI, was deleted from the transcript of pRC173F, indicating that this segment has a suppressive effect on IRES function. Further deletion study mapped the IRES suppression to the 46-nt RNA segment spanning from nt 425 to 471, which comprises the 5′ stem sequence of stem-loop VI. Results from KCl titration experiments suggest that the suppression of IRES activity is RNA structure-dependent.