A phylogenetically conserved stem-loop structure at the 5 ' border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation

Hepatitis C virus (HCV) initiates translation of its polyprotein under the control of an internal ribosome entry site (IRES) that comprises most of th e 341-nucleotide (nt) 5' nontranslated RNA (5'NTR). A comparative analysis of related flaviviral sequences suggested that an RNA segment for which sec ondary structure was previously ill defined (domain II, nt 44 to 118) forms a conserved stem-loop that is located at the 5' border of the HCV IRES and thus may function in viral translation. This prediction was tested by a mu tational analysis of putative helical structures that examined the impact o f both covariant and noncovariant nucleotide substitutions on IRES activity in vivo and in vitro. Results of these experiments provide support for pre dicted base pair interactions between nt 44 to 52 and 111 to 118 and betwee n nt 65 to 70 and 97 to 102 of the HCV 5'NTR. Substitutions at either nt 45 and 46 or nt 116 and 117 resulted in reciprocal changes in V1 nuclease cle avage patterns within the opposing strand of the putative helix, consistent with the predicted base pair interactions. IRES activity was highly depend ent on maintenance of the stem-loop II structure but relatively tolerant of covariant nucleotide substitutions within predicted helical segments. Sequ ence alignments suggested that the deduced domain II structure is conserved within the IRESs of pestiviruses as well as the novel flavivirus GB virus B. Despite marked differences in primary nucleotide sequence within conserv ed helical segments, the sequences of the intervening single-stranded loop segments are highly conserved in these different viruses. This suggests tha t these segments of the viral RNA may interact with elements of the host tr anslational machinery that are broadly conserved among different mammalian species.