Key result
The A368-G mutation in HCV RNA allowed a second RNase P cleavage at least 10 times more efficient than the original, forming a second tRNA-like structure without impairing IRES activity.
The conservation of the tRNA-like conformation and IRES activity in HCV RNA variants suggests an essential role for this motif in the viral life cycle.
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May support HCV RNA motif as antiviral target; leaves open clinical translation from animal models.
Hepatitis C virus (HCV) RNA is recognized and cleaved in vitro by RNase P enzyme near the AUG start codon. Because RNase P identifies transfer RNA (tRNA) precursors, it has been proposed that HCV RNA adopts structural similarities to tRNA. Here, we present experimental evidence of RNase P sensitivity conservation in natural RNA variant sequences, including a mutant sequence (A368-G) selected in vitro because it presented changes in the RNA structure of the relevant motif. The variation did not abrogate the original RNase P cleavage, but instead, it allowed a second cleavage at least 10 times more efficient, 4 nt downstream from the original one. The minimal RNA fragment that confers sensitivity to human RNase P enzyme was located between positions 299 and 408 (110 nt). Therefore, most of the tRNA-like domain resides within the viral internal ribosome entry site (IRES) element. In the variant, in which the mutation stabilizes a 4 nt stem-loop, the second cleavage required a shorter (60 nt) substrate, internal to the minimal fragment substrate, conforming a second tRNA-like structure with similarities to a 'Russian-doll' toy. This new structure did not impair IRES activity, albeit slightly reduced the efficiency of translation both in vitro and in transfected cells. Conservation of the original tRNA-like conformation together with preservation of IRES activity points to an essential role for this motif. This conservation is compatible with the presence of RNA structures with different complexity around the AUG start codon within a single viral population (quasispecies).
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María Pirón (2005) studied Hepatitis C virus (HCV). A368-G mutant sequence vs. Original HCV RNA sequence was evaluated on RNase P cleavage efficiency and IRES activity. The A368-G mutation in HCV RNA allowed a second RNase P cleavage at least 10 times more efficient than the original, forming a second tRNA-like structure without impairing IRES activity.
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