Detection of tRNA-like structure through RNase P cleavage of viral internal ribosome entry site RNAs near the AUG start triplet.

The 9600-base RNA genome of hepatitis C virus (HCV) has an internal ribosome entry site (IRES) in its first 370 bases, including the AUG start triplet at bases 342-344. Structural elements of this and other IRES domains substitute for a 5' terminal cap structure in protein synthesis. Recent work (Nadal, A., Martell, M., Lytle, J. R., Lyons, A. J., Robertson, H. D., Cabot, B., Esteban, J. I., Esteban, R., Guardia, J., and Gomez, J. (2002) J. Biol. Chem. 277, 30606-30613) has demonstrated that the host pre-tRNA processing enzyme, RNase P, can cleave the HCV RNA genome at a site in the IRES near the AUG initiator triplet. Although this step is unlikely to be part of the HCV life cycle, such a reaction could indicate the presence of a tRNA-like structure in this IRES. Because susceptibility to cleavage by mammalian RNase P is a strong indicator of tRNA-like structure, we have conducted the studies reported here to test whether such tRNA mimicry is unique to HCV or is a general property of IRES structure. We have assayed IRES domains of several viral RNA genomes: two pestiviruses related to HCV, classical swine fever virus and bovine viral diarrhea virus; and two unrelated viruses, encephalomyocarditis virus and cricket paralysis virus. We have found similarly placed RNase P cleavage sites in these IRESs. Thus a tRNA-like domain could be a general structural feature of IRESs, the first IRES structure to be identified with a functional correlate. Such tRNA-like features could be recognized by pre-existing ribosomal tRNA-binding sites as part of the IRES initiation cycle.

Recurring features of local tertiary structural elements in RNA molecules exemplified by hepatitis D virus RNA.

Elements of local tertiary structure in RNA molecules are important in understanding structure-function relationships. The loop E motif, first identified in several eukaryotic RNAs at functional sites which share an exceptional propensity for UV crosslinking between specific bases, was subsequently shown to have a characteristic tertiary structure. Common sequences and secondary structures have allowed other examples of the E-loop motif to be recognized in a number of RNAs at sites of protein binding or other biological function. We would like to know if more elements of local tertiary structure, in addition to the E-loop, can be identified by such common features. The highly structured circular RNA genome of the hepatitis D virus (HDV) provides an ideal test molecule because it has extensive internal structure, a UV-crosslinkable tertiary element, and specific sites for functional interactions with proteins including host PKR. We have now found a UV-crosslinkable element of local tertiary structure in antigenomic HDV RNA which, although differing from the E-loop, has a very similar pattern of sequence and secondary structure to the UV-crosslinkable element found in the genomic strand. Despite the fact that the two structures map close to one another, the sequences comprising them are not the templates for each other. Instead, the template regions for each element are additional sites for potential higher order structure on their respective complementary strands. This wealth of recurring sequences interspersed with base-paired stems provides a context to examine other RNA species for such features and their correlations with biological function.

Specific cleavage of hepatitis C virus RNA genome by human RNase P.

We have found that RNase P from HeLa cells specifically and efficiently cleaves hepatitis C virus (HCV) transcripts in vitro. The evidence includes identification of the 5'-phosphate polarity of the newly generated termini at position A(2860) as well as immunological and biochemical assays. Active cleavage has been shown in five dominant sequences of HCV "quasispecies" differing at or near the position of cleavage, demonstrating that this is a general property of HCV RNA. During the analysis, a second cleavage event was found in the 3' domain of the internal ribosome entry site. We have found that HCV RNA competitively inhibits pre-tRNA cleavage by RNase P, suggesting that HCV RNA has structural similarities to tRNA. This finding sets HCV apart from other pathogens causing serious human diseases and represents the first description of human RNase P-viral RNA cleavage. Here we discuss the possible meaning of these RNase P-accessible structures built into the viral genome and their possible role in vivo. Moreover, such structures within the viral genome might be vulnerable to attack by therapeutic strategies.