Abstract
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.
Highlights
The hepatitis C virus (HCV)1 is a flavivirus with a singlestranded RNA genome almost 9600 bases in length
In this paper we have demonstrated that there are placed recognition elements for the human pre-tRNA processing enzyme, RNase P, in several viral internal ribosome entry site (IRES) domains including HCV, classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV), cricket paralysis virus (CrPV), and EMCV
As first pointed out by Nadal et al [15] concerning human RNase P cleavage of the HCV IRES, it is likely that these events reflect neither the replication cycle of these viruses nor the canonical action of RNase P
Summary
Preparation of RNA Transcripts—HCV IRES RNAs were transcribed from Bluescript plasmid pN(1– 4728), which contains nucleotides 1– 4728 of hepatitis C virus under the T7 promoter (a gift from Dr Stanley Lemon, University of Texas, Galveston). This DNA template was cleaved by the SacII restriction enzyme. This plasmid, when linearized with the NcoI restriction enzyme, serves as template for a 32P-labeled RNA containing bases 1– 450 of the CSFV genome. This plasmid, when linearized with the NcoI restriction enzyme, encodes an RNA containing bases 1–220 of the CrPV IGR IRES. Digestion by calf alkaline phosphatase (Promega) was used to identify oligonucleotides containing 5Ј terminal phosphate residues produced by RNase P cleavage
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