Key result
Expression of the active RNase P ribozyme in cultured cells strongly inhibited foot-and-mouth disease virus IRES-dependent translation by inducing specific RNA cleavage.
The RNase P ribozyme can specifically cleave the FMDV IRES in cultured cells, inhibiting viral translation and providing a potential antiviral strategy against picornaviruses.
May inform ribozyme-based antivirals for picornaviruses; leaves open in vivo efficacy and clinical translation.
Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. We have previously shown that a structural motif within the foot-and-mouth-disease virus IRES is recognized in vitro as substrate for the Synechocystis sp. RNase P ribozyme. Here we show that this structure-dependent endonuclease recognizes the IRES element in cultured cells, leading to inhibition of translation. Inhibition of IRES activity was dependent on the expression of the active ribozyme RNA subunit. Moreover, expression of the antisense sequence of the ribozyme did not inhibit IRES activity, demonstrating that stable RNA structures located upstream of the IRES element do not interfere with internal initiation. RNAs carrying defective IRES mutants that were substrates of the ribozyme in vivo revealed an increased translation of the reporter in response to the expression of the active ribozyme. In support of RNA cleavage, subsequent analysis of the translation initiation manner indicated a switch from IRES-dependent to 5'-end-dependent translation of RNase P target RNAs. We conclude that the IRES element is inactivated by expression in cis of RNase P in the cytoplasm of cultured cells, providing a promising antiviral tool to combat picornavirus infections. Furthermore, our results reinforce the essential role of the structural motif that serves as RNase P recognition motif for IRES activity.
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Fernández et al. (2010) studied Picornavirus infection (in vitro model). RNase P ribozyme (Synechocystis sp. rnpB) vs. Inactive ribozyme mutant (RzP4) or antisense sequence was evaluated on IRES-dependent translation efficiency (luciferase activity). Expression of the active RNase P ribozyme in cultured cells strongly inhibited foot-and-mouth disease virus IRES-dependent translation by inducing specific RNA cleavage.
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