Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein translation by hammerhead ribozymes.

Abstract

To determine the effects of hammerhead ribozymes against hepatitis C virus (HCV) RNA on viral protein translation, a luciferase reporter gene vector, pCMV/T7-NCRC ⌬ -luc, was constructed containing the 5 Ј -noncoding region (5 Ј -NCR) and part of the core region of HCV. Four ribozymes, Rz1-Rz4, were designed to cleave at nucleotide positions 136-160, 313-337, 496-520, and 373-388, respectively. Each ribozyme cleaved the target RNA at expected positions under cell-free conditions. Rz2 and Rz4 significantly suppressed translation of NCRC ⌬ -luc RNA by 71 and 49%, respectively. Translation of control luciferase mRNA lacking viral elements was not affected by the ribozymes. Furthermore, when NCRC ⌬ -luc RNA and ribozymes were cotransfected into cells, Rz2 and Rz4 significantly suppressed expression by 73 and 56%, respectively. In contrast, cleavage-deficient ribozymes with a point mutation in the hammerhead domain had no significant effect. To determine the effects of endogenously produced ribozymes, eukaryotic expression vectors for Rz2 and Rz4 were constructed. Cotransfection of the vectors with CMV/T7-NCRC ⌬ -luc showed suppression of luciferase activities to 50 and 61%, respectively. Moreover, transfection of pCMV/T7-NCRC ⌬ -luc into stable Rz2 and Rz4 producer cells also showed substantial inhibition of luciferase activity. Ribozymes directed against the HCV genome can substantially and specifically inhibit viral gene expression under intracellular conditions. ( J. Clin. Invest. 1996. 98:2720-2728.)

Identification of eIF2Bγ and eIF2γ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

“…Hairpin ribozymes (10) as well as hammerhead ribozymes (15)(16)(17) targeting highly conserved regions of the 5Ј-end of the 9.5-kb HCV RNA molecule have been shown to significantly inhibit HCV core protein expression. In this study, we used a reverse functional genomics approach to discover potent ribozymes that specifically interfere with essential factors controlling viral replication (specifically IRES cofactors).…”

Small Interfering RNA Targeted to Hepatitis C Virus 5′ NontranslatedRegion Exerts Potent Antiviral Effect

“…Therefore, we plan to identify the sequences that are common among different HCV genotypes. The 5Ј NTR is one of the most conservative regions in the HCV genome and among the genotypes that differ from each other by 31% to 33% (43,47,56,60). The core is the most conservative sequence among the HCV proteins.…”

Translational Control of Viral Gene Expression in Eukaryotes

“…Ribozymes have been shown to be effective in blocking translation directed from the HCV IRES, although this probably occurs indirectly by RNA cleavage. Ribozymes constructed to target conserved sites within the viral 5Ј UTR blocked the translation of a luciferase reporter protein under control of the HCV IRES and 5Ј UTR in a tissue culture system, with little or no apparent toxicity to the host cell (402). Ribozymes have the added advantage of being efficiently packaged and expressed by various viral vectors, including vaccinia virus, adeno-associated virus, and various retroviruses.…”