CORE SPECIFIC ANTISENSE PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDES AS POTENT AND SPECIFIC INHIBITORS OF HEPATITIS-C VIRAL TRANSLATION

Antisense phosphorothioate oligodeoxynucleotides (S-ODN) complementary to sequence stretches in the 5' non coding region (NCR) of the hepati tis C virus (HCV) have recently been shown to effectively inhibit vira l gene expression. In order to further delineate the optimum target re gion in the highly conserved 5' end of the viral RNA, S-ODN complement ary to HCV core coding sequences were analysed in the present study. I n a rabbit reticulocyte lysate (RRL) in vitro translation assay S-ODN 5, complementary to the HCV-RNA nucleotides 340-353, and S-ODN-6, comp lementary to nucleotides 348-365, resulted in an inhibition of viral t ranslation of 90.4 +/- 1.3% and 93.7 +/- 5.1%, respectively at a conce ntration of 4.14 mu M. S-ODN 7, complementary to nucleotides 371-388, was relatively inefficient and showed a maximal inhibition of 42.4 +/- 12.2%. It has been suggested that in living cells an inhibition by S- ODN is mainly mediated by the action of RNAse H. Tn RRL the RNAseH con tent is very low; therefore, to simulate the situation in living cells inhibition experiments in RRL enriched with RNAse H were performed. U nder these conditions S-ODN 5, 6 and 7 inhibited viral translation by 45.6 +/- 6.3%, 80.3 +/- 2.8% and 70.9 +/- 5.7% at concentrations as lo w as 0.2 mu M. At this concentration no inhibition was observed in the standard RRL assay. In cell culture S-ODN 7 was by far the most effic ient inhibitor of viral translation, resulting in a specific inhibitio n of 89.4 +/- 3.6% at a concentration of 0.3 mu M. Taken together with the results of our previous study, nucleotides 326-348 comprising the 3' end of the NCR and nucleotides 371-388, located entirely in the co re coding region of the HCV RNA, are effective targets for S-ODN media ted inhibition of viral translation.