Identification and Characterization of a Highly Efficient Anti-HIV Pol Hammerhead Ribozyme

Abstract

The aim of this study was to identify and to characterize a highly active anti-HIV ribozyme. Therefore, the genome of HIV-1 IIIb was screened for not yet addressed GUC triplets within highly conserved sequences. Here we report the in vitro characteristics and the antiviral activity of the fittest identified anti-HIV hammerhead ribozyme, targeting the 13th GUC triplet within the HIV-1 pol gene (HHPol13). Multiple turnover kinetics were determined in vitro and revealed very promising kinetic data: V_max = 39 nM/minute, K_m = 576 nM, k_cat = 3.9/minute, and K_cat/K_m = 6.8/minute/μM. To analyze its antiviral activity the hammerhead ribozyme was expressed retrovirally in Hut78 cells followed by HIV-1 infection. The newly identified ribozyme conferred a long-term inhibition of HIV-1 replication until the end of the observation period at day 56. We were able to demonstrate that the antiviral activity was mainly due to a ribozyme effect combined with a limited antisense activity. Additionally, the effect of the identified ribozyme was compared with a retrovirally expressed siRNA directed against the same target in the HIV-1 pol gene. This siRNA (siPol13) showed no inhibition of HIV replication. In summary, the hammerhead ribozyme HHPol13 was demonstrated to confer superior cleavage and antiviral activity against HIV-1. These results suggest that even in the RNAi era ribozymes still have the potential as highly active antiviral agents.