Identification and characterization of a highly efficient anti-HIV pol hammerhead ribozyme.
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/microM. 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.
Müller-Kuller
Thea
T
Medizinische Klinik II, der Johann Wolfgang Goethe-Universität, Frankfurt, Germany.
Capalbo
Gianni
G
Klebba
Christian
C
Engels
Joachim W
JW
Klein
Stefan A
SA
D019380:Anti-HIV Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006801:Humans; D016337:RNA, Catalytic / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000378:metabolism*; D014161:Transduction, Genetic; D014779:Virus Replication / Q000187:drug effects; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors* / Q000235:genetics
D019380:Anti-HIV Agents; D016337:RNA, Catalytic; D034741:RNA, Small Interfering; C427204:hammerhead ribozyme; D054302:pol Gene Products, Human Immunodeficiency Virus