[40] Zidovudine resistance related connection mutations in HIV-1 reverse transcriptase cause selective dissociation from RNase H competent complexes

17th International HIV Drug Resistance Workshop

10-14 June 2008, Sitges, Spain

ZIDOVUDINE RESISTANCE RELATED CONNECTION MUTATIONS IN HIV-1 REVERSE TRANSCRIPTASE CAUSE SELECTIVE DISSOCIATION FROM RNASE H COMPETENT COMPLEXES

Antivir Ther. 2008; 13(Suppl. 3):A45 (abstract no. 40)

GL Beilhartz¹, M Ehteshami¹, B Scarth¹, E Tchesnokov¹, B Wynhoven², R Harrigan² and M Götte¹
¹McGill University, Montreal, Quebec, Canada; ²BC Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada

BACKGROUND: Thymidine analogue mutations (TAMs) in HIV-1 reverse transcriptase (RT) cause resistance to zidovudine (AZT) and other nucleoside reverse transcriptase inhibitors by increasing the rate of excision. Recent findings have identified mutations in the connection domain that augment AZT resistance, usually in a background of TAMs. It is widely accepted that these mutations may compromise RNase H cleavage, providing more time for AZT excision to occur. However, the underlying mechanism remains to be elucidated.

METHODS: In this study, we used connection mutations A360V and N348I as a model to address this problem with a combination of complementary biochemical tools. Previous studies revealed that TAMs in combination with A360V and N348I are associated with marked decreases in phenotypic susceptibility to AZT. Here, we employed enzyme kinetics and binding studies to elucidate the molecular mechanism.

RESULTS: Initial experiments confirm that N348I and A360V, in combination with TAMs, decrease the efficiency of RNase H cleavage and increase the amount of rescued DNA product following ATP-dependent excision. The A360T polymorphism is less likely to be associated with treatment and this mutation does not increase rates of excision. Band-shift assays show that the TAM/N348I/A360V mutant accumulates stable, transiently formed 10- to 12mer hybrids that can rebind to RT. These short hybrids dissociate from the RNase H competent complex and rebind to RT in the polymerase-competent mode that allows excision to occur. The connection mutations selectively reduce substrate binding in the RNase H competent mode. Furthermore, binding in the polymerase-competent mode appears to be facilitated, which is reflected in increases in processive DNA synthesis. As a consequence, mutant enzymes containing N348I, which show the strongest effects in this regard, enhance excision and rescue of DNA synthesis in the presence of pyrophosphate (PPi) or ATP.

CONCLUSION: Selective dissociation of transiently formed hybrids from RNase H competent complexes provides a mechanism for the increase in AZT excision associated with connection mutations N348I and A360V. The combination of diminished RNase H cleavage and increased processivity renders the use of both PPi and ATP advantageous, whereas classic TAMs solely enhance the ATP-dependent reaction.

2008-06-10
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