18th International HIV Drug Resistance Workshop Basic Principles & Clinical Implications June 9–13 2009, Fort Myers, Florida, USA

A NOVEL MOLECULAR MECHANISM OF DUAL RESISTANCE TO NUCLEOSIDE AND NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS

Antivir Ther 2009; 14 Suppl 1:A33 (abstract no. 31)

GN Nikolenko, KA Delviks-Frankenberry and VK Pathak
Viral Mutation Section, HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD, USA

BACKGROUND: It was recently shown by us and others that specific mutations in the connection subdomain (CN) and RNase H domain of HIV type-1 (HIV-1) reverse transcriptase (RT) exhibit dual resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). In this study, we propose a mechanism by which mutations in the CN and RNase H domain of HIV-1 RT confer dual resistance to NRTIs and NNRTIs.

METHODS: The levels of resistance to the NNRTIs nevirapine (NVP), delavirdine (DLV), efavirenz (EFV) and etravirine (ETR) were determined for RT mutants using a luciferase reporter gene-based drug susceptibility assay. RT template switching frequency was determined using a GFP reporter gene-based direct repeat deletion assay. RNase H activity was analysed using in vitro RNase H cleavage assay with virion-derived RTs.

RESULTS: Similarly to our previously described mechanism of NRTI resistance, we hypothesized that during NNRTI exposure, reduced RNase H cleavage of template allows more time for the NNRTI-RT-template/primer complex (NNRTI-RT-T/P) to remain competent for the resumption of DNA synthesis after NNRTI dissociation, resulting in increased NNRTI resistance. D549N substitution in RNase H alone increased resistance to NVP and DLV, but not to EFV and ETR, consistent with their decrease in Kd: NVP>DLV>EFV. Combination of D549N with mutations in the NNRTI-binding pocket (NNRTI-BP) L100I, K103N, V106A, Y181C and V179F, which presumably decrease the affinity of RT to NNRTIs, increased resistance from 3-to 30-fold to all four NNRTIs, emphasizing the role of affinity of NNRTIs to RT/T-P in our NNRTI resistance model. The affect of NNRTI exposure on RT template switching frequency is dependent on the affinity of each NNRTI to RT-T/P and can be modulated by the NNRTI resistance mutations in NNRTI-BP, which reduce affinity to the drugs, further supporting the importance of the relationships between NNRTI binding affinity and RNase H cleavage.

CONCLUSIONS: These experiments demonstrate that specific mutations in the cn and RNase H domain exhibit dual resistance to NRTIs and NNRTIs, provide insights into NNRTI resistance and suggest a novel mechanism by which mutations from treatment-experienced patients’ RT CN and RNase H domain can exhibit dual NRTI and NNRTI resistance.

2009-06-09
31

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