Hypersusceptibility mechanism of Tenofovir-resistant HIV to EFdA
1 Christopher Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65211, USA
2 Clinical Research Center, Department of Infectious Diseases and Immunology, National Hospital Organization Nagoya Medical Center, Nagoya 4600001, Japan
3 Division of Emerging Infectious Diseases, Tohoku University, Sendai 980-8575, Japan
4 Department of Internal Medicine, Kumamoto University, Kumamoto 860-8556, Japan
5 Experimental Retrovirology Section, HIV/AIDS Malignancy Branch, NIH, Bethesda, MD 20892, USA
6 Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA 15219, USA
7 Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA
Retrovirology 2013, 10:65 doi:10.1186/1742-4690-10-65Published: 24 June 2013
The K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF). 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials.
Here we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3’ primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent.
We have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.