title: Physiological Mg2+Conditions Significantly Alter the Inhibition of HIV-1 and HIV-2 Reverse Transcriptases by Nucleoside and Non-Nucleoside Inhibitors in Vitro.
abstract: Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg2+, whereas the free Mg2+concentration in cells is much lower. Artificially high Mg2+concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg2+. At low concentrations of Mg2+, NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg2+, whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg2+concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg2+concentrations resulted from a 3-9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on kcat/Km) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg2+concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC50values) more effective at low than at high Mg2+concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg2+-dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg2+conditions yielded results dramatically different from those from measurements using commonly employed high-Mg2+in vitro conditions. These results also emphasize differences in Mg2+sensitivity between the translocation inhibitor EFdATP and other NRTIs.
authors.affiliation: Cell Biology and Molecular Genetics, University of Maryland , College Park, Maryland 20742, United States.|Maryland Pathogen Research Institute , College Park, Maryland 20742, United States.
authors.forename: Vasudevan
authors.initials: V
authors.lastname: Achuthan
authors.affiliation: Christopher S. Bond Life Sciences Center, University of Missouri , Columbia, Missouri 65211, United States.|Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine , Columbia, Missouri 65211, United States.
authors.forename: Kamlendra
authors.initials: K
authors.lastname: Singh
authors.affiliation: Cell Biology and Molecular Genetics, University of Maryland , College Park, Maryland 20742, United States.|Maryland Pathogen Research Institute , College Park, Maryland 20742, United States.
authors.forename: Jeffrey J
authors.identifier: http://orcid.org/0000-0002-8710-7622
authors.initials: JJ
authors.lastname: DeStefano
mesh_terms: D003845:Deoxycytosine Nucleotides / Q000494:pharmacology; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D054306:Dideoxynucleotides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009154:Mutation; D009705:Nucleosides / Q000494:pharmacology*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013942:Thymine Nucleotides / Q000494:pharmacology; D016047:Zalcitabine / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; D009705:Nucleosides; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D015215:Zidovudine; C037406:2',3'-dideoxycytidine 5'-triphosphate; C031709:2',3'-dideoxyguanosine 5'-triphosphate; D016047:Zalcitabine; C052873:zidovudine triphosphate; C514824:reverse transcriptase, Human immunodeficiency virus 1; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D008274:Magnesium