title: Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors.
abstract: We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.
authors: Universität Bayreuth, Lehrstuhl Biopolymere und Forschungszentrum für Bio-Makromoleküle, Universitätsstrasse 30, 95447 Bayreuth, Germany.
authors: Anna
authors: A
authors: Schneider
authors: Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, SS 554, 09042, Monserrato, Cagliari, Italy.
authors: Angela
authors: A
authors: Corona
authors: Julius-Maximilians-Universität Würzburg, Institut für Virologie und Immunbiologie, Versbacher Strasse 7, 97078 Würzburg, Germany.
authors: Imke
authors: I
authors: Spöring
authors: Universität Bayreuth, Lehrstuhl Biopolymere und Forschungszentrum für Bio-Makromoleküle, Universitätsstrasse 30, 95447 Bayreuth, Germany.
authors: Mareike
authors: M
authors: Jordan
authors: Universität Heidelberg, Medizinische Fakultät Mannheim, Klinik für Kinder- und Jugendmedizin, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany.
authors: Bernd
authors: B
authors: Buchholz
authors: Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, SS 554, 09042, Monserrato, Cagliari, Italy.
authors: Elias
authors: E
authors: Maccioni
authors: Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti, "Sapienza" Università di Roma, Rome, I-00185, Italy.
authors: Roberto
authors: R
authors: Di Santo
authors: Julius-Maximilians-Universität Würzburg, Institut für Virologie und Immunbiologie, Versbacher Strasse 7, 97078 Würzburg, Germany.
authors: Jochen
authors: J
authors: Bodem
authors: Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, SS 554, 09042, Monserrato, Cagliari, Italy.
authors: Enzo
authors: E
authors: Tramontano
authors: Universität Bayreuth, Lehrstuhl Biopolymere und Forschungszentrum für Bio-Makromoleküle, Universitätsstrasse 30, 95447 Bayreuth, Germany birgitta.woehrl@uni-bayreuth.de.
authors: Birgitta M
authors: BM
authors: Wöhrl
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019380:Anti-HIV Agents / Q000737:chemistry / Q000494:pharmacology; D003001:Cloning, Molecular; D054306:Dideoxynucleotides / Q000737:chemistry / Q000494:pharmacology; D024921:Drug Resistance, Multiple, Viral / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D054309:Ribonuclease H, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013942:Thymine Nucleotides / Q000737:chemistry / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology
chemical_list: D019380:Anti-HIV Agents; D054306:Dideoxynucleotides; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; C052873:zidovudine triphosphate; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D054309:Ribonuclease H, Human Immunodeficiency Virus