title: Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis.
abstract: HIV-1 reverse transcriptase (RT) connection subdomain mutations at positions 348, 369 and 376 have been associated with resistance to non-nucleoside RT inhibitors (NNRTIs). N348I may interfere with the initiation of (+)-strand DNA synthesis by reducing polypurine tract (PPT) removal in the presence of nevirapine. The effect of NNRTIs on the RNase H-mediated cleavage of PPT-containing template-primers has been studied with wild-type HIV-1 RT and mutants N348I, T369I, T369V, T376S and N348I/T369I. In the presence of NNRTIs, all RTs were able to stimulate PPT cleavage after primer elongation. The enhancing effects of nevirapine and efavirenz were reduced in RTs carrying mutation N348I, and specially N348I/T369I. However, those mutations had no effect on rilpivirine-mediated cleavage. Prior to elongation, the PPT remains resilient to cleavage, although efavirenz and rilpivirine facilitate RNase H-mediated trimming of its 3'-end. The integrity of the 3'-end is essential for the initiation of (+)-strand DNA synthesis. In the presence of dNTPs, rilpivirine was the most effective inhibitor of (+)-strand DNA synthesis blocking nucleotide incorporation and preventing usage of available PPT primers. The N348I/T369I RT showed reduced ability to generate short RNA products revealing a cleavage window defect. Its lower RNase H activity could be attributed to enhanced rigidity compared to the wild-type enzyme.
authors: Centro de Biología Molecular 'Severo Ochoa' (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain.
authors: Gilberto
authors: G
authors: Betancor
authors: Centro de Biología Molecular 'Severo Ochoa' (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain.
authors: Mar
authors: M
authors: Álvarez
authors: Centro de Biología Molecular 'Severo Ochoa' (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain.
authors: Barbara
authors: B
authors: Marcelli
authors: Centro de Biología Molecular 'Severo Ochoa' (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain Laboratori de Retrovirologia, Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Badalona, 08916 Barcelona, Spain.
authors: Cristina
authors: C
authors: Andrés
authors: Laboratori de Retrovirologia, Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Badalona, 08916 Barcelona, Spain.
authors: Miguel A
authors: MA
authors: Martínez
authors: Centro de Biología Molecular 'Severo Ochoa' (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain lmenendez@cbm.csic.es.
authors: Luis
authors: L
authors: Menéndez-Arias
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis*; D024882:Drug Resistance, Viral / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009570:Nitriles / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011687:Purines / Q000378:metabolism; D011724:Pyridazines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012313:RNA; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D016914:Ribonuclease H / Q000378:metabolism; D000068696:Rilpivirine; D013698:Templates, Genetic
chemical_list: D019380:Anti-HIV Agents; D009570:Nitriles; D011687:Purines; D011724:Pyridazines; D011743:Pyrimidines; C034477:RNA primers; D018894:Reverse Transcriptase Inhibitors; C451734:etravirine; D012313:RNA; D004247:DNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D000068696:Rilpivirine