Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis

Nucleic Acids Research, Feb 2015

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.

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Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis

Nucleic Acids Research Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis Gilberto Betancor 1 2 Mar Alvarez 1 2 Barbara Marcelli 1 2 Cristina Andr es 0 1 2 Miguel A. Martnez 0 1 Luis Men endez-Arias 1 2 0 Laboratori de Retrovirologia, Fundaci o irsiCaixa, Hospital Universitari Germans Trias i Pujol , Badalona, 08916 Barcelona , Spain 1 Present addresses: Gilberto Betancor, Department of Infectious Diseases, King's College London, Guy's Hospital , London SE1 9RT , UK. Barbara Marcelli, Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , 9747 AG Groningen , The Netherlands. Cristina Andre s, Laboratorio de Retrovirolog a e Inmunopatogenia Viral, Institut d'Investigacions Biome`diques August Pi i Sunyer , 08036 Barcelona , Spain 2 Centro de Biolog a Molecular 'Severo Ochoa' (Consejo Superior de Investigaciones Cient ficas and Universidad Aut o noma de Madrid), c/Nicol a s Cabrera , 1, Campus de Cantoblanco, 28049 Madrid , Spain 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 templateprimers 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. - The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a major target of antiretroviral therapy (13). The HIV-1 RT is a DNA polymerase that can use either DNA or RNA strands as templates for DNA synthesis. The RT also possesses an RNase H activity responsible for the degradation of RNA in RNA/DNA complexes. RT DNA polymerase and RNase H activities are needed to convert the viral genomic RNA into double-stranded DNA that integrates into the genome of the host cell. During reverse transcription, the first DNA strand (i.e. ()-strand DNA) is synthesized by extending the 3 -end of a specific tRNA using the viral RNA as template. The RT RNase H activity degrades the RNA template, with the exception of two short polypurine tracts (PPTs), located at the center and at the 3 end of the viral RNA genome. These PPTs are used as major initiation sites for (+)-strand DNA synthesis. The tRNA and PPT primers are later removed by the RNase H activity of the RT (for reviews see (4,5)). Inhibitors of the DNA polymerization activity constitute the backbone of current therapies against HIV infection and AIDS. These drugs can be classified into nucleoside/nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). There are five NNRTIs approved for clinical use: nevirapine, delavirdine, efavirenz, etravirine and rilpivirine, although delavirdine is rarely used due to its inferior antiviral efficacy and inconvenient dosing schedule (3,6). The HIV-1 RT is a heterodimer composed of subunits of 66 and 51 kDa (p66 and p51, respectively). The NNRTI binding site is a hydrophobic pocket in the palm subdomain of p66, 10 A away from the polymerase active site. Leu100, Lys101, Lys103, Val106, Thr107, Val108, Val179, Tyr181, Tyr188, Val189, Gly190, Phe227, Trp229, Leu234, Pro236 and Tyr318 in p66 and Glu138 in p51 delineate the NNRTI binding pocket ((7,8); reviewed in (9)). NNRTIs have a low genetic barrier and single amino acid substitutions affecting residues at their binding pocket confer high-level resistance to nevirapine, delavirdine and efavirenz (e.g. K103N or Y181C) (reviewed in (3)). Kinetic studies have shown that NNRTIs slow the rate of the chemical reaction of nucleotide incorporation (10,11). Based on structural studies, it has been proposed that NNRTIs block reverse transcription by (i) changing the mobility of the RT thumb subdomain, (ii) distorting the catalytic triad formed by the side chains of Asp110, Asp185 (...truncated)


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Gilberto Betancor, Mar Álvarez, Barbara Marcelli, Cristina Andrés, Miguel A. Martínez, Luis Menéndez-Arias. Effects of HIV-1 reverse transcriptase connection subdomain mutations on polypurine tract removal and initiation of (+)-strand DNA synthesis, Nucleic Acids Research, 2015, pp. 2259-2270, 43/4, DOI: 10.1093/nar/gkv077