1. Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase
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Miguel Angel Martinez, Luis Menéndez-Arias, Mar Álvarez, Samara Martín-Alonso, Maria Nevot, Ministerio de Ciencia, Innovación y Universidades (España), Instituto de Salud Carlos III, and Fundación Ramón Areces more...
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0301 basic medicine ,DNA polymerase ,030106 microbiology ,Human immunodeficiency virus (HIV) ,medicine.disease_cause ,behavioral disciplines and activities ,strand displacement ,DNA polymerases ,03 medical and health sciences ,Strand displacement ,reverse transcriptase ,parasitic diseases ,Reverse transcriptase ,medicine ,Displacement (orthopedic surgery) ,antiretroviral drugs ,biology ,DNA synthesis ,Chemistry ,musculoskeletal, neural, and ocular physiology ,Thymidine analogue ,HIV ,DNA ,Molecular biology ,HIV Reverse Transcriptase ,030104 developmental biology ,Infectious Diseases ,Antiretroviral drugs ,Mutation ,HIV-1 ,biology.protein ,psychological phenomena and processes ,Thymidine - Abstract
Retroviral reverse transcriptases (RTs) have the ability to carry out strand displacement DNA synthesis in the absence of accessory proteins. Although studies with RTs and other DNA polymerases suggest that fingers subdomain residues participate in strand displacement, molecular determinants of this activity are still unknown. A mutant human immunodeficiency virus type 2 (HIV-2) RT (M41L/D67N/K70R/S215Y) with low strand displacement activity was identified after screening a panel of purified enzymes, including several antiretroviral drug-resistant HIV-1 and HIV-2 RTs. In HIV-1, resistance to zidovudine and other thymidine analogues is conferred by different combinations of M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q (designated as thymidine analogue resistance-associated mutations (TAMs)). However, those changes are rarely selected in HIV-2. We show that the strand displacement activity of HIV-2ROD mutants M41L/S215Y and D67N/K70R was only slightly reduced compared to the wild-type RT. In contrast, mutants D67N/K70R/S215Y and M41L/D67N/K70R/S215Y were the most defective RTs in reactions carried out with nicked and gapped substrates. Moreover, these enzymes showed the lowest nucleotide incorporation rates in assays carried out with strand displacement substrates. Unlike in HIV-2, substitutions M41L/T215Y and D67N/K70R/T215Y/K219Q had no effect on the strand displacement activity of HIV-1BH10 RT. The strand displacement efficiencies of HIV-2ROD RTs were consistent with the lower replication capacity of HIV-2 strains bearing the four major TAMs in their RT. Our results highlight the role of the fingers subdomain in strand displacement. These findings might be important for the development of strand-displacement defective RTs., Ministry of Science, Innovation and Universities of Spain through grants BIO2016-76716-R (AEI/FEDER, UE) to L.M.-A. and SAF2016-75277-R to M.A.M.; S.M.-A. is a predoctoral fellow of the Ministry of Science, Innovation and Universities of Spain (BES-2017-079836). M.N. was supported by the Instituto de Salud Carlos III through the Spanish AIDS network (RD16-0025/0041). An institutional grant of the Fundación Ramón Areces to the CBMSO is also acknowledged more...
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- 2020
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