Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance

Xu Hong-Tao, Martinez-Cajas, Ntemgwa, Coutsinos Dimitrios, Frankel, Brenner, Wainberg

Description

We investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT). Methods Recombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from Escherichia coli . Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3 Lys as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments. Results The efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT. Conclusion The diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2009
• Nombre de lectures: 5
• Langue: English

Extrait

Retrovirology

BioMedCentral

Open Access Research Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance 1 11,2 HongTao Xu, Jorge L MartinezCajas, Michel L Ntemgwa, 1,2,3 1,21,2,3 Dimitrios Coutsinos, Fernando A Frankel, Bluma G Brennerand 1,2,3 Mark A Wainberg*

1 2 Address: McGillUniversity AIDS Centre, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec H3T1E2, Canada,Department of 3 Medicine, McGill University, Montreal, Quebec H3A 2T5, Canada andDepartment of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2T5, Canada

Email: HongTao Xu  [e-mail]; Jorge L MartinezCajas  [e-mail]; Michel L Ntemgwa  [e-mail]; Dimitrios Coutsinos  [e-mail]; Fernando A Frankel  [e-mail]; Bluma G Brenner  [e-mail]; Mark A Wainberg*  [e-mail] * Corresponding author

Published: 11 February 2009Received: 24 October 2008 Accepted: 11 February 2009 Retrovirology2009,6:14 doi:10.1186/1742-4690-6-14 This article is available from: http://www.retrovirology.com/content/6/1/14 © 2009 Xu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background:We investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT). Methods:Recombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified fromEscherichia coli. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA Lys template and tRNA3as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/ km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV- terminated DNA synthesis was monitored in time-course experiments. Results:The efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8- and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT. Conclusion:The diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP.

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