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K70Q adds high-level tenofovir resistance to "Q151M complex" HIV reverse transcriptase through the enhanced discrimination mechanism.

Hachiya A, Kodama EN, Schuckmann MM, Kirby KA, Michailidis E, Sakagami Y, Oka S, Singh K, Sarafianos SG - PLoS ONE (2011)

Bottom Line: Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF.However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF.Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri, United States of America.

ABSTRACT
HIV-1 carrying the "Q151M complex" reverse transcriptase (RT) mutations (A62V/V75I/F77L/F116Y/Q151M, or Q151Mc) is resistant to many FDA-approved nucleoside RT inhibitors (NRTIs), but has been considered susceptible to tenofovir disoproxil fumarate (TFV-DF or TDF). We have isolated from a TFV-DF-treated HIV patient a Q151Mc-containing clinical isolate with high phenotypic resistance to TFV-DF. Analysis of the genotypic and phenotypic testing over the course of this patient's therapy lead us to hypothesize that TFV-DF resistance emerged upon appearance of the previously unreported K70Q mutation in the Q151Mc background. Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF. However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF. Biochemical experiments established that the increased resistance to tenofovir is not the result of enhanced excision, as K70Q/Q151Mc RT exhibited diminished, rather than enhanced ATP-based primer unblocking activity. Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain. Molecular dynamics simulations suggest that changes in the hydrogen bonding pattern in the polymerase active site of K70Q/Q151Mc RT may contribute to the observed changes in binding and incorporation of TFV-DP. The novel pattern of TFV-resistance may help adjust therapeutic strategies for NRTI-experienced patients with multi-drug resistant (MDR) mutations.

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NRTI resistance of HIVs with mutations at RT residue 70 in the background of WT or Q151Mc.Antiviral activities of HIV-1s carrying mutations at residue 70 (K70R, K70G, K70E, K70T, K70N, or K70Q) in the WT (A) or Q151Mc (B) background were determined by the MAGIC5 assay. The data for each clone were compared to WT (A) and Q151Mc (B) HIV-1 and are shown as fold increase; AZT (red), ddI (green), d4T (cyan), 3TC (orange), ABC (blue), and TFV-DF (purple). Error bars represent standard deviations from at least three independent experiments (see also Table S2 and Table S3). The asterisk indicates statistically significant in EC50 values (P<0.0001 by t-test).
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pone-0016242-g002: NRTI resistance of HIVs with mutations at RT residue 70 in the background of WT or Q151Mc.Antiviral activities of HIV-1s carrying mutations at residue 70 (K70R, K70G, K70E, K70T, K70N, or K70Q) in the WT (A) or Q151Mc (B) background were determined by the MAGIC5 assay. The data for each clone were compared to WT (A) and Q151Mc (B) HIV-1 and are shown as fold increase; AZT (red), ddI (green), d4T (cyan), 3TC (orange), ABC (blue), and TFV-DF (purple). Error bars represent standard deviations from at least three independent experiments (see also Table S2 and Table S3). The asterisk indicates statistically significant in EC50 values (P<0.0001 by t-test).

Mentions: To examine the effect of K70Q on drug susceptibility we generated a series of HIV variants with mutations at RT codon 70 (Figure 2A and also Table S2). The HIV-1K70Q variant exhibited marginal resistance to ddI and 3TC (5- and 3.3-fold, respectively), but no significant resistance to other NRTIs. We further examined whether the mutations at residue 70 affect susceptibility to NRTIs in the Q151Mc background (Figure 2B and also Table S3). HIV-1K70G/Q151Mc had enhanced resistance to d4T (4.6-fold) as compared to HIV-1Q151Mc. Notably, HIV-1K70Q/Q151Mc also showed enhanced resistance to ddI and d4T (2.4- and 4.4-fold, respectively, compared to HIV-1Q151Mc). In addition, HIV-1K70Q/Q151Mc displayed 5-fold increased resistance to TFV-DF compared to HIV-1Q151Mc. Other K70 mutations exhibited little or no resistance to TFV-DF.


K70Q adds high-level tenofovir resistance to "Q151M complex" HIV reverse transcriptase through the enhanced discrimination mechanism.

Hachiya A, Kodama EN, Schuckmann MM, Kirby KA, Michailidis E, Sakagami Y, Oka S, Singh K, Sarafianos SG - PLoS ONE (2011)

NRTI resistance of HIVs with mutations at RT residue 70 in the background of WT or Q151Mc.Antiviral activities of HIV-1s carrying mutations at residue 70 (K70R, K70G, K70E, K70T, K70N, or K70Q) in the WT (A) or Q151Mc (B) background were determined by the MAGIC5 assay. The data for each clone were compared to WT (A) and Q151Mc (B) HIV-1 and are shown as fold increase; AZT (red), ddI (green), d4T (cyan), 3TC (orange), ABC (blue), and TFV-DF (purple). Error bars represent standard deviations from at least three independent experiments (see also Table S2 and Table S3). The asterisk indicates statistically significant in EC50 values (P<0.0001 by t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020970&req=5

pone-0016242-g002: NRTI resistance of HIVs with mutations at RT residue 70 in the background of WT or Q151Mc.Antiviral activities of HIV-1s carrying mutations at residue 70 (K70R, K70G, K70E, K70T, K70N, or K70Q) in the WT (A) or Q151Mc (B) background were determined by the MAGIC5 assay. The data for each clone were compared to WT (A) and Q151Mc (B) HIV-1 and are shown as fold increase; AZT (red), ddI (green), d4T (cyan), 3TC (orange), ABC (blue), and TFV-DF (purple). Error bars represent standard deviations from at least three independent experiments (see also Table S2 and Table S3). The asterisk indicates statistically significant in EC50 values (P<0.0001 by t-test).
Mentions: To examine the effect of K70Q on drug susceptibility we generated a series of HIV variants with mutations at RT codon 70 (Figure 2A and also Table S2). The HIV-1K70Q variant exhibited marginal resistance to ddI and 3TC (5- and 3.3-fold, respectively), but no significant resistance to other NRTIs. We further examined whether the mutations at residue 70 affect susceptibility to NRTIs in the Q151Mc background (Figure 2B and also Table S3). HIV-1K70G/Q151Mc had enhanced resistance to d4T (4.6-fold) as compared to HIV-1Q151Mc. Notably, HIV-1K70Q/Q151Mc also showed enhanced resistance to ddI and d4T (2.4- and 4.4-fold, respectively, compared to HIV-1Q151Mc). In addition, HIV-1K70Q/Q151Mc displayed 5-fold increased resistance to TFV-DF compared to HIV-1Q151Mc. Other K70 mutations exhibited little or no resistance to TFV-DF.

Bottom Line: Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF.However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF.Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri, United States of America.

ABSTRACT
HIV-1 carrying the "Q151M complex" reverse transcriptase (RT) mutations (A62V/V75I/F77L/F116Y/Q151M, or Q151Mc) is resistant to many FDA-approved nucleoside RT inhibitors (NRTIs), but has been considered susceptible to tenofovir disoproxil fumarate (TFV-DF or TDF). We have isolated from a TFV-DF-treated HIV patient a Q151Mc-containing clinical isolate with high phenotypic resistance to TFV-DF. Analysis of the genotypic and phenotypic testing over the course of this patient's therapy lead us to hypothesize that TFV-DF resistance emerged upon appearance of the previously unreported K70Q mutation in the Q151Mc background. Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF. However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF. Biochemical experiments established that the increased resistance to tenofovir is not the result of enhanced excision, as K70Q/Q151Mc RT exhibited diminished, rather than enhanced ATP-based primer unblocking activity. Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain. Molecular dynamics simulations suggest that changes in the hydrogen bonding pattern in the polymerase active site of K70Q/Q151Mc RT may contribute to the observed changes in binding and incorporation of TFV-DP. The novel pattern of TFV-resistance may help adjust therapeutic strategies for NRTI-experienced patients with multi-drug resistant (MDR) mutations.

Show MeSH
Related in: MedlinePlus