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. 2016 Mar 18;44(5):2310-22.
doi: 10.1093/nar/gkw060. Epub 2016 Feb 4.

Biochemical Characterization of a Multi-Drug Resistant HIV-1 Subtype AG Reverse Transcriptase: Antagonism of AZT Discrimination and Excision Pathways and Sensitivity to RNase H Inhibitors

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Free PMC article

Biochemical Characterization of a Multi-Drug Resistant HIV-1 Subtype AG Reverse Transcriptase: Antagonism of AZT Discrimination and Excision Pathways and Sensitivity to RNase H Inhibitors

Anna Schneider et al. Nucleic Acids Res. .
Free PMC article

Abstract

We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.

Figures

Figure 1.
Figure 1.
Amino acid sequence alignment of subtype CRF02_AG WT and MR-RT. The positions of the amino acid substitutions in the MR-RT are highlighted as colored boxes. Red boxes: TAMs; purple boxes: Q151M MDR pathway; cyan box: EFV resistance mutation; green box: K65R mutation; yellow boxes: all other mutations. Compensatory mutations mentioned in the text that are associated with the major NRTI and NNRTI resistance mutations described for subtype B are labeled with dots in the same color as the main mutations. The sequence alignment was performed using the program Lalign (74). The polymerase domain is shown in gray, the connection subdomain in blue, and the RNase H domain in light brown.
Figure 2.
Figure 2.
Fidelity of subtype CRF02_AG WT and MR-RT. Primer extension reactions were performed with 20 nM P/T DNA, 0.08 U of pyrophosphatase, 1.25 μM of enzyme and 1.25 mM of the templated nucleotide (dTTP) or 1.25 mM of the mismatched nucleotide (dATP) at 37°C for 10 min. (A) Schematic representation of a primer extension reaction using T50dA as template. An end-labeled [32P]-P30/T50dA DNA/DNA substrate was used to perform a templated extension with dTTP or a mismatch extension in the presence of dATP. Extensions using the other templates were performed accordingly. Percentage of primer extension products after templated (light gray) and mismatched (dark gray) extensions of the P30 primer hybridized to the template T50dA (AA mismatch) (B), T50dC (CC mismatch) (C), T50dT (TT mismatch) (D) T50dG (GG mismatch) (E) by WT and MR-RT. Reaction products were separated on a 10% sequencing gel and quantified. Each diagram depicts the mean values and standard deviations (black bars) of three independent incorporation experiments. For quantification of the extended products, the total amount of labeled DNA per lane was set to 100%. P-values ≤ 0.05 represent statistically significant differences to the WT protein (*P-value ≤ 0.05; **P-value ≤ 0.01;***P-value ≤ 0.001).
Figure 3.
Figure 3.
DNA-dependent DNA polymerase activity of subtype CRF02_AG WT and MR-RTs. Reactions were carried out at 37°C for the times indicated on top of the gel with 30 nM [32P]-P17/M13mp18, 200 μM of each dNTP and 83 nM WT or MR-RT, or without RT (control) in a reaction volume of 10 μl. Extension products were analyzed by denaturing gel electrophoresis on a 10% sequencing gel and visualized by a phosphoimaging device. DNA size markers are shown on the left.
Figure 4.
Figure 4.
AZTMP excision. AZTMP excision reactions with the CRF02_AG WT and MR RTs were performed with 20 nM of a 5′ [32P] end-labeled and AZTMP terminated [32P]-P30-AZTMP/T50substrate in the presence of 5 mM ATP, 0.01 U/μl of pyrophosphatase and 600 nM RT as indicated. Reactions were started by RT addition and stopped after 5 min at 37°C. After separation of the reaction products and educt by denaturing gel electrophoresis, the bands were quantified densitometrically. The total amount of AZTMP-terminated primer per lane was set to 100% to calculate the percentage of excised AZTMP. Each diagram depicts the mean values and standard deviations (black bars) of three independent experiments. P-values ≤ 0.05 represent statistically significant differences to the WT protein (***P-value ≤ 0.001).
Figure 5.
Figure 5.
Discrimination between AZTTP and TTP during nucleotide incorporation. For comparison of AZTMP versus TMP incorporation by CRF02_AG WT and MR RTs, and AZTres-B RT, 100 nM of labeled [32P]-P30/T50 substrate and 1 μM of AZTTP or TTP were pre-incubated with 0.01 U/μl of pyrophosphatase for 5 min at 37°C. Reactions were started by the addition of 200 nM of RT as indicated and performed for 30 s at 37°C. Control, reaction without enzyme. Educts and products were separated by denaturing gel electrophoresis on a 10% sequencing gel.
Figure 6.
Figure 6.
Qualitative RNase H assay. Autoradiogram of a typical RNase H cleavage experiment. RNase H reactions with 100 nM 5′ labeled Cy5-RNA29/DNA29 hybrid and RT variants at concentrations of (i) 33.3 nM, (ii) 11.1 nM and (iii) 3.7 nM were performed for 10 min at 37°C. RNA ladder, Cy5-RNA29/DNA29 hybrid boiled, without RT, control, reaction without RT. RNA size markers are shown on the left.
Figure 7.
Figure 7.
Chemical structures of HIV-1 subtype B RNase H inhibitors.

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