Thymidine Analogue Excision and Discrimination Modulated by Mutational Complexes Including Single Amino Acid Deletions of Asp-67 or Thr-69 in HIV-1 Reverse Transcriptase
| Autor: | Miguel Angel Martinez, Mónica Kisic, Tania Matamoros, Jesús Mendieta, Luis Menéndez-Arias, Maria Nevot, Javier Martinez-Picado |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
DNA polymerase
Mutant medicine.disease_cause Biochemistry 03 medical and health sciences chemistry.chemical_compound Zidovudine medicine Humans Binding site Molecular Biology 030304 developmental biology 0303 health sciences Mutation biology 030306 microbiology Cell Biology Molecular biology HIV Reverse Transcriptase Reverse transcriptase 3. Good health Kinetics Stavudine chemistry DNA Viral Enzymology HIV-1 biology.protein Thymidine DNA medicine.drug |
| Zdroj: | Journal of Biological Chemistry; Vol 286 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m111.226100 |
| Popis: | Single amino acid deletions in the β3-β4 hairpin loop of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been identified in heavily treated patients. The deletion of Asp-67 together with mutations T69G and K70R (Δ67 complex) are usually associated with thymidine analog resistance mutations (TAMs) (e.g. M41L, T215Y, etc.) while the deletion of Thr-69 (Δ69) is rarely found in isolates containing TAMs. Here, we show that the complex Δ67/T69G/K70R enhances ATP-dependent phosphorolytic activity on primers terminated with 3'-azido-3'-deoxythymidine (AZT) or 2',3'-didehydro-2',3'-dideoxythymidine (d4T) both in the presence or absence of TAMs (i.e. M41L/T215Y), while Δ69 (or the complex S68G/Δ69/K70G) antagonize the effects of TAMs in ATP-mediated excision. These effects are consistent with AZT susceptibility data obtained with recombinant HIV-1 bearing the relevant RTs. Molecular dynamics studies based on models of wild-type HIV-1 RT and mutant Δ69, Δ67/T69G/K70R, and D67N/K70R RTs support a relevant role for Lys/Arg-70 in the excision reaction. In Δ69 RT, the side chain of Lys-70 locates away from the putative pyrophosphate binding site. Therefore, its participation in interactions required for the excision reaction is unlikely. Our theoretical studies also suggest a role for Lys-219 in thymidine analog excision/discrimination. However, pre-steady-state kinetics revealed only minor differences in selectivity of AZT-triphosphate versus dTTP between deletion-containing RTs and their homologous enzymes having the K219E mutation. K219E reduced both ATP- and pyrophosphate-mediated excision of primers terminated with thymidine analogues, only when introduced in RTs bearing Δ69 or S68G/Δ69/K70G, providing further biochemical evidence that explains the lack of association of Δ69 and TAMs in HIV-1 isolates. |
| Databáze: | OpenAIRE |
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