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http://dspace.mediu.edu.my:8181/xmlui/handle/10261/4923| Title: | Mutational Patterns Associated with the 69 Insertion Complex in Multi-drug-resistant HIV-1 Reverse Transcriptase that Confer Increased Excision Activity and High-level Resistance to Zidovudine |
| Authors: | Fundación para la Investigación y la Prevención del Sida en España Fondo de Investigación Sanitaria (España) Ministerio de Educación y Ciencia (España) Fundación Ramón Areces |
| Keywords: | HIV Reverse transcriptase Drug resistance Thymidine analogues Zidovudine |
| Publisher: | Elsevier |
| Description: | Human immunodeficiency virus type 1 (HIV-1) strains having dipeptide insertions in the fingers subdomain and other drug resistance-related mutations scattered throughout their reverse transcriptase (RT)-coding region show high-level resistance to zidovudine (AZT) and other nucleoside analogues. Those phenotypic effects have been correlated with their increased ATP-dependent phosphorolytic activity on chain-terminated primers. Mutations T69S and T215Y and a dipeptide insertion (i.e. Ser-Ser) between positions 69 and 70 are required to achieve low-level resistance to thymidine analogues. However, additional amino acid substitutions are necessary to achieve the high-level phenotypic resistance to AZT shown by clinical HIV isolates carrying a dipeptide insertion in their RT-coding region. In order to identify those mutations that contribute to resistance in the sequence context of an insertion-containing RT derived from an HIV clinical isolate (designated as SS RT), we expressed and purified a series of chimeric enzymes containing portions of the wild-type or SS RT sequences. ATP-mediated excision activity measurements using AZT- and stavudine (d4T)-terminated primers and phenotypic assays showed that molecular determinants of high-level resistance to AZT were located in the fingers subdomain of the polymerase. Further studies, using recombinant RTs obtained by site-directed mutagenesis, revealed that M41L, A62V and in a lesser extent K70R, were the key mutations that together with T69S, T215Y and the dipeptide insertion conferred high levels of ATP-dependent phosphorolytic activity on AZT and d4T-terminated primers. Excision activity correlated well with AZT susceptibility measurements, and was consistent with phenotypic resistance to d4T. Structural analysis of the location of the implicated amino acid substitutions revealed a coordinated effect of M41L and A62V on the positioning of the β3–β4 hairpin loop, which plays a key role in the resistance mechanism This work was supported in part by FIPSE (grant 36523/05) and Fondo de Investigación Sanitaria (through “Red Temática Cooperativa de Investigación en SIDA” G03/173). In addition, work in Madrid was supported by grant BIO2003/01175 (Ministerio de Educación y Ciencia) and an institutional grant from Fundación Ramón Areces. Grant BMC2003/2148 (Ministerio de Educación y Ciencia) (to M. A.M.) is also acknowledged Peer reviewed |
| URI: | http://dspace.mediu.edu.my:8181/xmlui/handle/10261/4923 |
| Other Identifiers: | Journal of Molecular Biology Vol. 365, Issue 2, 12 January 2007, Pages 298-309 0022-2836 (Print) 1089-8638 (Online) http://hdl.handle.net/10261/4923 10.1016/j.jmb.2006.09.073 |
| Appears in Collections: | Digital Csic |
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