Tesis doctoral de Monica Kisic Aguirre
The human immunodeficiency virus type 1 (hiv-1) reverse transcriptase (rt) is a multifunctional enzyme with rna- and dna- dependent dna polymerase, rnase h, strand transfer and strand displacement activities. Hiv-1 rt is an heterodimer composed of two subunits, p66 and p51, with subdomains termed fingers, palm, thumb and connection in both subunits and an rnase h domain in the larger subunit only. The íY3-íY4 hairpin loop of the hiv-1 rt fingers subdomain interacts with the incoming dntp and the template overhang, and is considered a hotspot for nucleoside analogue resistance mutations. Deletions in the íY3¿íY4 loop of hiv-1 rt are associated with the emergence of multidrug resistance. Common mutational patterns involve the deletion of asp67 (¿67) and mutations t69g and k70r, usually associated with thymidine analogue resistance-associated mutations (tams), while the deletion of thr69 (¿69) appears together with mutations of the q151m complex. We have studied the effect of ¿69 in a multidrug-resistant sequence background (including the q151m complex and substitutions k103n, y181c, m184v, and g190a) and in a wt sequence context, under steady- and pre-steady-state conditions. Furthermore, we studied the effect of deleting codons 67 and 69 in rts bearing tams m41l and t215y, or k219e. firstly, we show that the q151m complex accounts for most of the loss of susceptibility shown by the multidrug-resistant rts, while mutation k103n generates high-level resistance to efavirenz and other nnrtis. In addition, we demonstrate that mutations outside the nnrti binding pocket (e.G. A376s) can influence resistance. A376s confers low-level resistance to nevirapine, due to a reduced affinity for the inhibitor, and affects template-primer binding. transient kinetics have been used to demonstrate that ¿69 in a wt sequence context increases 3tc resistance through a 3tctp versus dctp discrimination mechanism. In comparison with the wt rt, the ¿69 rt showed decreased ability to excise primers terminated with aztmp in the presence of atp or ppi. These data support the role of the excision mechanism in mediating azt hypersusceptibility. We also demonstrate that the deletion has no effect on resistance to foscarnet. Furthermore, the introduction of deletion ¿69 in a sequence bearing tams m41l and t215y resulted in a lower atp-dependent phosphorolytic activity on primers terminated with aztmp or d4tmp. Interestingly, the ¿67 complex (¿67/t69g/k70r) showed significant atp-mediated excision. This complex also increased excision activity on thymidine analogue-terminated primers, when introduced in a sequence containing tams m41l and t215y. Secondary mutations, such as k219e (a tam-2 pathway mutation) had no effect on rescue reactions catalyzed by the ¿67 rt, while its introduction in the ¿69 rt led to a decrease of its ppi-dependent excision activity on both azt- and d4t-terminated primers. structural changes in the íY3-íY4 loop generated by these deletions can modify the accessibility of the ppi donor to the terminating nucleotide at the 3¿ end of the primer. We suggest that the evolutionary patterns followed by ¿69 and ¿67 can be explained by their role in the excision mechanism mediating resistance to nucleoside analogues.
Datos académicos de la tesis doctoral «Mecanismos moleculares de resistencia e hipersensibilidad a fármacos antirretovirales modulados por deleciones en la horquilla b3-b4 de la retrotranscriptasa del virus de la inmunodeficiencia humana tipo 1«
- Título de la tesis: Mecanismos moleculares de resistencia e hipersensibilidad a fármacos antirretovirales modulados por deleciones en la horquilla b3-b4 de la retrotranscriptasa del virus de la inmunodeficiencia humana tipo 1
- Autor: Monica Kisic Aguirre
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 16/12/2010
Dirección y tribunal
- Director de la tesis
- Luis Menendez Arias
- Tribunal
- Presidente del tribunal: josé Berenguer Carlos
- áfrica Holguín fernández (vocal)
- cecilio López galíndez (vocal)
- Miguel angel Martínez de la sierra (vocal)