Replication of HIV-1 is inhibited by azidothymidine (AZT), which leads to chain termination and inhibition of DNA synthesis. Resistance to AZT is frequently the result of mutations that increase the ability of RT to remove the chain-terminating nucleotides after they have been incorporated. It has been proposed that RNase H cleavage of the RNA template can occur when RT is stalled near the site of chain termination and contributes to the inhibition by causing the dissociation of the primer-template before the chain terminator can be excised. Mutations in the connection and RNase H domains of RT have been shown to increase excision. It has long been known that resistance to thymidine analogs is conferred by the mutations M41L, D67N, K70R, L210W, T215F/Y and T219Q/E in RT and that this resistance is suppressed by the additional presence of the M184V mutation. Changes in excision activity on DNA templates have been observed with these mutant RTs, but effects on RNase H cleavage resulting in indirect effects on excision activity is also possible with RNA templates. We used a 5'-labeled -3'-chain-terminated DNA primer annealed to either a DNA or RNA template to evaluate primer rescue activities, a 5'-labeled RNA template to evaluate RNA cleavage activity and a biotin-tagged chain-terminated oligodeoxynucleotide to monitor primer-template dissociation. We first investigated differences between RNA and DNA templates when the primers were chain terminated and observed a correlation between RNase H activities and template/primer (T/P) dissociation. An inverse correlation was observed between excision rescue rates and RNase H cleavages leading to T/P dissociation. We observed that the chain terminator (i.e. AZTMP or ddAMP) affected RNase H cleavages and excision rates with RNA template and dNTPs. When we investigated mutations in the N-terminal domain of RT associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance we found that primer rescue was decreased when M184V was present in combination with thymidine analog mutations (TAMs) and the template was RNA with either ATP or PPi as excision substrate. RNase H cleavage at secondary cleavage sites (-7, -8) was substantially reduced with M41L/T215Y RT in comparison with wild type RT, and primer-template dissociation was decreased. In contrast, when M184V was present, RNase H cleavage at the secondary cleavage sites and dissociation of the primer-template occurred at higher levels and excision rescue was decreased. The ability of RT to rescue an AZT terminated primer in the presence of the 184V mutation was restored when the RNase H activity was inactivated by the RNase H negative mutation E478Q. Electromobility shift assay (EMSA) analysis of AZT-resistant mutant RT with M184V showed an increased Kd for formation of the ternary complex. These results suggest that RNase H-mediated RNA-DNA template-primer dissociation is influenced by mutations associated with thymidine analog resistance, and that suppression of resistance to nucleoside RT inhibitors by M184V may be partly explained by effects on RNase H cleavage that decrease the time available for excision to occur. This is the first time that mutations in the polymerase domain are shown to affect excision rescue through an RNase H-dependent mechanism.

Introducción: En pacientes con VIH en TARV y fallo virológico a primera línea, establecer un esquema terapéutico tras haber identificado la mutación M184V, que confiere resistencia, representa una disyuntiva. Objetivo: Comparar la respuesta virológica de los esquemas terapéuticos prescritos a pacientes con VIH que presentaron la mutación M184V en dos hospitales nacionales de Lima, Perú durante los años 2008 a 2019 y determinar los factores de riesgo asociados a una mala respuesta virológica. Métodos: Se desarrolló un estudio de cohorte retrospectivo. Resultados: Un total de 175 participantes fueron elegibles para el estudio. El sexo masculino predominó (75.4%), la mediana de edad actual fue 41 años (IQR 35.84 ; 47.47) y tiempo en TARV fue 89 meses (IQR 57.7 ; 124.53). La mediana de carga viral inicial fue 4.5 log10 (IQR 3.97 ; 5.09) y el tiempo entre la genotipificación y el cambio de terapia fue 2 meses (IQR 0 ; 3.57). El esquema antirretroviral más utilizado fue IP + 2 INTR (55.4%). Con el esquema IP + INI se obtuvo 69% menos riesgo de mala respuesta virológica [p=0.019 (IC 95% 0.117 ; 0.825)]. Conclusiones: En los pacientes con VIH y mutación M184V, el esquema IP + INI ha demostrado una mayor disminución de la carga viral de control y, así, una buena respuesta virológica. Los factores de riesgo asociados a una mala respuesta virológica fueron la demora entre la genotipificación y el cambio de terapia, niveles elevados de carga viral inicial y mala adherencia. / Introduction: In patients with HIV in ART and virological failure to the first-line regimen, establishing a therapeutic regimen after having identified the M184V mutation, which confers ART resistance, represents a dilemma. Objective: To compare the virological response of the therapeutic regimens prescribed to patients with HIV who presented the M184V mutation in two national hospitals in Lima, Peru during the years 2008 to 2019, and to determine the risk factors associated with poor virological response. Methods: A retrospective cohort study was developed based on the information of the HIV program participants with the M184V mutation. Results: A total of 175 participants were eligible for the study. The male sex predominated (75.4%), the current median age was 41 years (IQR 35.84, 47.47) and the time on ART was 89 months (IQR 57.7, 124.53). The median initial viral load was 4.5 log10 copies/ml (IQR 3.97, 5.09) and the time between genotyping and the change of therapy was 2 months (IQR 0; 3.57). The most used antiretroviral regimen was PI + 2 NRTIs (55.4%). With the PI + INI ART, 69% less risk of poor virological response was obtained [p=0.019 (CI 95% 0.117 ; 0.825)]. Conclusions: In patients with HIV and the M184V mutation, the PI + INI ART has shown a greater decrease in control viral load and, thus, a good virological response. The risk factors associated with a poor virological response were the delay between genotyping and change of therapy, high levels of initial viral load, and poor adherence among the participants. / Tesis