Original Article Characterization of amino acids Arg, Ser and Thr at position 70 within HIV-1 reverse transcriptase S. Megens 1 , S. De Wit 2 , J. Bernatchez 3 , N. Dekeersmaeker 1 , L. Vinken 1 , K. Covens 1 , K. Theys 1 , R. J. Camacho 1,4 , A.-M. Vandamme 1,4 , M. Go ¨tte 3,5 , K. Van Laethem 1,6 1 KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, B-3000 Leuven, Belgium, 2 AIDS Reference Centre, Division of Infectious Diseases, CHU Saint-Pierre, Brussels, Belgium, 3 Department of Biochemistry, McGill University, Montre ´al, Quebec, Canada, 4 Centro de Mala ´ria e outras Doenc ¸as tropicais and Unidade de Microbiologia, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal, 5 Department of Microbiology and Immunology, McGill University, Montre ´al, Quebec, Canada, 6 AIDS Reference Laboratory, Universitaire Ziekenhuizen Leuven, Leuven, Belgium Objectives: The amino acid position 70 in HIV-1 reverse transcriptase (RT) plays an important role in nucleoside RT inhibitor (NRTI) resistance. K70R is part of the thymidine analog mutations, but also other amino acid changes have been associated with NRTI resistance, such as K70E and K70G. In this study, we investigated the in vivo selection of the HIV-1 RT mutations K70S and K70T and their in vitro effect on drug resistance and replication capacity. Methods: Recombinant viruses with RT mutations were generated to measure the in vitro drug susceptibility and replication capacity. Bayesian network analysis and three-dimensional modeling were performed to understand the selection and impact of the RT70 mutations. Results: K70S and K70T were found at a low frequency in RTI-experienced HIV-1 patients (0.10% and 0.20%). Baeyesian network learning identified no direct association with the in vivo exposure to any specific RTI. However, direct associations of K70S with mutations within the Q151M-complex and of K70T with K65R were observed. In vitro phenotypic testing revealed only minor effects of K70R/S/T as single mutations, associated with Q151M and within the context of the Q151M-complex. Discussion: These results suggest that the selection of K70S/T and their phenotypic impact are influenced by the presence of other mutations in RT. However, the low impact on in vitro phenotype here observed, alongside with the low in vivo prevalence, the exclusive direct association with known major RTI mutations and the unknown correlation with in vivo response, do not yet necessitate the inclusion of K70S/T in drug resistance interpretation systems. Keywords: HIV-1, Resistance, Mutation, Reverse transcriptase Introduction Nucleoside reverse transcriptase (RT) inhibitors (NRTIs) are analogs of deoxyribonucleosides that lack the 39-hydroxyl group and cause chain termina- tion during reverse transcription. NRTIs often constitute the backbone of combination antiretro- viral therapy (cART) in HIV-1 patients. Despite the success of cART in reducing morbidity and mortal- ity, its long-term activity can be compromised because of drug resistance development. Resistance to NRTIs generally occurs either by the excision or by the exclusion/discrimination mechanism. 1 The first mechanism consists of an enhanced ATP-mediated excision of the inhibitor at the dNTP binding site, as initially observed for the thymidine analog zidovu- dine (AZT) and typically caused by thymidine associated mutations (TAMs). The TAMs confer cross-resistance to most NRTIs and are associated with hypersusceptibility towards foscarnet (PFA). Second, resistance by exclusion consists of the selective discrimination against NRTIs but still allows incorporation of dNTPs, as described for several mutations and the multi-NRTI drug resis- tance pathway marked by Q151M. 2 Correspondence to: S. Megens, Rega Institute for Medical Research, Minderbroedersstraat 10, 3000 Leuven, Belgium. Email: sarah.megens@ rega.kuleuven.be 348 ß Acta Clinica Belgica 2014 DOI 10.1179/2295333714Y.0000000038 Acta Clinica Belgica 2014 VOL. 69 NO.5 brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Lirias