13th International HIV Drug Resistance Workshop


8–12 June 2004, Tenerife Sur-Costa Adeje, Canary Islands, Spain


NOVEL MECHANISMS INVOLVED IN NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR (NNRTI) RESISTANCE OF BOTH THE SUBTYPE D HIV-1 ISOLATE AND REVERSE TRANSCRIPTASE DERIVED FROM A DRUG-NAÏVE UGANDAN

Antiviral Therapy 2004; 9:S35

H Baird1, Y Gao1, E Paxinos2, J Galovich2, M Abreha1, P Mugyenyi3, C Petropoulos2 and EJ Arts1
1Case Western Reserve University, Cleveland, OH, USA; 2ViroLogic, Inc., South San Francisco, Calif., USA; and 3Joint Clinical Research Centre, Kampala, Uganda

BACKGROUND: Recent studies have shown that ARV treatment of non-subtype B HIV-1 infections can select for novel drug resistance mutations or substitutions considered ‘rare’ in drug-resistant subtype B isolates. We have recently identified a highly NNRTI-resistant subtype D HIV-1 isolate (D14-UG) from a drug-naïve Ugandan. NNRTI-resistant mechanism(s) was examined using mutated subtype D virus and recombinant RT.

METHODS: HIV-1 isolates derived from HIV infected, treatment-naïve Ugandan infants were propagated and tested for sensitivity to antiretroviral drugs. To investigate the possible sites responsible for resistance to NNRTIs, the reverse transcriptase (RT) coding region was sequenced and compared to that of NNRTI-resistant and sensitive subtype A, B and D HIV-1 isolates. Subtype D RTs containing substitution mutations at the putative NNRTI-resistant sites were then cloned (1) into the NL4-3 vector to test for NNRTI sensitivity with the Phenosense assay and (2) into RT expression vectors to study the mechanisms of this resistance.

RESULTS: D14-UG displayed high-level resistance to nevirapine in PBMC (>2000-fold) and in MT4 cultures (~800-fold), but weaker resistance to delavirdine (~13- fold) and efavirenz (~8-fold) in MT4 cultures. D14-UG did not contain the classic amino acid substitutions conferring NNRTI resistance (e.g. Y181C, K103N, G190A) but did have sites associated with drug resistance, I135L, T139V, and V245T. Phenosense assays confirmed that I135L and/or V245T mutations might confer resistance to nevirapine. Preliminary analyses of heterodimeric RT (p66/p51) consisting of every NNRTI-resistant D14-UG/sensitive D subunit combination suggests that the L135 and V139 residues are enacting their effect through p51 and T245 through p66.

CONCLUSIONS: Native sequences responsible for this NNRTI resistance (L135, V139, and T245) were extremely rare in any HIV-1 subtype but nonetheless stable considering wild-type fitness. Since nevirapine is ARV of choice for blocking perinatal transmission and for combination therapy in Uganda, any NNRTI resistance in the drug-naïve population is quite alarming.

PRESENTING AUTHOR: H Baird

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2004-06-08
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