XVIII International AIDS Conference

Abstract

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Natural polymorphisms in the protease of HIV-1 isolates explain hypersusceptibility to protease inhibitors

Presented by Andre Felipe Santos (Brazil).

A.F. Santos1, D.M. Tebit2, M.S. Lalonde2, A. Ratcliff2, M.A. Soares1,3, E.J. Arts2


1Universidade Federal do Rio de Janeiro, Genetica, Rio de Janeiro, Brazil, 2Case Western Reserve University, Division of Infectious Diseases, Cleveland, United States, 3Instituto Nacional de Câncer, Genetica, Rio de Janeiro, Brazil

Background: Antiretroviral therapy is becoming a reality in many African countries, where several HIV-1 non-B subtypes predominate. However, there are scarce studies on drug susceptibility in such subtypes. Here we evaluated the influence of protease polymorphisms in CRF02_AG on drug susceptibility and fitness.
Methods: Mutations G17E, I64M, K70R and I72V were introduced by site-direct mutagenesis in the BD6-15 CRF02_AG infectious molecular clone. All clones were transfected in 293T cells and used to infect MT-2 cell cultures. Phenotyping assays were conducted for six protease inhibitors (PI): amprenavir, atazanavir, indinavir, lopinavir, nelfinavir and saquinavir, in quadriplicate. IC50 values were calculated for each virus and for each drug and compared with the original BD6-15 clone through 2-tailed Student's T test, for which a p≤0.05 was considered significant. Head-to-head competitions were performed and after six days of infection, genomic DNA were extracted and the PR region PCR-amplified. The fragments were submitted to oligonucleotide ligation assay and the products were quantified by fluorescence.
Results: The double mutant 17E/64M presented hypersusceptibility (HS) to NFV (FC=0.4; p< 0.01) and a higher susceptibility to SQV and ATV (FC=0.5 and 0.6, respectively; p< 0.01 for both cases). The mutation K70R conferred HS to APV and IDV (FC=0.4 and 0.3, p< 0.03 for both cases). In the competition assays, the fitness ranking order was: 17E/64M>17E>64M>BD6>72V. The analysis of 1,034 CRF02 isolates from treatment-naive subjects retrieved from the Stanford database showed that 2.4% of them presented the polymorphisms 17E and 64M and 28.1% the K70R.
Conclusions: This study showed that the natural polymorphisms 17E and 64M in PR of HIV-1 CRF02-AG impact on PI susceptibility. Such finding could help directing PI-containing HAART regimens in countries where this CRF is predominant. In addition, our results provide proof-of-principle evidence that natural polymorphisms account for differences in susceptibility to ARV displayed by drug-nave HIV-1 isolates.


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