Abstract

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. Soares^1,3, E.J. Arts<sup>2

1</sup>Universidade 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.