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
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.
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.
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.
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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