Genetic diversity of simian immunodeficiency virus encoding HIV-1 reverse transcriptase persists in macaques despite antiretroviral therapy

Abstract

The impact of antiretroviral therapy (ART) on the genetics of simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV) populations has been incompletely characterized. We analyzed SIV genetic variation before, during, and after ART in a macaque model. Six pigtail macaques were infected with an SIV/HIV chimeric virus, RT-SHIV(mne), in which SIV reverse transcriptase (RT) was replaced by HIV-1 RT. Three animals received a short course of efavirenz (EFV) monotherapy before combination ART was started. All macaques received 20 weeks of tenofovir, emtricitabine, and EFV. Plasma virus populations were analyzed by single-genome sequencing. Population diversity was measured by average pairwise difference, and changes in viral genetics were assessed by phylogenetic and panmixia analyses. After 20 weeks of ART, viral diversity was not different from pretherapy viral diversity despite more than 10,000-fold declines in viremia, indicating that, within this range, there is no relationship between diversity and plasma viremia. In two animals with consistent SIV RNA suppression to <15 copies/ml during ART, there was no evidence of viral evolution. In contrast, in the four macaques with viremias >15 copies/ml during therapy, there was divergence between pre- and during-ART virus populations. Drug resistance mutations emerged in two of these four animals, resulting in virologic failure in the animal with the highest level of pretherapy viremia. Taken together, these findings indicate that viral diversity does not decrease with suppressive ART, that ongoing replication occurs with viremias >15 copies/ml, and that in this macaque model of ART drug resistance likely emerges as a result of incomplete suppression and preexisting drug resistance mutations.

FIG. 1.

Longitudinal changes in diversity of HIV-1 pol and plasma RNA level for animals in group 1 (A), group 2 (B), and group 3 (C). APDs are plotted for each animal (left y axis) as a function of time. Plasma viral RNA copies are plotted (right y axis) versus time for each animal. Samples with undetectable viral RNA (<15 copies/ml) are denoted by open symbols. Gray shading indicates EFV monotherapy (week 13) or ART (weeks 17 to 37). Animal M03250 experienced virologic failure and was euthanized (†). Circled points show samples included in Fig. 2.

FIG. 2.

Effect of ART on RT-SHIV_mne diversity in all animals. Genetic diversities were measured as APDs of sequences obtained from plasma samples collected from the six RT-SHIV_mne-infected macaques before initiating treatment (week 13), after a 1,000- to 10,000-fold decline in plasma RNA during ART (weeks 22 to 37), and after stopping ART (weeks 38 to 40). Results are plotted for each time period for animals in group 1, group 2, and group 3.

FIG. 3.

Effect of ART on RT-SHIV_mne divergence. The significance of divergence was assessed by a test for panmixia of single-genome sequences from the plasma of six RT-SHIV_mne-infected macaques before treatment (week 13), during combination ART (weeks 17.5 to 37), and after ART discontinuation (weeks 38 to 40). Shading indicates ART (weeks 17 to 37). The significance cutoff is shown by a dotted line at P values of <0.0001. Animals that received EFV monotherapy at week 13 are denoted by double daggers (‡). Animal M03250 experienced virologic failure and was euthanized at 26 weeks (†).

FIG. 4.

Effect of ART on HIV-1 pol evolution analyzed by neighbor-joining phylogenetic analyses of animals with complete suppression of viremia and showing no evidence of a population shift during ART. Trees were rooted on the transmitted viral variant (open squares). Single-genome sequences were from plasma samples obtained before treatment (week 13; open circles) and after a 1,000- to 10,000-fold decline in viremia during ART (week 17.5 to 37; closed circles). M04007 received EFV monotherapy at weeks 13 to 14, but M03430 did not.

FIG. 5.

Effect of ART on HIV-1 pol evolution analyzed by neighbor-joining phylogenetic analyses of animals with incomplete plasma virus suppression with evidence of population shifts during ART. Neither of these animals received EFV monotherapy prior to ART. Symbols are as described in the legend to Fig. 4.

FIG. 6.

Effect of ART on HIV-1 pol evolution analyzed by neighbor-joining phylogenetic analyses of animals with incomplete plasma virus suppression and drug resistance arising following monotherapy during ART. Both of these animals received EFV monotherapy prior to ART. Animal M03250 experienced virological failure and was euthanized at week 26. Symbols are as described in the legend to Fig. 4.

FIG. 7.

Interanimal evolution analyzed by neighbor-joining analyses of consensus sequences from week 1 and week 13, after a 1,000- to 10,000-fold decline in viremia during ART, and after discontinuing ART. Trees were rooted on the consensus sequence of virus in the cell culture supernatant used for the challenge.