Ultrasensitive Allele-Specific PCR Reveals Rare Preexisting Drug-Resistant Variants and a Large Replicating Virus Population in Macaques Infected with a Simian Immunodeficiency Virus Containing Human Immunodeficiency Virus Reverse Transcriptase

Abstract

It has been proposed that most drug-resistant mutants, resulting from a single-nucleotide change, exist at low frequency in human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) populations in vivo prior to the initiation of antiretroviral therapy (ART). To test this hypothesis and to investigate the emergence of resistant mutants with drug selection, we developed a new ultrasensitive allele-specific PCR (UsASP) assay, which can detect drug resistance mutations at a frequency of >0.001% of the virus population. We applied this assay to plasma samples obtained from macaques infected with an SIV variant containing HIV-1 reverse transcriptase (RT) (RT-simian-human immunodeficiency [SHIV] mne ), before and after they were exposed to a short course of efavirenz (EFV) monotherapy. We detected RT inhibitor (RTI) resistance mutations K65R and M184I but not K103N in 2 of 2 RT-SHIV-infected macaques prior to EFV exposure. After three doses over 4 days of EFV monotherapy, 103N mutations (AAC and AAT) rapidly emerged and increased in the population to levels of ϳ20%, indicating that they were present prior to EFV exposure. The rapid increase of 103N mutations from <0.001% to 20% of the viral population indicates that the replicating virus population size in RT-SHIV-infected macaques must be 10 6 or more infected cells per replication cycle. Studies have shown that human immunodeficiency virus type 1 (HIV-1) rapidly develops drug resistance in infected patients who undergo monotherapy or incompletely suppressive combination antiretroviral therapy (cART) (13,15,18,28). This rapid emergence of resistance lends support to the hypothesis that HIV-1 variants capable of conferring drug resistance are already present before therapy (8, 10). The high HIV-1 replication rate, high mutation rates (1,10,22), and strong host selective pressures that occur during infection and the resulting swarm of nonidentical viral variants (8, 24) also support this hypothesis. This high evolutionary rate of HIV-1 can be used to study the emergence of drug resistance, which in turn can provide insights into the size of the replicating population, information that is important for developing improved treatment strategies.In the present study, we assessed the levels of preexisting drugresistant mutants and their emergence, using as a model macaques that were infected with a simian immunodeficiency virus (SIV) containing HIV-1 reverse transcriptase (RT) (RT-simian-human immunodeficiency virus [SHIV] mne ) and treated with a short course of efavirenz (EFV) monotherapy followed by a commonly used cART regimen of tenofovir (TNV), emtricitabine (FTC), and EFV (2). As described by Ambrose et al. (2), plasma was collected and allele-specific PCR (ASP) and single-genome sequencing (SGS) were conducted on samples prior to and following EFV monotherapy and after cART. In samples collected after the initiation of cART, M184I and K65R, which encode FTC and TNV resistance, were detected in one of the macaques, which experienced virologic...

Low Frequency of Drug-Resistant Variants Selected by Long-Acting Rilpivirine in Macaques Infected with Simian Immunodeficiency Virus Containing HIV-1 Reverse Transcriptase

“…Despite waning RPV plasma concentrations and lack of complete virus suppression, which may be seen in noncompliant individuals and suggesting suboptimal in vivo drug inhibition, persistent DRMs were not selected in the plasma or tissues of either animal after RPV LA administration. This is in contrast to our previous studies, in which EFV monotherapy was administered over 4 days in RT-SHIV-infected macaques and rapidly selected the NNRTI resistance mutation K103N in the plasma virus that affected the efficacy of subsequent combination therapy containing EFV, particularly in two animals with high plasma viremia levels similar to the animals in this study (30,40,62). Another study using a different strain of RT-SHIV in rhesus macaques also showed that K103N and other DRMs arise during EFV monotherapy (34).…”

Effect of Suberoylanilide Hydroxamic Acid (SAHA) Administration on the Residual Virus Pool in a Model of Combination Antiretroviral Therapy-Mediated Suppression in SIVmac239-Infected Indian Rhesus Macaques

“…This time point was chosen to allow seeding of the residual viral reservoir while limiting the opportunity of the virus to develop and archive drug resistance variants, as has been shown for SHIVmne-infected animals prior to the initiation of ART (77) and may be more likely in the setting of the very high replication rates of SIVmac239. As shown in Fig.…”

Quantifying Selection against Synonymous Mutations in HIV-1 env Evolution

“…The number of cells infected by HIV-1 per day is on the order of 10 7 (23), but the number of viruses contributing to the next generation might be considerably smaller due to the burstiness of replication. Recent evidence points to a relevant population size in excess of the inverse mutation rate (27). In fact, the evolutionary dynamics depends only weakly on the actual value of the population size N once the product N is of order one or larger (28).…”