Because of its high mutation rate, large population size, and rapid replication cycle, HIV-1 is able to diversify into a complex genetic population after transmission to a new host. Its pathogenicity in a tractable animal model with a well characterized challenge inoculum, and its sensitivity to widely-used RT inhibitors make the RT-SHIV model a valuable tool for modeling HIV-1 diversity and evolution of resistance to RT inhibitors. Our results showed that RT-SHIV populations in the infected macaques comprised both dominant and minor subpopulations. Similar genetic structures have been revealed by analysis of HIV-1 populations within and between different patient anatomical compartments [22, 23]. In most cases, we observed one or two dominant subpopulations and many minor subpopulations in each plasma sample. The dominant subpopulations usually accounted for at least 20% of each virus population.
All three macaques were treated identically, with short course EFV, followed by combination therapy 4 weeks later. Nevertheless, three different patterns of virological response were observed. In M03250, at least 4 subpopulations that encode EFV resistance appeared that contained either of the K103N alleles (AAT or AAC). This occurred following monotherapy. This animal subsequently failed combination therapy, at which time the virus population was characterized by the appearance of viruses with additional mutations, initially K65R (conferring TNF resistance) followed by a clonal subpopulation containing K103N and M184I (conferring FTC resistance), which rapidly became dominant. M04008 had a similar response to the initial monotherapy, with similar proportions of multiple subpopulations containing both AAT and AAC detected by week 17. However, the plasma viremia remained low in this animal, and no new subpopulations containing additional resistance mutations were detected during ART. Macaque M04007, by contrast, contained no subpopulations with drug resistance mutations, despite having been treated identically to the other two animals. M02350 had a much higher viral load than the other two macaques prior to therapy. Although the other two animals had similar viral loads at the time of monotherapy, M04008 had much higher viremia (more than 1 log) at weeks 2 to 10 post-infection. We hypothesize that the patterns observed reflect the relative population sizes of productively infected cells in these animals, with higher viremia in M03250 correlating with the presence and selection of multiple subpopulations of K103N variants by EFV monotherapy and the appearance of additional drug resistance mutations (K65R and M184I/V) within the population containing one of the K103N alleles. The amount of virus replication prior to week 13 in M04007 may have been too small for any K103N mutants to be present in the replicating population at that time or the frequency of K103N in week 13 was too low to be detected with our sampling size. However, this mutation was also not detected by allele specific real-time PCR (ASP) assay .
Subpopulations with both K103N alleles were present as early as one week after treatment with EFV in M03250 and M04008. These subpopulations comprised about 20% of the total virus population, as detected by allele specific PCR . However, none of these subpopulations increased in frequency and persisted as a stable subpopulation like the dominant wild-type subpopulation (sub3 in Figure 3) during combination therapy. This persistent stability indicated that a single drug resistance mutation either does not confer a significant selective advantage under this condition or a potential reduced replicative capacity caused by the drug resistance mutation [1, 24] allowed additional compensatory mutations to accumulate. For example, in M03250 at weeks 23 and 25, 6-8 weeks after initiation of combination therapy, 11 of 23 minor subpopulations contained K103N mutations and totaled 25-30% of the entire population. A similar phenomenon was observed in monkey M04008, which did not fail therapy. Therefore, even during drug treatment, when virus replication was suppressed, the dominant subpopulations were still wild type. For the most part, there was little change in the composition of the subpopulations during ART in the three animals. Prior to therapy failure, the dominant subpopulations were wild type even during ART. This analysis is supported by other reports, indicating that wild type virus may be preserved during therapy and reemerges after selective pressure is stopped (4)
The presence of a variety of RT-SHIV subpopulations containing K103N in M02350 and M04008 following EFV monotherapy (Additional files 1 and 2), which never became dominant, indicates that K103N alone did not confer a growth advantage to the virus in either the presence or absence of therapy. The existence of multiple minor subpopulations carrying either K103N AAC or AAT suggests that different subpopulations acquired them independently, rather than from a common ancestor, implying that a large effective population size must have been present pre-therapy. By contrast, the outgrowth of a single clonal subpopulation resistant to both EFV and FTC that resulted in therapeutic failure implies that the K103N population may have been so small that the M184I variant was present at a low frequency at the time of initiation of combination therapy. Similarly, a singe clonal population containing both K103N and K65R was present only briefly during combination therapy.
Remarkably, before the doubly resistant population became dominant in macaque M02350, a wild type subpopulation (sub4, Figure 3) present at low frequency before week 22 became the dominant species. Indeed its growth in the population between weeks 23 and 24 was both in relative terms (about 18 to 50% of all populations) and in absolute terms (1620 to 33810 c/ml, Figure 4), indicative of replication and not simply due to population shift. This subpopulation then declined rapidly (at least relatively) to 5% at week 25 and 2% at week 26. It could be that there were beneficial features not directly involved in drug resistance in this variant. Understanding the reason for this phenomenon will await further experimentation.
All monkeys in this study were inoculated with a cell culture supernatant containing RT-SHIV, which was a mixture containing a dominant subpopulation that accounted for 80% of the virus in the challenges. In the two monkeys, M03250 and M04008, this dominant cell supernatant subpopulation was rapidly replaced by new dominant subpopulations (Figures 3 and 5) characterized by the V75L mutation not detected in the inoculum. In M04007, the dominant cell supernatant subpopulation persisted throughout the study (Figure 6). The different fates of the challenge virus within the different animals are perhaps due to differences in host genetics or immunity. V75 is polymorphic in untreated HIV-1 infected patients and it has been suggested that its side chain stabilizes the fingers domain of RT and that its peptide backbone interacts with single-stranded DNA templates . It was also reported in other macaque RT-SHIV studies [26, 27], without quantitative analysis. While V75T causes resistance to dideoxyribonucleoside RT inhibitors , V75L has not been reported to be a drug resistance mutation. It has, however, been implicated as a secondary mutation for quinoxaline (an NNRTI inhibitor) in vitro . V75L appeared in that study after the introduction of the quinoxaline resistant mutation G190Q. In our study, V75L appeared before the emergence of any drug resistant mutations, and it spread to almost all subpopulations in later time points. This pattern suggests that V75L probably conferred a selective advantage to the virus on its own, rather than being secondary to known drug resistant mutations. In M04007 V75L was not detected at week 13 and the only V75L subpopulation found in week 17 did not persist or spread to other subpopulations at later time points, suggesting that the selective advantage it confers may be host specific. Since ultradeep sequencing showed that this mutation was present at less than 0.01% of the genomes in the inoculum, it must have arisen de novo and been selected in all three macaques. V75 has been shown to be within a human A3 supertype CTL epitope (Los Alamos HIV Immunology Database). Further studies are needed to investigate if this mutation is also within a macaque CTL epitope.
We observed a rapid increase in the frequency of another common polymorphism, L214F, in M03250 from 0% at week 0 to 41% at week 10, and 100% at week 25. The frequency of 214F increased much more slowly in M04008, and 214F was not observed at all in M04007. The 214F mutation is associated with nucleoside analogue mutation cluster 2 (D67N+K70R+K219Q+T215F) and negatively associated with nucleotide analogue mutation cluster 1 (M41L+L210W+T215Y) [30, 31]. Our data indicate that 214F might be associated with a negative virological response to NNRTI treatment because of its low frequency in M04008 and M04007, which responded well to the NNTRI treatment, and its rapidly increasing frequency in M03250, which failed the treatment. L214F was reported in previous RT-SHIV studies [26, 27], although no quantitative analysis was reported.