Despite antiretroviral therapy (ART), a latent reservoir of replicationcompetent HIV-1 persists in resting memory CD4+ T cells and precludes a cure 1–6. Lorenzo-Redondo et al. 7 analysed HIV-1 sequences collected from three individuals during the first six months of ART, discovered specific patterns of sequence evolution, and concluded that viral replication persists during therapy. We believe that these evolutionary patterns are artefacts of rapidly decaying viral subpopulations present during the first months of therapy and are not characteristic of the long-lived reservoir. We therefore argue that ref. 7 does not provide evidence that ongoing replication is an additional barrier to a cure for treated individuals who consistently maintain low viral loads. There is a Reply to this Comment by Lorenzo-Redondo, R. et al. Nature 551, http://doi. org/10.1038/nature24635 (2017). Lorenzo-Redondo et al. 7 collected samples before and three and six months after treatment initiation, when labile viral populations dominate and change rapidly. Before treatment, most HIV-1 DNA in resting CD4+ T cells exists in an unintegrated state, decaying with a half-life of days 8, 9. Another major population of infected resting cells decays with a half-life of weeks 10. The latent reservoir of integrated proviruses, observed in blood and lymphoid tissue 1, is smaller and decays with half-life of approximately four years 5, 6. Lifelong persistence of this reservoir is determined by the longevity and proliferation of the infected cells 11. Initiation of ART blocks new infection from replenishing these populations, revealing their different lifespans. Differential decay causes marked shifts in infected cell populations in the first six months of ART, bringing into question any conclusions about viral evolution gleaned from this period. Below, we support our claim by simulating differential decay and replicating the analysis of ref. 7 on the simulated data. We find that false signals of viral evolution—and ongoing viral replication—often appear. To estimate the size and decay of labile compartments, we examined a cohort of seven early-treated individuals, which we consider comparable to the two early-treated participants in ref. 7. The quantitative viral outgrowth assay (qVOA) has been used on blood samples 10 to detect resting CD4+ T cells harbouring replication-competent HIV-1. At the onset of ART, infected cell frequencies greatly exceeded those of individuals on long-term ART. A multi-log, multi-phasic decay over the first year of therapy reduced frequencies to levels typically observed during long-term ART. Fitting to the most extensively sampled individual, we inferred a large, fast-decaying population, a smaller population with slower decay, and a very small, persistent reservoir, which was approximated as constant (Fig. 1). At zero, three, and six months after ART initiation, labile populations comprised 99.99%, 96.2%, and 76% of infected resting cells, respectively, masking the persistent reservoir. The RNA-based assays performed by Lorenzo-Redondo et al. 7 on lymphoid tissue paint a similar picture: the infection decays rapidly over the first three to six months, eventually dwindling to a more stable state that is more than three orders of magnitude smaller than the pretreatment population (extended data fig. 1 of ref. 7). Regardless of sequencing depth, the limited number of infected cells in a blood draw or tissue biopsy is likely to prevent the persistent reservoir from being sequenced at early time points. The genetic diversity of this reservoir emerges only later. Latency studies are therefore generally restricted to participants who have received suppressive ART for more than six months, a precaution not …