HIV-1 DNA from cell samples taken at the initiation of antiretroviral therapy (ART) will be dominated by virus variants that have very recently infected short-lived cells (blue). The short-lived cells will die within a few months of therapy, and the HIV-1 DNA will be dominated by infected cells with longer life spans (red). The HIV-1 DNA in these cells will contain a larger fraction of sequences corresponding to virus that replicated earlier during the course of the infection. This shift in the cell populations can result in a false signal of evolution (Panel B). This illustration assumes that 90% of the infected cells were short-lived (half-life of 21 days). The remaining 10% of the cells were assigned a half-life of 350 days. To mimic the HIV-1 DNA samples obtained from the cells sampled at 0, 3, and 6 months after initiation of ART (the sampling times used by Lorenzo-Redondo et al. (2016), we randomly sampled sequences obtained from the HIV-1 RNA present before treatment (distribution illustrated in panel A). From these sequences, we calculated the apparent divergence of the pseudo-samples at 3 and 6 months from the pseudo-sample taken when the ART was started. There was large variation in this backward evolution signal among patients. Averaged over patients, the observed shift corresponded to a rate of evolution of 0.0025 substitutions per year.