HIV: Persistence through division

HIV infection continues to be a global epidemic, with almost 37 million individuals infected worldwide. Despite considerable effort, there is no vaccine and, once contracted, there is no cure for HIV infection (Escolano et al., 2017). HIV-1 infection can be suppressed for decades by combination antiretroviral therapy, which targets multiple steps in the viral life cycle. However, HIV-1 persists in a latent state as a stably integrated provirus, and it rapidly reemerges from this latent reservoir within 2–3 wk after therapy is interrupted. The reservoir is established very early in infection and has an estimated half-life of 44 mo, making it the major barrier to curing HIV-1 infection (Siliciano and Greene, 2011). How the reservoir is maintained for such long periods of time and the precise cellular and molecular nature of the reservoir are not well defined. In this issue of JEM, Hosmane et al. describe a new technique for investigating the reservoir and use it to uncover a fundamental element that contributes to its persistence. Understanding the reservoir has been a difficult challenge in part because replication-competent latent cells are only a small fraction of all infected cells. It is estimated that 0.1–10 × 10−6 CD4 T cells in individuals on suppressive combination therapy harbor intact latent viruses. The most accurate measurements are made by viral outgrowth assays (VOA), in which peripheral CD4 T cells from virally suppressed patients are activated to produce virus in vitro, and the number of latent cells quantified by limiting dilution. However, this assay can vary by up to 1 log (Laird et al., 2013), and in addition, it significantly underestimates the size of the latent reservoir because not all latent cells are reactivated to produce virus in vitro (Ho et al., 2013; Bruner et al., 2016). In addition, the cells circulating in blood may or may not be entirely representative of what could be a diverse latent compartment. Although faster and simpler assays that detect HIV-1 nucleic acids have been developed to measure the reservoir, they are problematic because only a very small percentage of the integrated viral DNA in antiretrovirally suppressed individuals is intact and able to produce infectious particles. Thus, DNA measurements that only account for a small segment of the virus cannot easily distinguish between intact and defective proviruses. Moreover, the defective proviruses can be transcribed, and therefore RNA-based assays are similarly flawed (Imamichi et al., 2016). Viral outgrowth assays indicate that most latent cells reside in resting CD4 memory T cells. This finding is consistent with the idea that HIV reservoir persistence is associated with long-lived CD4 T cells and with the observation that actively infected T cells die by apoptosis or pyroptosis (Doitsh et al., 2014). However, several recent studies, including Hosmane et al. (2017) in this issue, suggest that clonal proliferation of infected cells may also play a role in maintaining the reservoir (Maldarelli et al., 2014; Wagner et al., 2014; Cohn et al., 2015; Kearney et al., 2015; Simonetti et al., 2016). Examination of the HIV-1 integration sites in circulating CD4 T cells showed that a large number of HIV-1 proviruses are found in expanded clones of cells (Maldarelli et al., 2014; Wagner et al., 2014; Cohn et al., 2015). As might be expected based on the relative rarity of intact proviruses integrated into CD4 T cell DNA, the majority of the CD4 T cell clones examined contained defective proviruses (Imamichi et al., 2014; Cohn et al., 2015). However, a unique patient with a history of metastatic squamous cell carcinoma had what appeared to be an expanded clone of CD4 T cells carrying a replication-competent virus (Simonetti et al., 2016). Unfortunately, definitive proof of clonal expansion of CD4 T cells carrying latent virus could not be obtained in that case because the virus was integrated in a region of the genome that could not be mapped with certainty. Therefore, although the possibility of multiple independent integration events was very unlikely, it could not be formally ruled out. Given that isolation of latent cells is not yet feasible, Hosmane et al. (2017) and Lorenzi et al. (2016) independently developed methods that combine limiting dilution viral outgrowth with viral sequencing to simultaneously quantitatively and qualitatively characterize replication-competent viruses in the latent reservoir (QVOA; Lorenzi et al., 2016; Hosmane et al., 2017). By sequencing a large number of unique, replication-competent viruses, both groups found that >50% of the viruses emerging from the reservoir are identical to other viral isolates from the same individual. Moreover, the same groups of viruses could be found at two time points separated by 6 mo, indicating they are stable over time (Lorenzi et al., 2016). Because HIV-1 mutates rapidly, finding identical viruses in multiple single cells is strongly