HIV rebounds from latently infected cells, rather than from continuing low-level replication

Abstract

Rapid rebound of plasma viremia in patients after interruption of long-term combination antiretroviral therapy (cART) suggests persistence of low-level replicating cells or rapid reactivation of latently infected cells. To further characterize rebounding virus, we performed extensive longitudinal clonal evolutionary studies of HIV env C2-V3-C3 regions and exploited the temporal relationships of rebounding plasma viruses with regard to pretreatment sequences in 20 chronically HIV-1-infected patients having undergone multiple 2-week structured treatment interruptions (STI). Rebounding virus during the short STI was homogeneous, suggesting mono- or oligoclonal origin during reactivation. No evidence for a temporal structure of rebounding virus in regard to pretreatment sequences was found. Furthermore, expansion of distinct lineages at different STI cycles emerged. Together, these findings imply stochastic reactivation of different clones from long-lived latently infected cells rather than expansion of viral populations replicating at low levels. After treatment was stopped, diversity increased steadily, but pretreatment diversity was, on average, achieved only >2.5 years after the start of STI when marked divergence from preexisting quasispecies also emerged. In summary, our results argue against persistence of ongoing low-level replication in patients on suppressive cART. Furthermore, a prolonged delay in restoration of pretreatment viral diversity after treatment interruption demonstrates a surprisingly sustained evolutionary bottleneck induced by punctuated antiretroviral therapy.

Fig. 1.

Time relationship of rebound sequences to the MRCA. (Left) Results from phylogenetic trees (Fig. S2) containing the clonal sequences from pretreatment (pre) and from late time point after treatment stop (post). For each patient, the MRCA was inferred, and sampling times of the clones with the highest and lowest MRCA distance are indicated. (Right) Analysis of trees obtained from nine patients who were sampled intensively (Fig. S3) including also the clonal sequences obtained during the structured treatment interruptions (STI) and frequently thereafter. (Two identical clones from different time points had the highest distance to the MRCA in patient 106.)

Fig. 2.

Changes over time in viral diversity of clonal C2-V3-C3 sequences (A), average genetic distances (net divergence) from the pretreatment time point (B), and of average branch lengths to the MRCA (C) from pretreatment to the first, second to fifth interruption, 1–2 year time points, and to time points later than 2.5 years estimated by Tamura-Nei six-parameter model. Asterisks indicate the level of statistical significance (*, P < 0.05; **, < 0.01; ***, < 0.001; Mann–Whitney test with Bonferroni correction for multiple comparisons).

Fig. 3.

Predictions of coreceptor use in the aligned V3 loop amino acid sequences by position-specific scoring matrix (PSSM, subtype B SI/NSI matrix). Scores of individual clones are shown longitudinally from pretreatment (blue dots), during the short STI (green) to the last time point (red). Scores above approximately −2 are predictive of X4 coreceptor phenotype. Bars denote mean and SD.