Abstract
In order to design strategies for eradication of HIV-1 from infected individuals, detailed insight into the HIV-1 reservoirs that persist in patients on suppressive antiretroviral therapy (ART) is required. In this regard, most studies have focused on integrated (proviral) HIV-1 DNA forms in cells circulating in blood. However, the majority of proviral DNA is replication-defective and archival, and as such, has limited ability to reveal the dynamics of the viral population that persists in patients on suppressive ART. In contrast, extrachromosomal (episomal) viral DNA is labile and as a consequence is a better surrogate for recent infection events and is able to inform on the extent to which residual replication contributes to viral reservoir maintenance. To gain insight into the diversity and compartmentalization of HIV-1 under suppressive ART, we extensively analyzed longitudinal peripheral blood mononuclear cells (PBMC) samples by deep sequencing of episomal and integrated HIV-1 DNA from patients undergoing raltegravir intensification. Reverse-transcriptase genes selectively amplified from episomal and proviral HIV-1 DNA were analyzed by deep sequencing 0, 2, 4, 12, 24 and 48 weeks after raltegravir intensification. We used maximum likelihood phylogenies and statistical tests (AMOVA and Slatkin-Maddison (SM)) in order to determine molecular compartmentalization. We observed low molecular variance (mean variability ≤0.042). Although phylogenies showed that both DNA forms were intermingled within the phylogenetic tree, we found a statistically significant compartmentalization between episomal and proviral DNA samples (P<10−6 AMOVA test; P = 0.001 SM test), suggesting that they belong to different viral populations. In addition, longitudinal analysis of episomal and proviral DNA by phylogeny and AMOVA showed signs of non-chronological temporal compartmentalization (all comparisons P<10−6) suggesting that episomal and proviral DNA forms originated from different anatomical compartments. Collectively, this suggests the presence of a chronic viral reservoir in which there is stochastic release of infectious virus and in which there are limited rounds of de novo infection. This could be explained by the existence of different reservoirs with unique pharmacological accessibility properties, which will require strategies that improve drug penetration/retention within these reservoirs in order to minimise maintenance of the viral reservoir by de novo infection.
Highlights
In the majority of HIV-1 infected individuals antiretroviral therapy (ART) is able to sustain suppression of plasma viral load to undetectable levels (,50 copies HIV RNA/ml plasma) for sustained intervals
Novel antiviral agents targeting the viral integrase offer tools to explore the viral reservoirs that persist in the face of ART and we have shown that raltegravir perturbs these reservoirs as evidenced by an accumulation of episomal DNA upon rategravir intensification (Buzon et al, 2010)
Through ‘‘deep sequencing’’ technology, we have longitudinally analyzed the genotypes of HIV episomes and integrated HIV DNA to evaluate whether they represent interrelated sequences or whether they have distinct origins
Summary
In the majority of HIV-1 infected individuals antiretroviral therapy (ART) is able to sustain suppression of plasma viral load to undetectable levels (,50 copies HIV RNA/ml plasma) for sustained intervals. It has been suggested that low level viremia in ART-suppressed patients represents release of viral particles by long-lived latently infected CD4+ T-cells [3,4,5] or virions produced as a result of low-level, residual viral replication [6,7,8,9,10,11]. The nature of this residual viremia, remains poorly understood, mainly because the very low number of virions in plasma limits its molecular characterization [3,12,13]
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