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. 2011 Oct;7(10):e1002314.
doi: 10.1371/journal.ppat.1002314. Epub 2011 Oct 27.

Deep molecular characterization of HIV-1 dynamics under suppressive HAART

Maria J Buzón  1 , Francisco M Codoñer, Simon D W Frost, Christian Pou, Maria C Puertas, Marta Massanella, Judith Dalmau, Josep M Llibre, Mario Stevenson, Julià Blanco, Bonaventura Clotet, Roger Paredes, Javier Martinez-Picado
Affiliations

Deep molecular characterization of HIV-1 dynamics under suppressive HAART

Maria J Buzón et al. PLoS Pathog. 2011 Oct.

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phylogenetic tree of patient 1.
A neighbor-joining approach, as implemented in MEGA4, was used to construct a phylogenetic tree with the best evolutionary model found in jModeltest v0.1.1. Circles and squares represent longitudinal episomal and integrated DNA sequences, respectively. Legends of phylogenetic trees represent weeks available for further analysis. Sizes of the symbols represent the different percentages of clonal sequences. 1,000 bootstrap replicates were performed; only values greater than 50% are shown at tree nodes.
Figure 2
Figure 2. Phylogenetic tree of patient 2.
A neighbor-joining approach, as implemented in MEGA4, was used to construct a phylogenetic tree with the best evolutionary model found in jModeltest v0.1.1. Circles and squares represent longitudinal episomal and integrated DNA sequences, respectively. Legends of phylogenetic trees represents weeks available for further analysis. Size of the symbols represent the different percentages of clonal sequences. 1,000 bootstrap replicates were performed; only values greater than 50% are shown at tree nodes.
Figure 3
Figure 3. Phylogenetic tree and proportions of episomal and integrated HIV-1 sequences in patient 1.
A neighbor-joining approach, as implemented in MEGA4, was used to construct a phylogenetic tree with the best evolutionary model found in jModeltest v0.1.1. a. Circles represent longitudinal episomal DNA sequences. b. Squares represent longitudinal integrated DNA sequences. Legend of phylogenetic trees represents weeks available for further analysis. Sizes of the symbols represent the different percentages of clonal sequences. c. Longitudinal representation of the clonal variability of each episomal sample. d. Longitudinal representation of the clonal variability of each integrated sample. Areas of pie charts in white shading indicate sequences present with a frequency below 1%; gray shading indicates sequences with a frequency above 1% present in only one sample throughout the study period; colors represent sequences with a frequency above 1% present in two or more samples throughout the study period. 1,000 bootstrap replicates were performed; only values greater than 50% are shown at tree nodes.
Figure 4
Figure 4. Phylogenetic tree and proportions of episomal and integrated HIV-1 sequences in patient 2.
A neighbor-joining approach as implemented in MEGA4 was used to construct a phylogenetic tree with the best evolutionary model found in jModeltest v0.1.1. a. Circles represent longitudinal episomal DNA sequences. b. Squares represent longitudinal integrated DNA sequences. Legend of phylogenetic trees represents weeks available for further analysis. Sizes of the symbols represent different percentage of clonal sequences. c. Longitudinal representation of clonal variability of each episomal sample. d. Longitudinal representation of clonal variability of each integrated sample. Areas of pie charts in white shading indicate sequences present with a frequency below 1%; gray shading indicates sequences with a frequency above 1% present in only one sample throughout the study period; colors represent sequences with a frequency above 1% present in two or more samples throughout the study period. 1,000 bootstrap replicates were performed; only values greater than 50% are shown at tree nodes.
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