Current address: Infectious Disease Division, Massachusetts General Hospital, and Ragon Institute, Boston, Massachusetts, United States of America
Current address: Lifesequencing S.L., Parc Cientific Universitat de Valencia, Paterna, Valencia, Spain
Conceived and designed the experiments: MJB JB RP JMP. Performed the experiments: MJB MM CP. Analyzed the data: MJB FMC SDWF. Contributed reagents/materials/analysis tools: MCP JD JML. Wrote the paper: MJB MS BC JMP.
The authors have declared that no competing interests exist.
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 (
In the majority of HIV-1 positive patients, antiretroviral therapy (ART) effects a sustained reduction in plasma viremia to below detectable levels. Despite this, replication competent viruses persist and fuel viremia if antiretroviral treatment is interrupted. This viral persistence stands in the way of viral eradication through ART. While this ability to persist in the face of therapy is generally considered to be attributable to a reservoir of latently infected cells, there is debate as to how this reservoir is maintained and in particular, whether there is replenishment of the reservoir by low level, residual replication. 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. Statistical methods showed molecular compartmentalization, among and within episomal and integrated HIV-1 DNA samples, and suggest that episomal DNA in PBMC originates from a cellular/anatomic reservoir that is not revealed by sequencing of proviral DNA in PBMC in this study. These, and other data, suggest that ongoing replication, which can be blocked by adding raltegravir, occurs from proviruses that are genetically distinguishable from those detected at >1% frequency in these circulating blood cells.
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. However, viremia resumes if treatment is interrupted. Therefore, HIV-1 is able to persist in the face of suppressive ART. In addition low-level residual viremia has been detected with ultrasensitive assays that are able to measure down to several copies of HIV RNA/ml plasma
Intensification protocols employing integrase inhibitors have been used to probe the viral reservoirs that persist in ART-suppressed patients. When viral integration is inhibited, the linear viral genome, which is the precursor to the integrated provirus, is converted to episomes
The study sample comprised two participants from our previously reported raltegravir-intensification study
We constructed a phylogenetic tree for each patient to assess if episomal and integrated HIV-1 DNA sequences belonged to different genetic populations or to one intermixed population. We used a neighbor-joining approach, as implemented in MEGA4
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.
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.
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1 | W0 | 0.0091 | <0.0001 | 0.7287 | <10−6 | 1 | 0.105 |
W2 | 0.0019 | <0.0001 | 0.9889 | <10−6 | 1 | 0.056 | |
W4 | 0.0265 | 0.0063 | 0.1891 | <10−6 | 2 | 0.026 | |
W12 | 0.0313 | 0.0019 | 0.8412 | <10−6 | 2 | 0.033 | |
W24 | 0.0422 | 0.0198 | 0.5160 | <10−6 | 4 | 0.578 | |
Total | 0.0278 | 0.0201 | 0.1684 | <10−6 | 12 | 0.001 | |
2 | W0 | 0.0057 | 0.0081 | 0.1679 | <10−6 | 5 | 0.584 |
W2 | <0.0001 | 0.0067 | 0.1092 | <10−6 | 1 | 0.058 | |
W4 | <0.0001 | 0.0084 | 0.7680 | <10−6 | 1 | 0.057 | |
W12 | NA | 0.0054 | NA | NA | NA | NA | |
W24 | <0.0001 | NA | NA | NA | NA | NA | |
W48 | 0.0019 | NA | NA | NA | NA | NA | |
Total | 0.0076 | 0.0079 | 0.1457 | <10−6 | 9 | 0.001 |
Mean internal diversity for each time point and DNA source is shown for each patient. Genetic diversity (π), defined as the average number of nucleotide differences per site between any two sequences chosen randomly from the sample population, is calculated with the best evolutionary model found in the jmodeltest Tamura and Nei model
*Probability that a migration event is random after 10,000 resampling replicates.
NA, indicates that the test or the data is not applicable or available.
We next assessed whether longitudinal episomal and integrated DNA sequences had a temporal structure. Firstly, we constructed a separate neighbor-joining phylogenetic tree for each patient and each viral DNA form (
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 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.
AMOVA test - Episomal HIV-1 DNA | ||||||
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The longitudinal comparisons of patient 1 are indicated in the upper-right hemi-matrix of the table (
AMOVA test - Integrated HIV-1 DNA | ||||||
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Slatkin-Maddison test - Episomal HIV-1 DNA | ||||||
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The longitudinal comparisons of patient 1 are indicated in the upper-right hemi-matrix of the table (
Slatkin-Maddison test - Integrated HIV-1 DNA | ||||||
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The longitudinal comparisons of patient 1 are indicated in the upper-right hemi-matrix of the table (
Our results revealed that in both episomal and integrated HIV-1 DNA samples, distinct genetic populations appeared at different time-points, suggesting that the appearance of each viral DNA form in blood could be the result of stochastic mobilization of different HIV-infected cells. This effect has been observed for residual viremia
A population structure can occur at two levels: (i) different composition at the sequence level and (ii) different proportions of specific haplotypes. Therefore, the percentage of each clonal sequence of each DNA sample was represented (
Previous reports have shown evidence of compartmentalization between residual plasma viremia and proviruses in fractionated and unfractionated PBMC
The lack of population structure or evolution in integrated HIV-1 genomes is best explained by the fact that proviruses are predominantly defective and archival. Although the provirus is the molecular precursor for all virions, only a very small percentage of proviruses are replication competent and only a small percentage of these would exist in a latent state- one capable of producing replication-competent virions. Therefore, temporal structure might simply be a result of continuous seeding of new cells over time. In this regard, multiple monotypic HIV-1 sequences have been observed across the uterine cervix and in blood, presumably as a result of the proliferation of cells harboring proviruses
Proviral HIV sequences are currently thought to be representative of archival HIV infection in an infected patient. Based on this hypothesis, sources of residual viremia other than CD4+ T-cells have been postulated as long-lived viral reservoirs
Our results collectively suggest 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 a limited cellular/anatomic reservoir in which de novo infection continues during HAART because some antiretroviral drugs do not effectively inhibit replication in this compartment. However, evidence that episomes transiently increase after raltegravir intensification suggests that this cellular/anatomic reservoir may be accessible to raltegravir, in contrast to other drugs. If proven in future work, the concept that ongoing replication during successful HAART originates from proviruses that are not detectable in peripheral blood mononuclear cells has important implications for the design of strategies aimed at viral eradication or functional cure. It indicates the need to further define the limited and covert cellular/anatomic reservoir in which ongoing HIV replication may occur during suppressive HAART.
The study was approved by the Germans Trias i Pujol hospital review board and informed consent was obtained in writing from study participants.
We extensively analyzed longitudinal samples from 2 HIV-infected patients whose plasma viral load had been suppressed to <50 HIV-1 RNA copies/ml for 2 years on a stable HAART regimen. Both patients had participated in a previously reported raltegravir-intensification study
A median of 6×107 PBMC were obtained at weeks 0, 2, 4, 12, 24 and 48 after intensification and purified by Ficoll centrifugation and resuspended in 350 µl of P1 buffer (Qiaprep miniprep kit, Qiagen). 250 µl of cell suspensions were used for extrachromosomal HIV-1 DNA extraction (Qiaprep miniprep kit, Qiagen) using a modification for the isolation of low-copy-number plasmids. Total cellular DNA was purified from 100 µl of cell resuspension with a standard protocol (QIAamp DNA Blood Kit, Qiagen) as previously described
Analysis of HIV genomes from a sample containing a low copy number of HIV, such as PBMC from patients with undetectable viral load, can result in a high probability of resampling. The probability of resampling is related to both the number of target molecules during the amplification step and the number of sequenced clones. Therefore the higher the input of target molecules in the PCR and the higher the number of sequenced clones, the less likely the probability of resampling
We used a two-step PCR to amplify the RT region of episomal and integrated HIV-1 DNA. Primers
Pooled, purified PCR products were used as template to generate a single amplicon covering codons 150 to 250 from the RT region. The amplicon library was generated in triplicate during 20 cycles of PCR amplification (Platinum Taq DNA Polymerase High Fidelity, Invitrogen, Carlsbad, CA) followed by pooling and purification of triplicate PCR products using magnetic beads (Agencourt AMPure Kit (Beckman Coulter, Benried, Germany) to eliminate primer-dimers. The number of molecules was quantified by fluorometry (Quant-iT PicoGreen dsDNA assay kit, Invitrogen, Carlsbad, CA). The quality of each amplicon was analyzed by spectrometry using a BioAnalyzer (Agilent Technologies Inc., Santa Clara, CA). Deep Sequencing (DS) was performed in-house on a 454 Life Science/Roche platform. The error rate of the
454 DNA amplicon sequences were aligned with an HXB2 reference sequence using Muscle v3.7
jModeltest v0.1.1
In order to detect differences in sequence composition between episomal and integrated DNA at different time-points, we performed two analyses, (i) an analysis of molecular variance (AMOVA) as implemented in the Arlequin software package
(TIF)
We thank the IntegRal Collaborative Group: E Grau, R Ayen, T Gonzalez (IrsiCaixa Foundation, Badalona, Spain), R Escrig, I Bravo, J Puig (Fundació Lluita contra la SIDA, Badalona, Spain), M Larus, JM Gatell (Hospital Clínic-Idibaps, Barcelona, Spain), P Domingo (Hospital Sant Pau, Barcelona, Spain). Also, we thank Jorge Carrillo (IrsiCaixa Foundation, Badalona, Spain) for his technical support.