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Quantitative deep sequencing reveals dynamic HIV-1 escape and large population shifts during CCR5 antagonist therapy in vivo.

Tsibris AM, Korber B, Arnaout R, Russ C, Lo CC, Leitner T, Gaschen B, Theiler J, Paredes R, Su Z, Hughes MD, Gulick RM, Greaves W, Coakley E, Flexner C, Nusbaum C, Kuritzkes DR - PLoS ONE (2009)

Bottom Line: Profound baseline V3 loop sequence heterogeneity existed; predicted CXCR4-using populations were identified in a largely CCR5-using population.Greater V3 diversity was observed post-selection.This previously unreported degree of V3 loop sequence diversity has implications for viral pathogenesis, vaccine design, and the optimal use of HIV-1 CCR5 antagonists.

View Article: PubMed Central - PubMed

Affiliation: Massachusetts General Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
High-throughput sequencing platforms provide an approach for detecting rare HIV-1 variants and documenting more fully quasispecies diversity. We applied this technology to the V3 loop-coding region of env in samples collected from 4 chronically HIV-infected subjects in whom CCR5 antagonist (vicriviroc [VVC]) therapy failed. Between 25,000-140,000 amplified sequences were obtained per sample. Profound baseline V3 loop sequence heterogeneity existed; predicted CXCR4-using populations were identified in a largely CCR5-using population. The V3 loop forms associated with subsequent virologic failure, either through CXCR4 use or the emergence of high-level VVC resistance, were present as minor variants at 0.8-2.8% of baseline samples. Extreme, rapid shifts in population frequencies toward these forms occurred, and deep sequencing provided a detailed view of the rapid evolutionary impact of VVC selection. Greater V3 diversity was observed post-selection. This previously unreported degree of V3 loop sequence diversity has implications for viral pathogenesis, vaccine design, and the optimal use of HIV-1 CCR5 antagonists.

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Neighbor-joining (NJ) tree over time for subject 18.All unique V3 forms found are included, and the frequency of the 3 most common nucleotide forms indicated at each time point. The most common sequence at the first time point was used as an out-group for the trees. Week 0, yellow; week 2, red; week 16 (virologic failure), blue. We have indicated the location and frequency of the most common sequence at each time point, and the predominant lineage after selection.
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pone-0005683-g003: Neighbor-joining (NJ) tree over time for subject 18.All unique V3 forms found are included, and the frequency of the 3 most common nucleotide forms indicated at each time point. The most common sequence at the first time point was used as an out-group for the trees. Week 0, yellow; week 2, red; week 16 (virologic failure), blue. We have indicated the location and frequency of the most common sequence at each time point, and the predominant lineage after selection.

Mentions: Figure 3 shows a neighbor joining (NJ) tree of all unique forms found in subject 18, selected as a representative case; the NJ trees of the other 3 cases are provided in the supplement (Figure S3). This tree illustrates both the clear evolutionary trajectory of the virus over 16 weeks of sampling, and the complexity of the V3 loop at baseline. There were 1,910 unique variants in this 39-amino acid fragment of the virus in these samples. A remarkable diversity of forms was already present in the baseline sample. The majority of these forms are expected to represent true biological variants, rather than PCR or sequencing artifact, given the relatively small fraction of experimental error that remained in the sequences after processing (see above discussion and compare Fig 3 with the control tree shown in Fig S2). The most common form of the V3 loop at week 0 was found in only 0.4% of the sequences at week 2, and was lost by week 16. There was significant selection for a single lineage at the second time point (week 2), with one X4 sequence from that lineage making up 91% of the sample; this form of the V3 loop continued to be the most common form at the third time point (week 16), persisting at 70%. Interestingly, despite a genetic bottleneck imposed by VVC, many minor related variants within the selected lineage persisted at low levels and continued to evolve alongside the dominant X4 form. This point can be visualized in the tree by the acquisition and building of new variants from the many variants present at week 0 within the selected lineage, and by the left to right progression from week 0 to 2 to 16 (yellow to red to blue) within that lineage. Two distinctive novel lineages were detected at week 16, constituting 4% and 5% of the sample, respectively. The branch lengths after VVC selection are relatively long compared to relationships among baseline sequences. These findings suggest that rather than rapidly replacing the susceptible form of the virus with a single X4 variant, new levels of diversity in the V3 loop were explored under the selective pressure of VVC. Many variants were carried forward and continuing evolutionary pressure drove de novo exploration of the sequence space.


Quantitative deep sequencing reveals dynamic HIV-1 escape and large population shifts during CCR5 antagonist therapy in vivo.

Tsibris AM, Korber B, Arnaout R, Russ C, Lo CC, Leitner T, Gaschen B, Theiler J, Paredes R, Su Z, Hughes MD, Gulick RM, Greaves W, Coakley E, Flexner C, Nusbaum C, Kuritzkes DR - PLoS ONE (2009)

Neighbor-joining (NJ) tree over time for subject 18.All unique V3 forms found are included, and the frequency of the 3 most common nucleotide forms indicated at each time point. The most common sequence at the first time point was used as an out-group for the trees. Week 0, yellow; week 2, red; week 16 (virologic failure), blue. We have indicated the location and frequency of the most common sequence at each time point, and the predominant lineage after selection.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682648&req=5

pone-0005683-g003: Neighbor-joining (NJ) tree over time for subject 18.All unique V3 forms found are included, and the frequency of the 3 most common nucleotide forms indicated at each time point. The most common sequence at the first time point was used as an out-group for the trees. Week 0, yellow; week 2, red; week 16 (virologic failure), blue. We have indicated the location and frequency of the most common sequence at each time point, and the predominant lineage after selection.
Mentions: Figure 3 shows a neighbor joining (NJ) tree of all unique forms found in subject 18, selected as a representative case; the NJ trees of the other 3 cases are provided in the supplement (Figure S3). This tree illustrates both the clear evolutionary trajectory of the virus over 16 weeks of sampling, and the complexity of the V3 loop at baseline. There were 1,910 unique variants in this 39-amino acid fragment of the virus in these samples. A remarkable diversity of forms was already present in the baseline sample. The majority of these forms are expected to represent true biological variants, rather than PCR or sequencing artifact, given the relatively small fraction of experimental error that remained in the sequences after processing (see above discussion and compare Fig 3 with the control tree shown in Fig S2). The most common form of the V3 loop at week 0 was found in only 0.4% of the sequences at week 2, and was lost by week 16. There was significant selection for a single lineage at the second time point (week 2), with one X4 sequence from that lineage making up 91% of the sample; this form of the V3 loop continued to be the most common form at the third time point (week 16), persisting at 70%. Interestingly, despite a genetic bottleneck imposed by VVC, many minor related variants within the selected lineage persisted at low levels and continued to evolve alongside the dominant X4 form. This point can be visualized in the tree by the acquisition and building of new variants from the many variants present at week 0 within the selected lineage, and by the left to right progression from week 0 to 2 to 16 (yellow to red to blue) within that lineage. Two distinctive novel lineages were detected at week 16, constituting 4% and 5% of the sample, respectively. The branch lengths after VVC selection are relatively long compared to relationships among baseline sequences. These findings suggest that rather than rapidly replacing the susceptible form of the virus with a single X4 variant, new levels of diversity in the V3 loop were explored under the selective pressure of VVC. Many variants were carried forward and continuing evolutionary pressure drove de novo exploration of the sequence space.

Bottom Line: Profound baseline V3 loop sequence heterogeneity existed; predicted CXCR4-using populations were identified in a largely CCR5-using population.Greater V3 diversity was observed post-selection.This previously unreported degree of V3 loop sequence diversity has implications for viral pathogenesis, vaccine design, and the optimal use of HIV-1 CCR5 antagonists.

View Article: PubMed Central - PubMed

Affiliation: Massachusetts General Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
High-throughput sequencing platforms provide an approach for detecting rare HIV-1 variants and documenting more fully quasispecies diversity. We applied this technology to the V3 loop-coding region of env in samples collected from 4 chronically HIV-infected subjects in whom CCR5 antagonist (vicriviroc [VVC]) therapy failed. Between 25,000-140,000 amplified sequences were obtained per sample. Profound baseline V3 loop sequence heterogeneity existed; predicted CXCR4-using populations were identified in a largely CCR5-using population. The V3 loop forms associated with subsequent virologic failure, either through CXCR4 use or the emergence of high-level VVC resistance, were present as minor variants at 0.8-2.8% of baseline samples. Extreme, rapid shifts in population frequencies toward these forms occurred, and deep sequencing provided a detailed view of the rapid evolutionary impact of VVC selection. Greater V3 diversity was observed post-selection. This previously unreported degree of V3 loop sequence diversity has implications for viral pathogenesis, vaccine design, and the optimal use of HIV-1 CCR5 antagonists.

Show MeSH
Related in: MedlinePlus