Phylogenetic analysis of RT sequences from free plasma virus and from provirus in resting CD4⁺ T cells of patient 147. (A) Sampling timeline. After an initial blood draw for baseline analysis of plasma and latent reservoir sequences, the patient began a period of intensive sampling with visits every 2 to 3 days, as indicated by colored vertical lines and symbols. Note that each color represents five or six consecutive study visits. The number of clones obtained at each time point was roughly constant for each patient. A single follow-up sample was obtained 1 month after the intensive sampling period. (B) Maximum likelihood phylogenetic tree of RT sequences from plasma (triangles) and from resting CD4⁺ T cells (circles). The sequences represent codons 38 to 219 of RT. Clusters of five or more identical sequences are boxed. Individual taxa are numbered from top to bottom, and numbers are shown for taxa that illustrate coclustering of plasma and cellular sequences or release of wild-type virus into the plasma (Table 1). All sequences from patient 147 clustered together, away from those of other study patients and reference sequences (HXB2, JRFL, and SF2). Patient-specific clustering was observed for all patients studied. (C) RT genotype of plasma and cellular sequences. This patient was initially treated with AZT, 3TC, and indinavir for approximately 1 year. Then, 65 months prior to entry, he began a regimen consisting of EFV along with the PIs ritonavir (RTV) and saquinavir (SQV) and various nucleoside/nucleotide RT inhibitors (NRTIs). RTV was given at full dose. Although there were some treatment interruptions early on, the patient maintained good suppression of viremia on these regimens. At 15 months prior to entry, the patient began a regimen of EFV, RTV, and SQV along with the NRTIs 3TC and tenofovir (TDF). This was taken throughout the study. Colored boxes indicate the mutations conferring resistance to NRTIs (red) or NNRTIs (blue). (D) Predicted phenotype of each RT sequence in the presence of RT inhibitors used in prior regimens or the regimen taken during the study period. Predictions were made using a well-characterized, rules-based algorithm developed by R. W. Shafer and colleagues (http://hivdb.stanford.edu/). Colors indicate predicted levels of resistance to the indicated drugs. d4T, stavudine.

Phylogenetic analysis of protease sequences from free plasma virus and from provirus in resting CD4⁺ T cells of patient 148. (A) Sampling timeline. (B) Maximum likelihood phylogenetic tree of protease sequences from plasma (triangles) and from resting CD4⁺ T cells (circles). Note that protease and RT sequences were obtained from independent PCRs. Therefore, different numbers of positive reactions were obtained, and these represented different viral genomes present in vivo. Thus, the shape of the tree is not identical to that of the RT tree shown in Fig. 2B. Nevertheless, there is a PPS that constitutes most of the residual viremia. (C) Protease genotype of plasma and cellular sequences. See the legend to Fig. 2 for treatment history. Colored boxes indicate the mutations conferring resistance to PIs. (D) Predicted phenotype of each protease sequence in the presence of PIs used in prior regimens or the regimen taken during the study period. IDV, indinavir; NFV, nelfinavir; SQV, saquinavir; RTV, ritonavir; APV, amprenavir.

Phylogenetic analysis of RT and env sequences from free virus and from provirus in various cell types in patient 154. (A) Sampling timeline. (B) Maximum likelihood phylogenetic tree of RT sequences from free virus in the plasma (triangles) and CSF (inverted triangles) and from provirus in resting CD4⁺ T cells (circles), activated CD4⁺ T cells (diamonds), monocytes (hexagons), and unfractionated PBMC (squares). Some resting cell sequences (indicated with an I through the circle) were obtained using a special assay that definitively detects integrated HIV-1 DNA that is competent for virion production following cellular activation (32). Sequences with documented R5 tropism are indicated (see Fig. S8 in the supplemental material). Three independent plasma sequences obtained from long amplicons that include both the RT and env genes are indicated. (C) RT genotype of plasma and cellular sequences. Prior to study entry, the patient took AZT monotherapy and then failed a regimen consisting of ddI, stavudine (d4T), and EFV. At 21 months prior to study entry, the patient started a suppressive HAART regimen consisting of AZT, 3TC, tenofovir (TDF), the nucleoside/nucleotide RT inhibitor (NRTI) abacavir (ABC), and the protease inhibitor lopinavir boosted with ritonavir. Colored boxes indicate the mutations conferring resistance to NRTIs (red) and NNRTIs (blue). Note that the top portions of Fig. 2C and D are truncated. However, no RT resistance mutations were present for taxa 1 to 45. (D) Predicted phenotype of each RT sequence in the presence of RT inhibitors used in prior regimens or the regimen taken during the study period. (E) Regions of the HIV-1 genome analyzed to show that the PPS in RT represents a PPC. Colored arrows indicate the locations of nested primers used for amplification of RT (red) and the C2-V4 region of env (blue). These two regions were linked by long-distance RT-PCR (green). Additional sequence information was obtained by amplification of the full-length env gene (orange). Products from this reaction were also used in phenotypic analysis of coreceptor usage. LTR, long terminal repeat. (F) Maximum likelihood phylogenetic tree of sequences of the C2-V4 region of the env gene from free virus in the plasma (triangles) and provirus in resting CD4⁺ T cells (circles). Three independent plasma sequences obtained from long amplicons that also include the RT gene are indicated. Sequences obtained by amplification of the full-length env gene are indicated by outlined triangles. Colors indicate sampling times, as shown in panel A.

Phylogenetic analysis of RT sequences from free plasma virus and from provirus in resting CD4⁺ T cells and monocytes of patient 148. (A) Sampling timeline. (B) Maximum likelihood phylogenetic tree of RT sequences from free virus in the plasma (triangles) and provirus in resting CD4⁺ T cells (circles) and monocytes (hexagons). Some resting cell sequences (indicated with an I through the circle) were obtained using a special assay that definitively detects integrated HIV-1 DNA that is competent for virion production following cellular activation (32). Clusters of five or more identical sequences are boxed. Individual taxa are numbered from top to bottom, and numbers for taxa with particular properties are shown. (C) RT genotype of plasma and cellular sequences. Prior to study entry, the patient was treated with monotherapy and dual-nucleoside analogue therapy involving AZT, stavudine (d4T), and 3TC for a total of 59 months. The patient then started a three-drug regimen consisting of d4T, 3TC, and indinavir but failed after the protease inhibitor was switched to nelfinavir. Then, 66 months before study entry, the patient began a suppressive regimen consisting of ddI, EFV, ritonavir (RTV), quinavir (SQV). Subsequently, the NNRTI was changed to nevirapine (NVP), and SQV was replaced with lopinavir boosted with RTV (LPR/r). The patient maintained excellent suppression of viremia throughout the study on ddI, NVP, and LPR/r. At approximately day 570, ddI was replaced with tenofovir (TDF) and 3TC. Colored boxes indicate the mutations conferring resistance to nucleoside/nucleotide RT inhibitors. No NNRTI resistance was seen. (D) Predicted phenotype of each RT sequence in the presence of RT inhibitors used in prior regimens or the regimen taken during the study period.

Phylogenetic analysis of RT sequences from free plasma virus and from provirus in resting CD4⁺ T cells of patient 135. (A) Sampling timeline. (B) Maximum likelihood phylogenetic tree of RT sequences from free virus in the plasma (triangles) and provirus in resting CD4⁺ T cells (circles). (C) RT genotype of plasma and cellular sequences. Prior to study entry, the patient was treated with AZT and stavudine (d4T) monotherapy for a total of 31 months and then with d4T and 3TC for 5 months. At 79 months prior to entry, the patient started a suppressive HAART regimen consisting of AZT, 3TC, ritonavir (RTV) (full dose), and saquinavir (SQV). At day 470, RTV and SQV were replaced with lopinavir boosted with ritonavir, and 2 months later, AZT was replaced with tenofovir. Nucleoside/nucleotide RT inhibitor (NRTI) resistance mutations are indicated by red boxes. No NNRTI resistance was seen. (D) Predicted phenotype of each RT sequence in the presence of RT inhibitors used in prior regimens or the regimen taken during the study period.

The PPC is underrepresented among integrated proviruses in resting CD4⁺ T cells from the blood and among replication-competent viruses in resting CD4⁺ T cells from the blood. (A) Comparison of plasma and resting CD4⁺ T-cell sequences from patient 154 at day 410. Resting cell sequences were analyzed by direct sequencing or by a method that detects only integrated proviruses (32). Plasma sequences matching the PPC in Fig. 5B are indicated. (B) Comparison of plasma sequences and replication-competent virus in resting CD4⁺ T cells from patient 154 at day 634. Plasma sequences matching the PPC in Fig. 5B are indicated.