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HIV Cure: T Memory Stem Cells -
Hideouts Of HIV Against Any Antiviral Treatment - new
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Original article follows below
By Ryan Inoyori | January 13, 2014
T memory stem cells have been discovered as possible long-term viral HIV reservoirs that can be used as potential targets of advanced treatments. Experts found that these infected cells prevent antiviral therapy effectiveness even for several years of treatment.
T Memory Stem Cells Infection
Experts from Massachusetts General Hospital and the Ragon Institute of MGH, MIT and Harvard found where HIV can also hide inside the body, a small group of recently identified T cells with stem-cell like properties.
HIV damages the human race by attacking the immune cells and making patients vulnerable against opportunistic infections such as Kaposi's sarcoma and pneumonia. Antiviral drugs can only suppress replication but can't remove the viral load in cellular scale.
"Most human cells are short lived so it has been unclear how HIV manages to stick around for decades in spite of very effective antiviral treatment. This question led to the hypothesis that HIV might infect stem cells - the most long-lasting cells in the body - but traditional organ-specific stem cells, even those that give rise to all immune and blood cells, are resistant to HIV infection. We have discovered that a new group of T cells, called T memory stem cells, are susceptible to HIV and likely represent the longest lasting cellular niche for the virus," Dr. Mathias Lichterfeld of the MGH Infectious Disease Division said in a statement, as quoted by Eureka Alert.
Targeting Specific Cells
According to MGH/Ragon team, blood samples taken from patients under long-term antiviral treatment revealed levels of HIV DNA at the highest point inside T memory stem cells. Also, the amount of HIV DNA in these infected stem-cell like cells remained relatively stable over time even with long-term medication which means ART is ineffective in cellular level to kill the virus.
"Our findings suggest that novel, specific interventions will have to be designed to target HIV-infected T memory stem cells. Methods of inhibiting stem cell pathways are being studied to eliminate cancer stem cells - persistent cells that are responsible for tumour recurrence after conventional treatments kill proliferating tumour cells. We are now investigating whether any of the drugs that target cancel stem cells might be effective against HIV-infected T memory stem cells," Lichterfeld explained.
Exposing HIV reservoirs are very critical in the research for cure or vaccine which will enable new drugs to specifically target cells within and free them from viral load then ends the long-term, aching antiretroviral drugs therapy.
Discovering where HIV persists in spite of treatment
Recently discovered T memory stem cells may be long-term viral reservoir, potential targets for future treatment

January 12, 2014
By Sue McGreevey, Massachusetts General Hospital Public Affairs
HIV antiviral therapy lets infected people live relatively healthy lives for many years, but the virus doesn't go away completely. If treatment stops, the virus multiplies again from hidden reservoirs in the body. Now, investigators from the Harvard-affiliated Massachusetts General Hospital (MGH) and the Ragon Institute of MGH, MIT, and Harvard may have found HIV's viral hiding place in a small group of recently identified T cells with stem cell-like properties. "Most human cells are short-lived, so it has been unclear how HIV manages to stick around for decades in spite of very effective antiviral treatment," said Mathias Lichterfeld of the MGH Infectious Disease Division, an assistant professor at Harvard Medical School and corresponding author of a report on the findings receiving advance online publication in Nature Medicine.
Though HIV normally attacks immune system cells called T cells, those cells are short-lived. The virus' ability to survive years of therapy caused researchers to wonder whether it might also infect stem cells, regenerative cells that are the longest-lasting in the body, Lichterfeld said. The problem with that idea is that organ-specific stem cells are resistant to HIV infection.
"We have discovered that a new group of T cells, called T memory stem cells, are susceptible to HIV and likely represent the longest-lasting cellular niche for the virus," Lichterfeld said.
HIV's devastating impact on the human immune system is due to its habit of infecting the CD4-positive T cells that direct and support the infection-fighting activities of other immune cells. Subtypes of CD4 T cells have different functions and all are capable of being infected by HIV. With antiviral treatment, drugs keep the virus in infected cells from replicating; because most CD4 T cells are short-lived, they die relatively soon. CD4 T memory stem cells, however, can live for decades, and give rise to several types of T cells. This means HIV-infected T memory stem cells could continuously regenerate new HIV-infected cells, fueling HIV persistence in the body.
The MGH/Ragon team found that T memory stem cells express both CD4 and CCR5 the receptor proteins HIV uses to enter cells suggesting that these long-lived cells could be the much-sought HIV reservoir. They then found that thesecells can be readily infected with HIV, which was unexpected because traditional stem cells resist HIV infection. Importantly, the investigators found that levels of HIV DNA in patients receiving long-term antiviral treatment were highest in T memory stem cells.
Blood samples taken from patients soon after initial infection and several years later revealed that the viral sequences found in T memory stem cells after six to 10 years of treatment were similar to those found in circulating T cells soon after infection, indicating that HIV had persisted relatively unchanged in T memory stemcells. In addition, the amount of HIV DNA in thesecells remained relatively stable over time, even after long-term treatment caused viral levels to drop in other T cell subsets.
"Our findings suggest that novel, specific interventions will have to be designed to target HIV-infected T memory stem cells," said Lichterfeld. "Methods of inhibiting stem cell pathways are being studied to eliminate cancer stem cells persistent cells that are responsible for tumor recurrence after conventional treatments kill proliferating tumor cells. We are now investigating whether any of the drugs that target cancer stem cells might be effective against HIV-infected T memory stem cells.
"Identifying the reservoirs for HIV persistence is a critical step toward developing interventions that could induce a long-term remission without the need for antiviral medication, or possibly eliminate the virus entirely," he said. "Although a real cure for HIV has been elusive, it is not impossible." Maria Buzon of MGH Infectious Diseases and the Ragon Institute is lead author of the Nature Medicine paper. Additional co-authors are Hong Sun, Chun Li, Amy Shaw, Katherine Seiss, Zhengyu Ouyang, Enrique Martin-Gayo, Jin Leng, Florencia Pereyra, Xu Yu, and Bruce Walker, Ragon Institute; Eric Rosenberg, MGH Infectious Diseases Division; Timothy Henrich and Jonathan Li, Brigham and Women's Hospital; and Ryan Zurakowski, University of Delaware. Walker, the director of the Ragon Institute, is also a Howard Hughes Medical Institute investigator.
HIV-1 persistence in CD4+ T cells with stem cell-like properties
Nature Medicine Published online 12 January 2014
Maria J Buzon1,2, Hong Sun2,3, Chun Li2, Amy Shaw2, Katherine Seiss2, Zhengyu Ouyang2, Enrique Martin-Gayo2, Jin Leng2, Timothy J Henrich4, Jonathan Z Li4, Florencia Pereyra2,4, Ryan Zurakowski5, Bruce D Walker2,6, Eric S Rosenberg1, Xu G Yu2 & Mathias Lichterfeld1
1Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA. 2Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, USA. 3Key Laboratory of AIDS Immunology, Department of Laboratory Medicine, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning Province, China. 4Division of Infectious Diseases, Brigham and Women's Hospital, Boston, Massachusetts, USA. 5Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA. 6Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
Cellular HIV-1 reservoirs that persist despite antiretroviral treatment are incompletely defined. We show that during suppressive antiretroviral therapy, CD4+ T memory stem cells (TSCM cells) harbor high per-cell levels of HIV-1 DNA and make increasing contributions to the total viral CD4+ T cell reservoir over time. Moreover, we conducted phylogenetic studies that suggested long-term persistence of viral quasispecies in CD4+ TSCM cells. Thus, HIV-1 may exploit the stem cell characteristics of cellular immune memory to promote long-term viral persistence.
Antiretroviral combination therapy effectively suppresses HIV-1 replication, but replication-competent virus can persist in memory CD4+ T cells despite treatment1, 2. The memory CD4+ T cell compartment includes long-lasting central memory (TCM) cells, which undergo a sequential developmental program with progressive commitment to more differentiated, short-lived effector memory (TEM) and terminally differentiated T cell types3, 4. The presence of a more immature memory T cell population with stem cell-like properties has previously been hypothesized on the basis of animal studies5, 6, 7, 8, 9, and recently, small proportions of T cells with stem cell characteristics have been discovered in humans10, 11, mice12 and nonhuman primates13. These cells, termed TSCM cells, seem to represent the earliest and most long-lasting developmental stage of memory T cells, and can differentiate into large numbers of TCM, TEM and terminally differentiated T cells while maintaining their own pool size through homeostatic self-renewal. We hypothesized that HIV-1 can use CD4+ TSCM cells as a preferred niche for promoting long-term viral persistence.
To test this concept, we initially investigated the susceptibility of CD4+ TSCM cells to HIV-1 infection. These experiments demonstrated that CD4+ TSCM cells, phenotypically defined as described in previous studies10, 14 and in Supplementary Figure 1, were approximately as susceptible as CD4+ TCM cells to infection with an R5-tropic HIV-1 isolate (Fig. 1a), although their surface expression of CCR5 was slightly lower (Supplementary Fig. 2a,b). In addition, CD4+ TSCM cells were highly susceptible to infection with a vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1 viral particles (Fig. 1a and Supplementary Fig. 3), despite their comparatively low expression of T cell activation makers (Supplementary Fig. 4). We also observed that HIV-1 RNA was readily detectable in CD4+ TSCM cells from untreated HIV-1-infected patients (Supplementary Fig. 2c). CD4+ TSCM cells had low sensitivity to the cytopathic effects associated with HIV-1 infection (Supplementary Fig. 2d) and expressed reduced levels of the cell-intrinsic HIV-1 restriction factors TRIM5α, APOBEC3G and SAMHD1 (Supplementary Fig. 2e). Together, these data indicate that CD4+ TSCM cells are permissive to HIV-1 infection and can serve as physiologic target cells for HIV-1.
We next determined the levels of HIV-1 DNA in sorted CD4+ TSCM cells from HIV-1-infected patients who had been treated with suppressive highly active antiretroviral therapy (HAART) for a median of 7 years (Supplementary Table 1). The proportions of CD4+ TSCM cells in these patients did not differ from those in an HIV-1-negative control cohort (Supplementary Fig. 5). In these HAART-treated patients, per-cell levels of HIV-1 DNA were highest in CD4+ TSCM cells, but their average contribution to the total viral reservoir in CD4+ T cells was only approximately 8% (Fig. 1b). Notably, in this cross-sectional analysis, the contribution of CD4+ TSCM cells to the total viral reservoir in CD4+ T cells varied considerably among different HAART-treated patients and was inversely associated with HIV-1 DNA levels in the entire CD4+ T cell compartment (Fig. 1c). We observed this negative association selectively in the CD4+ TSCM cell compartment (Supplementary Fig. 6), and it resulted in a disproportionately increased contribution of CD4+ TSCM cells to the total viral CD4 T+ cell reservoir in patients with a smaller viral reservoir in CD4+ TCM and TEM cells.
This suggests that HIV-1-infected CD4+ TSCM cells represent a not necessarily large but very stable and durable component of the viral CD4+ T cell reservoir that becomes increasingly prominent when viral reservoirs in alternative CD4+ T cell subsets are limited. HIV-1 DNA was also detectable in CD4+ TSCM cells from elite controllers, a small group of HIV-1-infected individuals who maintain undetectable levels of HIV-1 replication in the absence of antiretroviral therapy15, although at significantly lower levels than in CD4+ TSCM cells from HAART-treated patients (Supplementary Fig. 7).
As only a small proportion of CD4+ T cell-associated HIV-1 DNA encodes replication-competent virus16, we performed viral outgrowth assays from three study subjects who had been on continuous suppressive antiretroviral combination therapy with HAART for a median of 28 months (range 14-42 months). We were able to retrieve replication-competent virus from CD4+ TSCM cells in all three cases, and the estimated frequency of cells harboring replication-competent HIV-1 in CD4+ TSCM cells exceeded the corresponding frequencies in CD4+ TCM and TEM cells in two of the three patients (Fig. 1d). These findings indicate that HIV-1 DNA in CD4+ TSCM cells is functionally capable of resuming active viral gene expression.
Given their stem cell-like properties, CD4+ TSCM cells may represent a privileged site for long-term viral persistence. To better investigate this, we longitudinally analyzed HIV-1 DNA in sorted CD4+ T cell subsets from eight individuals who started antiretroviral therapy during primary infection and then remained on suppressive antiretroviral therapy without treatment interruptions. Using pair-wise comparisons between cell-associated HIV-1 DNA during earlier stages of antiretroviral therapy (median of 1 year, range: 10-14 months) and during later stages of treatment (median of 9 years, range 7-11 years), we observed stable or mildly decreasing viral DNA levels in CD4+ TSCM cells; the viral DNA decline in CD4+ TCM and naive CD4+ T cells was slightly more pronounced (Fig. 1e). In contrast, in the more short-lived CD4+ TEM and terminally differentiated T cell populations, we noticed a significant reduction in per-cell levels of total HIV-1 DNA over time (Fig. 1e). Notably, among all CD4+ T cell subsets, the relative longitudinal decline in total HIV-1 DNA at per-cell levels was smallest in CD4+ TSCM cells, although differences between CD4+ TSCM cells and naive CD4+T cells and TCM cells did not reach statistical significance in our small study cohort (Fig. 1f). Of note, CD4+ TSCM cells made a relatively small contribution to the total CD4+ T cell reservoir after the first year of suppressive antiretroviral therapy (Fig. 1g). Yet, after long-term antiretroviral treatment, there was a significant increase in the contribution of CD4+ TSCM cells to the total viral reservoir in CD4+ T cells, despite the fact that the numeric contribution of CD4+ TSCM cells to the total CD4+ T cell pool did not change. The contribution of CD4+ TCM cells to the total viral CD4+ T cell reservoir also slightly increased over time, but this did not reach the level of statistical significance. In contrast, the contribution of CD4+ TEM cells to the viral CD4+ T cell reservoir declined, despite a numerically increased proportion of TEM cells in the total CD4+ T cell pool (Fig. 1g). These data, although collected from a limited number of patients, suggest that CD4+ TSCM cells can support long-term viral persistence in patients treated with HAART.
We subsequently sequenced the proviral Env gene in DNA samples isolated from longitudinally sorted CD4+ T cell subsets from three HIV-1-infected patients who did not receive antiretroviral therapy during the initial years of disease, followed by continuous treatment with suppressive antiretroviral agents (Fig. 2a). We observed substantial intraindividual variability between viral sequences from CD4+ TSCM cells collected at the beginning of antiretroviral therapy and several years later, probably reflecting sampling of cells infected with different circulating viral strains during early disease stages (Fig. 2b). Yet, in CD4+ TSCM and CD4+ TCM cells (which were sampled in approximately 10- to 30-fold higher frequencies than CD4+ TSCM cells), we noticed several identical HIV-1 sequences in samples collected at the beginning of antiretroviral therapy and after 4-8 years of continuous treatment, which is consistent with long-term viral persistence in these CD4+ T cell subsets (Fig. 2b). Notably, identical proviral sequences during early and later stages of antiretroviral therapy were not detected in naive or more terminally differentiated CD4+ T cell subsets. We subsequently analyzed phylogenetic relationships between proviral HIV-1 Env sequences from CD4+ TSCM cells and circulating viral RNA amplified from plasma samples collected during early untreated disease stages and from residual HIV-1 viremia at the time of suppressive antiretroviral therapy. Notably, we observed that among all viral sequences from CD4+ T cell subsets collected during suppressive antiretroviral therapy at later disease stages (6-12 years after infection), HIV-1 DNA isolated from CD4+ TSCM and TCM cells was phylogenetically most closely related to circulating plasma sequences from early infection; this suggests that the viral strains circulating in early disease seem more likely to persist long term upon infection of CD4+ TCM and TSCM cell subsets (Fig. 2c). In addition, pair-wise sequence comparisons revealed that the genetic distance between early plasma HIV-1 RNA sequences and HIV-1 DNA sequences from CD4+ T cell subsets collected during later stages of infection was lowest for HIV-1 DNA sequences from CD4+ TSCM and CD4+ TCM cells (Supplementary Fig. 8). Sequences from CD4+ TSCM cells also showed phylogenetic associations with contemporaneous and ensuing sequences isolated from plasma during suppressive antiretroviral therapy, which is consistent with a possible interchange between viral strains in CD4+ TSCM cells and circulating viral species (Fig. 2c). Finally, we noted viral sequences from CD4+ TSCM cells at early stages of antiretroviral therapy that were identical to those from CD4+ TCM, TEM and terminally differentiated T cells isolated several years later, supporting the role of CD4+ TSCM cells as precursor cells for more differentiated CD4+ T cell subsets (Fig. 2c). Although these phylogenetic studies were performed in a limited number of patients, they suggest that CD4+ TSCM and TCM cells may comprise a long-term reservoir for HIV-1.
This study indicates that CD4+ TSCM cells, despite their low frequencies, stand out among other memory CD4+ T cell subsets as the cell population in which long-term HIV-1 persistence is particularly evident, probably owing to intrinsic cellular programs of these cells that give them superior abilities to self-renew, resist apoptosis and survive for extremely long periods of time10, 13. Recently, stem cell-like functional properties were also observed in certain effector CD4+ T cell subsets, such as T helper type 17 cells17, and it will be important to analyze HIV-1 persistence in these long-lasting effector cell populations. Moreover, future studies will be necessary to determine whether a low viral reservoir in CD4+ TSCM cells represents a distinguishing feature of nonpathogenic simian immunodeficiency virus infection in natural hosts, as previously demonstrated for CD4+ TCM cells18. Interestingly, pharmaceutical inhibition of stem cell-specific molecular pathways is being investigated for targeting cancer stem cells19, and the specific targeting of cellular pathways responsible for the stem cell-like properties of CD4+ TSCM cells may also have adjunct or additive effects on reducing the persistence of HIV-1-infected CD4+ TSCM cells. Thus, our increasing understanding of how stem cell-like properties of cellular immune memory maintain HIV-1 persistence despite HAART may be translatable into improved clinical strategies for inducing HIV-1 eradication and cure20.

Peripheral blood mononuclear cell (PBMC) samples from HIV-infected individuals or HIV-1-negative study subjects were used for this study according to protocols approved by the Institutional Review Board of Massachusetts General Hospital in Boston. All study participants gave written informed consent.
Cell sorting and flow cytometry.
CD4+ TSCM cells and other CD4+ T cell subsets were isolated according to a previously described protocol14 with minor modifications. At least 100 million PBMCs were stained with monoclonal antibodies directed against CD4 (clone RPA-T4, BD Biosciences, 1:25 dilution), CD3 (clone UCHT1, Biolegend, 1:50 dilution), CD45RA (clone MEM-56, Life Tech, 1:50 dilution), CCR7 (clone 3D12, BD Biosciences, 1:25 dilution), CD62L (clone SK11, BD Biosciences, 1:25 dilution), CD122 (clone TU27, Biolegend, 1:25 dilution) and CD95 (clone DX2, BD Biosciences, 1:25 dilution). After 20 min, CCR7+CD45RA+ naive CD4+ T cells, CCR7+CD45RA-central memory CD4+ T cells, CCR7-CD45RA- effector memory CD4+T cells, CCR7-CD45RA+ terminally differentiated CD4+ T cells and CCR7+CD45RA+CD62L+CD95+CD122+CD4+ T memory stem cells were live-sorted in a specifically designated biosafety cabinet (Baker Hood), using a FACS Aria cell sorter (BD Biosciences) at 70 pounds per square inch. Cell sorting was performed by the Ragon Institute Imaging Core Facility at Massachusetts General Hospital and resulted in isolation of live lymphocytes with the defined phenotypic characteristics of >95% purity, as determined by three dedicated experiments in which sorted cells were subjected to repeat flow cytometric analysis (Supplementary Fig. 1b). For phenotypic characterization, cells were additionally stained with antibodies specific to CCR5 (clone 2D7/CCR5, BD Biosciences, 1:25 dilution) CXCR4 (clone 12G5, Biolegend, 1:25 dilution), CD38 (clone 90, Biolegend, 1:25 dilution) or HLA-DR (clone HIT2, Biolegend, 1:25 dilution) or annexin V and acquired on an LSRII flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (Treestar).
Assessment of cell-associated HIV-1 DNA.
Isolated CD4+ T cells were digested as previously described2 to extract cell lysates. We amplified total HIV-1 DNA with primers and probes previously described21. As a standard curve, we amplified serial dilutions of chronically infected 293T cells (kindly provided by F. Bushman). Proviral HIV-1 DNA copy numbers were calculated relative to CCR5 gene copy numbers according to standard procedures.
Analysis of cell-associated HIV-1 RNA.
Cell-associated HIV-1 RNA in sorted CD4+ T cells was quantified by real-time RT-PCR, using primers and probes previously described22. HIV-1 RNA copy numbers were determined according to a standard HIV-1 RNA sample run in serial dilutions, and final results were expressed as the number of HIV-1 RNA copies per microgram of total RNA. The assay used had a detection threshold of 1 HIV-1 RNA copy per μg of total RNA.
Gene expression analysis.
Expression of selected gene transcripts in individual CD4+ T cell subsets was analyzed by semiquantitative RT-PCR using Taqman gene expression assays with standardized primers and probes and normalized to the expression of the housekeeping gene Actb (encoding ß-actin) in each CD4+ T cell subset. In vitro HIV-1 infection assays.
Unselected PBMCs from HIV-1-negative donors were cultured in RPMI medium supplemented with 10% FCS and 50 U/ml of recombinant human interleukin-2 (rhIL-2). A total of 10x106 PBMCs were infected with a GFP-encoding, VSV-G-pseudotyped virus (multiplicity of infection (MOI) = 1, unless otherwise indicated) or a GFP-encoding R5-tropic viral strain (Ba-L, MOI = 1); both isolates were kindly provided by D. Littman. Cells were then washed twice with PBS and cultured at 200,000 cells/well in 96-well round-bottom plates for 5 d. On day 5, cells were stained with surface antibodies to identify individual CD4+ T cell subsets, washed and analyzed on a LSRII flow cytometer.
Analysis of HIV-1 replication products.
HIV-1 reverse transcripts were amplified from cell lysates with primers hRU5-F2 and hRU5-R and probe hRU5-P (early RT products) or with primers GagF1 and GagR1 and probe P-HUS-103 (late RT products)23. Integrated HIV-1 DNA was detected using nested PCR with Alu-1/Alu-2 primers and HIV-1 LTR primer L-M667 for the first-round PCR and LTR primer AA55M, Lambda T primers and MH603 probe for the second-round quantitative PCR, as described previously24. Serial dilutions of HIV-1 DNA from cell lysates of the HIV-1-infected cell line 293T (provided by F. Bushman, University of Pennsylvania) were used for reference purposes. Proviral HIV-1 DNA copy numbers were calculated relative to the CCR5 gene previously quantified with the same standard curve. 2-LTR HIV-1 DNA was quantified as previously described25.
Viral outgrowth assays.
Sorted CD4+ T cell populations were seeded at 10,000 cells/well (TSCM cells) or 20,000 cells/well (TCM and TEM cells) in round-bottom 96-well plates. Subsequently, cells were stimulated with PHA (2μg/ml), rhIL-2 (100 units/ml) and irradiated allogeneic PBMCs from HIV-negative healthy donors. CD8-depleted, PHA-stimulated PBMCs from HIV-negative donors were added to each well on day 3 and again on day 7 and 14 of culture. Latently HIV-1-infected ACH-2 cells (NIH AIDS Reagent Program) were run as positive control cells, and CD4+ cell-depleted PBMC samples from HAART-treated patients that were otherwise treated identically served as negative controls. The cultures were subjected to removal of 33% of the cell suspension every 7 d and replenished with fresh rhIL-2-containing medium. After 14-21 d, cell supernatant from each well was harvested, and the number of wells containing infectious HIV-1 was assessed by incubation of the supernatant with TZM-bl cells (NIH AIDS Reagent Program), a permissive HeLa cell clone that contains HIV-1 Tat-responsive reporter genes for firefly luciferase under control of the HIV-1 LTR, permitting sensitive and accurate measurements of infection. Luciferase activity was quantified by luminescence and is directly proportional to the number of infectious virus particles present in the initial inoculum. Estimated frequencies of cells with replication-competent HIV-1 were calculated using limiting-dilution analysis as described in ref. 26; all data were consistent with a single-hit Poisson distribution, as determined using a goodness-of-fit analysis based on a likelihood ratio test26.
Viral sequencing.
Cell lysates from sorted T cell populations and plasma were used for HIV-1 envelope sequencing encompassing the V3 region. For plasma samples, 6 mL of plasma from each time point were ultracentrifuged at 170,000g for 30 min before proteinase K digestion and RNA isolation by acid guanidiniumisothiocyanate. One-step RT-PCR reaction was then performed in triplicates using outer primers envA/LA11 (ref. 27). PCR products were used as a template to generate an amplicon by nested PCR with inner primers LA12 and LA13 (ref. 27). For V3 amplification from HIV-1 DNA in cell lysates, two-step nested PCR was performed with the same primer pairs. For amplification of HIV-1 RNA and DNA sequences, two to four separate reactions were conducted for each sample during first-round PCR; these PCR products were then pooled and used as templates for second-round PCR. Amplification products were inserted into TOPO cloning vectors and used to transform One Shot Stbl3 chemically competent E. coli (Life Technologies). Individual bacterial colonies were amplified by overnight culture, and extracted DNA was ligated and directly sequenced by T7 or T3 primers on an ABI 3100 PRISM automated sequencer, without prior PCR-based amplification. Sequences were aligned with an HXB2 reference sequence using BioEdit V7.1.9. A neighbor-joining method, as implemented in MEGA4 (ref. 28), was used to construct phylogenetic trees with phylogenetically informative HIV-1 nucleotide sequences. These sequences omit nucleotide mutations that occur only once and may therefore possibly be introduced by polymerase-induced errors during PCR29.
Phylogenetically informative sites were identified as described before29 (http://indra.mullins.microbiol.washington.edu/DIVEIN/insites.html). This conservative approach may slightly underestimate nucleotide diversity relative to single-template amplification methods, but a direct comparison between HIV-1 sequences derived by PCR/cloning and single-genome amplification in a number of our samples demonstrated equivalent population structure (Supplementary Fig. 9), consistent with prior studies30. For comparison purposes, viral sequences were analyzed by single-genome amplification according to a protocol described before31.
Statistical analyses.
Data are summarized as individual data plots with horizontal lines reflecting the median or as box and whisker plots indicating the median, interquartile range, and minimum and maximum values. Spearman's correlation coefficient was calculated to analyze correlations. Differences were tested for statistical significance using Wilcoxon's rank-sum tests, Mann-Whitney U test, Kruskal-Wallis or Fisher's exact test, followed by Bonferroni's correction or Dunn's test for multiple comparisons where applicable.

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