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Lifespan of effector memory CD4+ T cells determined by replication-incompetent integrated HIV-1 provirus

Imamichi, Hiromia; Natarajan, Venb; Adelsberger, Joseph W.b; Rehm, Catherine A.a; Lempicki, Richard A.b; Das, Biswajitb; Hazen, Allisonb; Imamichi, Tomozumib; Lane, H. Clifforda

Author Information
doi: 10.1097/QAD.0000000000000223

Introduction

Achieving an understanding of the lifespan of human T cells and their subsets is of critical importance in achieving a better understanding of the functioning of the human immune system in health and disease [1–3]. Human CD4+ T cells can be separated into naive, central memory and effector memory subsets [4–6]. The conventional wisdom is that the naive CD4+ T cells are long-lived resting cells [7–9], whereas the subsets of memory CD4+ T cells are antigen-experienced cells with rapid rates of cell turnover [9–12]. In particular, the effector memory CD4+ T cells are thought to be terminally differentiated [3,6,13–17] and short-lived cells [18] with half-lives ranging from 3 to 6 days [11,12]. The present study takes advantage of the chance observation of the presence of a unique replication-incompetent provirus in the effector memory CD4+ T cells of an HIV-infected individual who has been followed for over 20 years at the clinic at the NIH to address the question of the lifespan of these cells. Following mapping of the integration site of this provirus to confirm that it represented a single integration event, we were then able to follow its path over a period of 17 years. Using this approach, we demonstrate that effector memory CD4+ T cells can persist for much longer than previously reported without any evidence of reversion to central memory or naive phenotypes during the time of observation.

Materials and methods

Study participants

All participants were enrolled in National Institute of Allergy and Infectious Diseases Institutional Review Board approved HIV-1 clinical research protocols and provided written informed consent before study participation. Study participant 1 is a 59-year-old white American man who came to the NIAID AIDS clinic in 1983 for experimental treatment of HIV-1 infection with the reverse transcriptase inhibitor Suramin. Since then, he has been continuously treated with a variety of experimental and licensed therapies. These are detailed in Fig. 1a and have included lymphocyte and bone marrow transfers from his HIV-seronegative syngeneic twin. His plasma HIV-RNA levels have been maintained at below 50 copies/ml since 1996, and total CD4+ cell counts have ranged between 236 and 1617 (median 887) cells/μl during the same time. Study participants 2–5 were on combination antiretroviral therapy (cART) and had maintained their HIV-RNA levels below 50 copies/ml for at least 1 year at the time of peripheral blood mononuclear cell (PBMC) collections in 1998 and 1999.

Fig. 1
Fig. 1:
Long-term persistence of an integrated HIV-1 provirus containing a pretermination stop codon at position 42 of the HIV-1 protease (W42Stop) in a patient with HIV-1 infection.(a) The clinical characteristics of the study participant and chromosomal locations of HIV-1 integration sites are shown. Four different proviruses in different integration sites (9q21, 6p15, 7q36 and 19q13) are highlighted in red, blue, purple and green, respectively. The numbers inside the parentheses indicate the frequency of the specific integration site detected at a given time point. (b) Frequencies of the W42Stop provirus in CD4+ T-cell subsets. The numbers inside the parentheses indicate the total number of HIV-1 protease gene sequences examined at a given time point. (c) The HIV-1 provirus is integrated in the opposite orientation to the SMC5 gene. The integration site is located within an intron between exons 11 and 12 of SMC5. Position of TGG (W)-to-TAG (Stop) change: W42Stop is at nt 2376 of HXB2. Bcl I cuts only once in most HIV-1 isolates at nt 2429 in the protease. (d) Sequence analysis of the inverse PCR products. A total of 17 sequences were obtained and all were identical. Result of highlighter analysis (Highlighter v2.2.1, www.hiv.lanl.gov) is shown.
Fig. 1
Fig. 1:
Long-term persistence of an integrated HIV-1 provirus containing a pretermination stop codon at position 42 of the HIV-1 protease (W42Stop) in a patient with HIV-1 infection.(a) The clinical characteristics of the study participant and chromosomal locations of HIV-1 integration sites are shown. Four different proviruses in different integration sites (9q21, 6p15, 7q36 and 19q13) are highlighted in red, blue, purple and green, respectively. The numbers inside the parentheses indicate the frequency of the specific integration site detected at a given time point. (b) Frequencies of the W42Stop provirus in CD4+ T-cell subsets. The numbers inside the parentheses indicate the total number of HIV-1 protease gene sequences examined at a given time point. (c) The HIV-1 provirus is integrated in the opposite orientation to the SMC5 gene. The integration site is located within an intron between exons 11 and 12 of SMC5. Position of TGG (W)-to-TAG (Stop) change: W42Stop is at nt 2376 of HXB2. Bcl I cuts only once in most HIV-1 isolates at nt 2429 in the protease. (d) Sequence analysis of the inverse PCR products. A total of 17 sequences were obtained and all were identical. Result of highlighter analysis (Highlighter v2.2.1, www.hiv.lanl.gov) is shown.

Purification of CD4+ T-cell subsets

Cryopreserved PBMCs were obtained from indvidual 1 between 1989 and 2009, and from individuals 2–5 between 1998 and 1999. CD4+ T cells were isolated from PBMCs by negative selection using CD4+ T-cell isolation kit II (Miltenyi Biotec Incorporated, Auburn, California, USA). CD4+ T-cell subsets (naive, central memory and effector memory) were obtained by FACS-sorting with the BD FACSAria II SORP (BD Biosciences, San Jose, California, USA) cell sorter on the basis of the expression of CD27 and CD45RO. The mAbs used were CD3 Pacific Blue (UCHT1); CD4 allophycocyanin (APC)-Cy7 (RPA-T4); CD45RO APC (UCHL1); and CD27 FITC (L128). All antibodies were obtained from BD Biosciences. Gating strategy used for the FACS-sorting of CD4+ T-cell subsets is depicted in Supplemental Figure 1, http://links.lww.com/QAD/A489.

Analysis of HIV-1 integration sites

Analysis of integration sites was performed by an inverse PCR method as previously described [19], except that BclI [20] was used for digestion of genomic DNA. Primer pairs used for the inverse PCR are listed in supplemental Table 1, http://links.lww.com/QAD/A489. The inverse PCR was performed with the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, Indiana, USA). Each round of PCR consisted of 25 cycles, with the initial denaturation at 94°C for 2 min, followed by 25 cycles of denaturation at 94°C for 15 s, annealing at 50°C for 30 s and extension at 72°C for 2 min, with the final extension at 72°C for 7 min. The amplicons captured the junction between the 5′ end of HIV-1 genome and host cell DNA. On average, 11 independent inverse PCR reactions (range: 3–34) were performed for each time point. The DNA was sequenced with the ABI BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA) and analysed with the ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). The human genomic sequence in each inverse PCR product was identified as a unique best-hit by BLAT search at the UCSC Bioinformatics Human Genome database (http://www.genome.ucsc.edu; Feb. 2009: GRCh37/hg19). The integration site in the SMC5 gene was independently confirmed by a custom-designed PCR that specifically amplifies the gene segment spanning the integration site in the host genome and the HIV-1 protease gene using the primer sets listed in supplemental Table 1, http://links.lww.com/QAD/A489.

Measurements of levels of HIV-DNA in CD4+ T-cell subsets

Limiting dilution analysis was performed on serial five-fold dilutions of CD4+ T-cell genomic DNA by a nested PCR method as previously described [21]. Location of the PCR primers is shown in Supplemental Figure 3, http://links.lww.com/QAD/A489. The number of HIV-DNA copies was calculated according to the Poisson distribution formula. The HIV-1 copies were then normalized to total genomic DNA input (assuming a cellular genome weights 6 pg) and expressed as HIV-1 copies per 106 CD4+ T cells.

Hypermutation analysis in the HIV-1 protease region of the pol gene

Sequences were screened for evidence of human APOBEC3G-mediated editing/hypermutation, defined as a mutational process in which G-to-A transitions far exceed all other mutations [22], using the HYPERMUT software (http://www.hiv.lanl.gov) [23].

Site-directed mutagenesis and preparation of virus stocks

Construction of an HIV-1 variant containing a stop codon (TGG-to-TAG change) at codon 42 (W42Stop) in the protease gene was conducted by site-directed mutagenesis as previously described [24,25]. Additional experimental details are included in the supplementary information, http://links.lww.com/QAD/A489.

Detection of HIV-1 proteins by western blot

Viral lysates for the wild-type and the W42Stop mutant were adjusted to yield equivalent levels of protein (2 μg). Antibodies used in the western blot assay were a rabbit anti-PR antibody (kindly provided from Dr Robert Gorelick, Leidos Biomedical Research Incorporated); mouse monoclonal antip24 antibody (PerkinElmer, Santa Clara, California, USA, NEA9306); mouse monoclonal anti-RT antibody (PerkinElmer; NEA9304); rabbit anti-INT (kindly provided from Dr Robert Gorelick); and goat anti-ENV antibody (Abcam, Cambridge, Massachusetts, USA, ab21179).

Electron microscope images

Prior to transfection, HEK293T cells (0.1 × 106 cells/well) were seeded onto six-well plates in DM-10 media and incubated at 37°C over night. Equal amounts (2.5 μg of DNA) of pNL4.3WT or pNL4.3W42Stop were used for transfection of HEK293T cells with 7.5 μl of the Transit LT. Approximately 24 h after transfection, the cells were fixed with PBS containing 2% glutaraldehyde and 0.1 mol/l sodium cacodylate trihydrate for 1 h at room temperature and then processed for electron microscopy analysis as previously described [26].

Assessment of replication competency of the wild-type and mutant HIV-1 recombinant viruses

Virus stocks for the wild-type and the mutant HIV-1 containing the W42Stop mutation were treated with DNase I (Life Technologies, Grand Island, New York, USA) to remove contaminating plasmid DNA. An aliquot of virus that was heat-inactivated at 99°C for 15 min served as a negative control for infection. A total of 8 × 106 PHA-stimulated CD4+ T cells were exposed to equal amounts of the wild-type or the W42Stop viruses (adjusted by virion protein levels to 100 ng) at 37°C for 2 h. At days 1 and 7 postinfection, cells were harvested and washed with ice-cold PBS. The cells were then treated with a 25 mmol/l glycine-150 mmol/l NaCl solution for 10 min on ice in order to remove any extracellular virions. The presence of the HIV-DNA in target cells was determined by PCR amplification of the HIV-1 protease gene as described above. Levels of p24 in culture supernatants were measured by a PerkinElmer HIV-1 p24 antigen ELISA kit.

Results

Long-term persistence of an integrated HIV-1 provirus containing a pretermination stop codon at position 42 of the HIV-1 protease

In 1998, while monitoring the presence of cell-associated HIV-RNA in PBMCs from HIV-infected individuals with plasma viral load less than 50 copies/ml to look for an evidence of residual viral replication, we coincidentally identified one individual whose cell-associated HIV-RNA contained an unusually high percentage (∼50% of amplicons examined) of a stop codon at position 42 of the HIV-1 protease. We also found the same stop codon, TGG (W, tryptophan) to TAG (stop) change, at the exactly same position in cell-associated HIV-DNA from the matching time point and named this provirus ‘W42Stop’. This provirus was found in PBMCs of this patient from 1991 to 2005. Given the fact that this naturally occurring replication-incompetent provirus would initially only have been able to infect a single cell, we sought to determine whether or not we could prove this assumption, and, if true, use this provirus to follow the fate of this cell and its progeny (Fig. 1a). Utilizing an inverse PCR technique [19], we were able to map the integration site to an intron between exons 11 and 12 of the SMC5 (Structural maintenance of chromosome 5) gene on chromosome 9 (9q21.12) (Fig. 1c). The same site was identified in 17 independent experiments utilizing samples that spanned 15 years (Fig. 1a and d). In contrast, analysis of integration sites for three other proviral species revealed three different chromosomal sites of integration: 6q15 (BACH2), 19q13 (RPS19) and 7q36 (intergenic region) (Fig. 1a). Having established that the W42Stop provirus had a single integration site and thus represented the marking of a single cell at a discrete point in time, we looked to see whether we could follow the fate of that cell and its progeny.

Naive, central memory and effector memory CD4+ T-cell subsets were examined for the presence of W42 and W42Stop HIV-DNA at various time points (Fig. 1b). HIV-DNA was detected in all three subsets examined. The frequency (copies/106 cells) of total HIV-DNA (integrated and unintegrated forms combined) was the highest in effector memory CD4+ T cells, followed by central memory, and the lowest in naive CD4+ T cells at most time points (Fig. 1b). The W42Stop provirus was only detected in the effector memory subset during the entire 17-year observational period (1993–2009). The frequency of effector memory cells harbouring the W42Stop was initially 100 copies/106 effector memory cells (0.01%) in 1993 and increased to 10 000 copies/106 effector memory cells (1% of the cells) in 1999, coincident with 3 years of effective cART (Fig. 2a and b). Following this time, the frequency of cells harbouring the W42Stop mutation underwent a logarithmic decay with a functional half-life of 11.1 months. Given that human effector memory CD4+ T cells have previously been reported to be short-lived (14–21 days) [18] with half-lives ranging from 3 to 6 days [11,12], our finding is quite surprising. Examination of the HIV-1 protease sequences (297 bp long) derived from the effector memory cells during 1993–2009 revealed that the nucleotide sequences for the W42Stop provirus (a total of 98 sequences) were identical (Supplemental Figure 1, http://links.lww.com/QAD/A489). This finding further confirms our hypothesis that the HIV-1 provirus with the W42Stop originated from a single event and was then replicated through somatic cell division.

Fig. 2
Fig. 2:
The frequency of effector memory CD4+ T cells harbouring the W42Stop mutation.(a) The total number of effector memory CD4+ T cells containing the W42Stop HIV-1 provirus at different points in time indicating a functional half-life of 11.1 months. (b) The total number of effector memory CD4+ T cells containing the W42Stop and change in plasma HIV-RNA over time.

The W42Stop mutation leads to a noninfectious form of HIV-1

To further prove that the W42Stop provirus did not encode for a replication-competent virus, we next conducted a series of experiments using site-directed mutagenesis to determine the impact of the W42Stop mutation on the replication competency of the encoded virus. A schematic diagram depicting the experiment design is shown in Fig. 3a. Unlike the wild-type virus with its dense core (Fig. 3b), the mutant viruses had a ring or doughnut-shaped structure, due to the failure of protease to cleave the gag-pol precursor (Fig. 3c). The protein compositions of the wild-type and the mutant viral particles were analysed by western blotting of sucrose-purified virions (Fig. 3d). The wild-type virions expressed the envelope, Gag-Pol precursor and cleaved Gag-Pol proteins. The mutant virions expressed envelope but only contained unprocessed and/or partially processed forms of Gag-Pol precursors. Thus, the lack of an intact HIV-1 protease in the W42Stop virions led to severe disruption of proteolytic processing of the Gag-Pol polyproteins. The proteolytic processing of the Env glycoprotein precursor, which is carried out by the host cell proteases, was unaffected in both the wild-type and the mutant virions. Of note was the observation that although RNA was contained in both wild-type and mutant viruses (Fig. 3e), only wild-type virus was able to replicate. The replication-competencies of the wild-type and the mutant viruses were assessed by culturing these viruses with CD4+ T cells and measuring levels of HIV-1 p24 antigen in the culture supernatants (Fig. 3f). No HIV-1 p24 antigen was detected in the culture supernatants of the mutant virus. The inability of the mutant virus to replicate was further confirmed by the absence of amplifiable HIV-DNA in the target CD4+ T cells (Fig. 3g). Taken together, these data demonstrated that the W42Stop virus is replication-incompetent.

Fig. 3
Fig. 3:
The W42Stop mutation leads to a noninfectious form of HIV-1.(a) A schematic diagram depicting the design of the experiments using recombinant NL4.3 viruses. Heat-inactivated viruses were used as a control for virus infection. (b, c) Electron microscopy images of the wild-type (b) and the W42Stop mutant (c) viruses. (d) Expression of HIV-1 proteins in the viral lysates of the wild-type and the W42Stop mutant viruses. Expected band sizes (kDa) of the indicated proteins on western blots are indicated. (e) Viral RNA was contained in both wild-type and mutant viruses. (f, g) Detection of HIV-1 p24 antigen in HIV-1 infected cell culture supernatants by ELISA (at day 7 postinfection); and the HIV-DNA in infected cells by nested PCR of the HIV-1 protease gene.

A high prevalence of lethal mutations in the peripheral blood pool of HIV-DNAs and cell-associated HIV-RNAs

Although the primary focus of this study was to examine the fate of an integrated provirus with a particular stop codon mutation in the protease gene (the W42Stop) in effector memory CD4+ T cells, we also observed through additional detailed sequencing that such genetic defects could be detected in HIV-1 proviral DNA in high percentages of all sequenced proviruses from peripheral blood lymphocytes. Analysis of the 297-bp HIV-1 protease gene amplified from the cells of the study participant (Individual 1) collected in 1997 revealed that 14% (three of 22) of HIV-DNA from naive CD4+ T cells and 38% (eight of 21) of HIV-DNA from central memory CD4+ T cells contained premature stop codons within this 297-bp region (Fig. 4). A detailed examination of the dideoxynucleotide content of the stop-codons within the region protease revealed that all of the premature stop codons detected in naive and central memory CD4+ T cells were consistent with the action of APOBEC-mediated hypermutation [27–29]. Examination of date-matched plasma samples found no evidence of premature stop codons within the plasma viral RNA sequences (Fig. 4). In individual 1, premature stop codons were detected as early as 1993 and persistently detected for over a 17-year period in all three CD4+ T-cell subsets examined (Fig. 5a). Overall, 21% of HIV-DNA sequences derived from this 3% of the HIV-1 provirus genome (Fig. 5c) in naive and central memory CD4+ T cells contained premature stop codons. An additional four patients (individuals 2–5), all of whom were on suppressive antiretroviral therapy with plasma HIV-RNA levels less than 50 copies/ml, were chosen for analysis of the presence of cell-associated HIV-RNA with genetic defects. All four harboured cell-associated HIV-1 RNA containing premature stop codons in this same 297-bp region at frequencies ranging from 10 to 21% (Fig. 5b). A fairly high prevalence of HIV-1 proviruses containing lethal mutations has also been reported by others in patients with various virological profiles [30–36]. Therefore, the occurrence of high levels of lethal mutations in the peripheral blood pool of HIV-1 proviruses is likely to be a generalized phenomenon.

Fig. 4
Fig. 4:
HIV-1 proviruses containing lethal mutations can be found in naive, central memory and effector memory CD4+ T cells.A total of 57 amino acid sequences of the HIV-1 protease derived from naive, central memory (CM), effector memory (EM) CD4+ T cells and plasma (Pla) of individual 1 in 1997. ‘Freq.’: the frequency of a given amino acid sequence. An asterisk (*): a premature stop codon. ‘HypMut’ indicates a group of sequences that contain an APOBEC-mediated hypermutation; ‘HypMut/Stop’ indicates a group of sequences that contain a premature stop codon introduced by an APOBEC-mediated hypermutation; and ‘W42Stop’ indicates a group of sequences that contain a TGG-to-TAG change at codon 42 of HIV-1 protease and lack the APOBEC-mediated hypermutation.
Fig. 5
Fig. 5:
Prevalence of lethal mutations in the peripheral blood pool of HIV-DNAs and cell-associated HIV-RNAs in HIV-infected patients with prolonged viral suppression.(a) Frequencies of sequences containing a premature stop codon in the HIV-1 protease gene derived from HIV-DNA of naive, central memory and effector memory CD4+ T cells of individual 1. The numbers inside the parentheses indicate the total number of the HIV-1 protease gene sequences examined at a given time point. An asterisk (*): time points at which the plasma HIV-RNA <50 copies/ml. The designations for ‘HypMut’, ‘HypMut/ Stop’ and ‘W42Stop’ are the same as those in Fig. 4. (b) Percentages of HIV-1 protease sequences derived from HIV-RNA in peripheral blood mononuclear cells (PBMCs) from four additional HIV-infected individuals (individuals 2–5). Sequences are from samples collected in 1998 and 1999. (c) Frequencies of HIV-1 quasispecies containing premature stop codons were determined on the basis of the protease gene (red rectangle), which is 297 bp and accounts for approximately 3% of the entire HIV-1 genome.

Discussion

In this study, taking advantage of an experiment of nature, we have demonstrated the long-term persistence of effector memory CD4+ T cells in a patient with HIV-1 infection and a lack of reversion of these cells to either central memory or naive CD4+ T cells during their decay. We also report the occurrence of high levels of lethal mutations in the peripheral blood pool of HIV-1 proviral DNAs.

Determining the lifespan of human T cells is challenging and the reports in the literature in this area are inconsistent [1–3]. The majority of studies have utilized pulse-chase assays with markers such as BrdU or 2H-glucose [8–12] or relied upon following cells bearing lethal chromosomal lesions induced by therapeutic radiation [7]. Although these methods are powerful in analysing kinetic behaviour of the labelled cells, they are unable to distinguish dividing cells from dying cells, as both lead to dilution of the marker. The present study took advantage of the chance observation of the presence of a unique replication-incompetent provirus in the effector memory cells of an HIV-infected individual who has been followed for over 20 years to address the question of the lifespan of these cells. Following mapping of the integration site of this provirus to confirm that it represented a single integration event, we were able to follow its path over a period of 17 years. As this virus had become part of the genome of the cell and faithfully replicated during each cell division, it allowed us to fully monitor the fate of this cell and its progeny from the time of proviral integration. Using this approach, we demonstrate that effector memory CD4+ T cells can persist over 17 years (longer than previous estimates of 2–3 weeks) [18]. In the present study, we estimated the functional half-life for the effector memory CD4+ T-cell clone to be 11.1 months. However, the 11.1-month half-life could be an overestimate if the effector memory clone was continuing to divide after 1999 when we detected decay of the clone. Currently, we do not have the ability to assess whether continuing cell division took place for the effector memory clone after 1999. As demonstrated by this study, measuring the exact ‘intermitotic’ half-life of individual clones remains a challenge. We also demonstrate that human effector memory CD4+ T cells undergo substantial clonal expansion and can exist for years without detectably reverting to naive or central memory phenotypes. It is theoretically possible that central memory CD4+ T cells residing in lymphoid tissue harbour this same provirus and thus we cannot definitively state that there has been no reversion from effector memory to central memory.

Given the temporal relationship between the expansion of this clone and levels of the HIV-1 provirus, it is likely that the target of this cell is HIV-1 or an accompanying opportunistic pathogen such as cytomegalovirus (CMV). These results indicate that active immune responses may in some instances persist over decades and are consistent with studies of primary CMV infection in renal transplants. In those studies, the frequency of CMV-specific CD4+ T cells increased from less than 0.05 to 1.24% (range: 0.21–1.60%, n = 7) during primary infection, and the circulating CMV-specific CD4+ T cells were predominantly an effector memory phenotype and persisted for at least 120 weeks [37].

An additional finding of interest was the identification of lethal mutations in HIV-1 peripheral blood proviral DNA occurring at a high frequency. It is important to point out that our study only examined 297 bp of a 9800-bp genome. Given that we observed that approximately 20% of proviruses harboured a stop codon within these 279 base pairs, only equivalent to 3% of the entire HIV-1 genome, it is quite plausible that far more proviruses would be found to harbour a stop codon or other genetic defects somewhere in their full-length HIV-1 genome. Many of these defective proviruses cannot encode a replication-competent virus but retain the ability to encode the production of RNA (as shown in this study) and protein. It is tempting to speculate that the ongoing production of these ‘foreign materials’ within CD4+ T cells, even in patients with plasma virus loads less than 50 copies/ml, may play some role in the ongoing immune activation that is characteristic of HIV-1 infection. Using extensive sequencing, it has been recently suggested that the size of the latent reservoir of HIV-1 in peripheral blood may be 60 times greater than previously thought [38]. Combined with our observation of long-term persistence of replication-incompetent integrated HIV-1 provirus and associated RNA transcripts, it is increasingly clear that estimation of the size of HIV-1 reservoir, the characterization of viral latency and the decay dynamics of HIV-1 may be more complex than currently appreciated. More detailed analyses utilizing genome-wide sequencing of HIV-1 may provide a clearer understanding of the genetic integrity of HIV-1 proviruses in the peripheral blood pool.

Acknowledgements

We thank Richard Davey, Colleen Hadigan, Joseph Kovacs, Frank Maldarelli and Mary Wright for providing patient specimens; Robin Dewar, Helene Highbarger, Akram Shah for running the Abbott HIV-1 assay; Xiaojun Hu and Da Wei Huang for helpful discussion; Robert Gorelick for providing antibodies for western blot; and Kunio Nagashima for electron microscopy analysis.

This work was funded through the intramural research programme of the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (Bethesda, Maryland) and in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the U.S. Government.

H.I. and H.C.L. designed the study, analysed the data and prepared this manuscript. H.I., T.I. and A.H. performed experiments. V.N., R.A.L. B.D. and T.I. contributed to the design of some experiments; J.W.A. performed T-cell sorting. C.A.R. led the identification of patient specimens.

Conflicts of interest

The authors declare no competing financial interests.

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Keywords:

genetic marker; HIV-1; in-vivo persistence of CD4-positive T lymphocytes; integration; provirus; replication-incompetent

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