Abstract Highly active antiretroviral therapy (HAART) suppresses human immunodeficiency virus (HIV) replication to undetectable levels but cannot fully eradicate the virus because a small reservoir of CD4+ T cells remains latently infected. Since HIV efficiently infects only activated CD4+ T cells and since latent HIV primarily resides in resting CD4+ T cells, it is generally assumed that latency is established when a productively infected cell recycles to a resting state, trapping the virus in a latent state. In this study, we use a dual reporter virus—HIV Duo-Fluo I, which identifies latently infected cells immediately after infection—to investigate how T cell activation affects the estab-lishment of HIV latency. We show that HIV latency can arise from the direct infection of both resting and activated CD4+ T cells. Importantly, returning productively infected cells to a resting state is not associated with a significant silencing of the integrated HIV. We further show that resting CD4+ T cells from human lymphoid tissue (tonsil, spleen) show increased latency after infection when compared to peripheral blood. Our findings raise significant questions regarding the most commonly accepted model for the establishment of latent HIV and suggest that infection of both resting and activated primary CD4+ T cells produce latency.

Author Summary The study of HIV latency has been hindered because there are few latently infected cells in vivo, and we cannot distinguish latently infected cells from uninfected cells prior to reactivation of the latent provirus. In general, HIV latency is quantitatively studied by reactivating latently infected cells after latency has been established. However, this practice limits the investigation of how latency is established and how latent provirus can be reactivated. Our recently developed dual reporter virus, HIV Duo-Fluo I, can identify latently infected cells early after infection. In this study, we use HIV Duo-Fluo I to investigate how T cell activation affects the outcome of HIV infection.

Citation: Chavez L, Calvanese V, Verdin E (2015) HIV Latency Is Established Directly and Early in Both Resting and Activated Primary CD4 T Cells. PLoS Pathog 11(6): e1004955. https://doi.org/10.1371/journal.ppat.1004955 Editor: Michael Emerman, Fred Hutchinson Cancer Research Center, UNITED STATES Received: October 3, 2014; Accepted: May 13, 2015; Published: June 11, 2015 Copyright: © 2015 Chavez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: Funding for this project was provided by the National Institute on Drug Abuse (NIDA) Avant-Garde award program,1DP1 DA031126 (http://www.drugabuse.gov); NIDA, 1R01 DA030216; and the Collaboratory of AIDS Researchers for Eradication (CARE), UNC/NIH-Federal-5-31532 (https://www.delaneycare.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Once highly active antiretroviral therapy (HAART) became available in 1995, HIV infection was transformed from a deadly disease into a chronic lifelong condition [1]. The antiretroviral drugs used in HAART target multiple stages of the viral lifecycle, which can reduce patient viremia to undetectable levels [2–4]. However, HAART cannot eradicate HIV [5] because infected individuals harbor a small reservoir of latently infected cells that contain a transcriptionally silent but reactivatable provirus [6]. Because this latent reservoir prevents viral eradication, there is an urgent need to study and better understand the mechanisms of latency. HIV infection primarily targets CD4+ T cells, and the most extensively studied latent reservoir resides within resting CD4+ T cells [7–9]. During infection, HIV enters a target cell and reverse-transcribes its genomic viral RNA into a double-stranded cDNA that then enters the nucleus and integrates into the host genome, where it becomes controlled by the host transcriptional machinery. In most cases, integration of the viral genome leads to productive infection, in which viral genes are transcribed followed by virion production. However, in rare instances, latency occurs instead of productive infection and is characterized by a provirus that produces little-to-no viral transcripts [10]. Because the latently infected cell is not producing viral proteins, it escapes the viral cytopathic effects and is ignored by the immune system. Furthermore, since antiretroviral drugs only target active viral replication, they are ineffective against latent proviruses. Latent HIV is primarily found within memory CD4+ T cells, which have a long half-life in vivo [11, 12], allowing latent virus to persist within infected individuals for decades [13]. However, when latently infected memory CD4+ T cells encounter an antigen or are exposed to specific cytokines or chemokines, proviral transcription is activated, leading to productive infection [8, 14]. This “reactivation” is likely the cause of viral rebound after a patient stops HAART, and it explains why infected individuals must take antiretroviral drugs for life. HIV latency has proven difficult to study because latently infected cells are very rare in vivo (~1 in 1 × 106 cells) [11], and they cannot be distinguished from uninfected cells [15]. Despite these challenges, several in vitro latency models exist, which have led to important observations about how latently infected cells are maintained and reactivated (reviewed in references [16, 17]). However, it is not clear how the latent reservoir is established because current technologies only quantify latently infected cells by reactivating them from latency. We recently developed a dual reporter virus, HIV Duo-Fluo I, that can distinguish between cells that are productively infected, latently infected, or uninfected, and allows us to purify each population [18]. Using this new reporter virus, we can study the kinetics of HIV latency immediately after infection by employing two separate fluorescent markers: an LTR-driven eGFP marker (productive infection) and an LTR-independent mCherry marker (latent infection) driven by an EF1α promoter (Fig 1A). It should be noted that we use the term “productive infection” here and throughout the manuscript to indicate an infection resulting in the expression of the LTR-driven GFP reporter. Since the virus used in this manuscript is env-deficient, these infections are not truly productive. However, they are behaving like a productive infection in terms of virus expression levels. Using this dual reporter virus, we have studied how HIV latency is established with a unique focus on the role of T cell activation. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Resting primary CD4+ T cells support both productive and latent HIV infection. (A) Diagram of the HIV Duo-Fluo I virus, in which eGFP has replaced the nef gene, and a whole transcription unit—consisting of an EF1α promoter driving the expression of an mCherry fluorescent marker—has been inserted downstream. Upon infection with the HIV Duo-Fluo I virus, cells that express GFP alone or GFP and mCherry are considered productively infected; cells that express only mCherry are considered latently infected; cells that lack expression of either fluorescent marker are considered uninfected. (B) Expression of the activation markers CD69 and CD25 in resting primary CD4+ T cells either left untreated or stimulated with CCL19, IL-7, or αCD3/αCD28 activating beads for 72 h. Mean Fluorescence Intensity (MFI) for CD25 expression is also shown. (C) Infection profiles of untreated or stimulated primary CD4+ T cells 6 days after infection via flow cytometry. Untreated resting CD4+ T cells were infected with either HIV Duo-Fluo I virus alone or the Vpx-containing HIV Duo-Fluo I virus. Stimulated cells were infected with HIV Duo-Fluo I alone. Productive infection (GFP+ and GFP/mCherry double-positive) and latent infection (mCherry+) were analyzed by flow cytometry. Data shown are from a single donor but are representative of three separate donors. (D) Quantified values of latent infection and productive infection from panel C. Data represents the average of three donors. (E) Ratios of latent infection to productive infection were calculated using data from panel D. Data represent the average of three donors. (F) Quantified values for reactivation of pre-integration latent virus and post-integration provirus calculated from the isolated uninfected populations (GFP/mCherry double-negative) of untreated and stimulated primary CD4+ T cells via flow cytometry (S4 Fig). Six days after infection, uninfected cells were isolated via fluorescence-activated cell sorting (FACS) and were either left unstimulated or stimulated with αCD3/αCD28 activating beads alone or αCD3/αCD28 activating beads in the presence of raltegravir for 48 h. Reactivatable pre-integration latent virus was calculated by subtracting the amount of productive infection from cells treated with αCD3/αCD28 activating beads alone and cells treated with αCD3/αCD28 activating beads in the presence of raltegravir. Reactivatable post-integration latent provirus was calculated by subtracting the amount of productive infection from unstimulated cells and cells treated with αCD3/αCD28 activating beads in the presence of raltegravir. Data represent the average of three donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant. https://doi.org/10.1371/journal.ppat.1004955.g001 Based primarily on in vitro evidence, it is generally accepted that HIV predominantly replicates in activated CD4+ T cells [19–22]. Conversely, resting CD4+ T cells present several barriers to HIV infection (reviewed in reference [23]), as they do not support efficient nuclear import [24] or integration of the viral cDNA [22, 25]. However, the most notable obstacle to infection of resting CD4+ T cells occurs at the stage of reverse transcription [26, 27]. Resting CD4+ T cells do not support reverse transcription nearly as efficiently as activated cells because, at least in part, they contain the restriction factor SAMHD1 [28, 29]. Additionally, in vivo, most HIV-infected resting CD4+ T cells exhibit a memory phenotype, suggesting that they arose from the infection of previously activated CD4+ T cells. Based on this evidence, a leading theory postulates that latency is established from infected activated CD4+ T cells that revert back to a resting memory state. According to this model, the transition to a resting memory state is associated with a decrease in NFκB and pTEFb activity, two critical factors for HIV transcription, and with a concomitant silencing of the HIV genome [30]. However, for this type of latency to occur, the infected cell would have to survive the virus-induced cytopathic effects and the host immune response that usually kill productively infected cells very quickly (cells survive ~1 day) [31, 32]. Another possibility is that infection occurs at a “sweet spot” in the trajectory that activated CD4+ T cells taken from full activation to a fully rested state. This sweet spot would be characterized by permissivity for HIV reverse transcription and integration but not for HIV transcription [33]. Interestingly, previous studies have reported that resting CD4+ T cells can be directly infected, with the strongest evidence coming from in vivo and ex vivo studies of both SIV and HIV infection [34–39]. Most studies that show resting CD4+ T cells can be directly infected have been performed with cells isolated from primary lymphoid tissues. In vivo studies have found that resting CD4+ T cells in lymphoid tissue harbor viral RNA [35], and ex vivo studies have shown that directly infecting resting CD4+ T cells from lymphoid tissue results in productive infection [40]. Strikingly, a subsequent study found that resting CD4+ T cells in ex vivo lymphoid cells isolated from tonsillar tissue can support HIV infection, but purified CD4+ T cells isolated from that same lymphoid tissue could not [41], suggesting that the lymphoid tissue microenvironment is critical for rendering resting CD4+ T cells permissive to HIV infection. Indeed, several lymphoid tissue–associated factors, including cytokines [42], chemokines [43], extracellular matrixes [44], and cell surface markers [45], enhance HIV infection in resting CD4+ T cells. Therefore, HIV latency may be established by the direct infection of resting CD4+ T cells when they are exposed to soluble factors that do not induce classic T cell activation. In this study, we use the dual reporter virus, HIV Duo-Fluo I, to investigate the role of T cell activation on the establishment of HIV latency in primary CD4+ T cells. We also use HIV Duo-Fluo I to explore the theories of how HIV latency is established; namely, whether it occurs through infection of activated CD4+ T cells that return to a resting state or through the direct infection of resting CD4+ T cells. We find that both resting and activated primary CD4+ T cells can support both productive and latent infection. In the case of activated T cells, the latent state is established very early in the infection and is not significantly influenced by the return of that activated cell to a resting state. We further observed that the fraction of cells that become latent (latent/productive) is higher in resting CD4+ T cells than in activated CD4+ T cells.

Discussion The role that T cell activation plays in establishing HIV latency within CD4+ T cells is still not fully understood. HIV replication is clearly most efficient in activated CD4+ T cells [19–22], and the largest in vivo latent reservoir is within memory CD4+ T cells [11, 12]. This evidence suggests that HIV latency is established in one of two ways: 1) Activated CD4+ T cells become productively infected but survive viral cytopathic effects and evade elimination by the immune system long enough for the cell to transition to a resting memory state; or 2) CD4+ T cells that are transitioning from an activated to a resting memory state are infected by HIV while the cellular environment can still support integration of viral cDNA but cannot support proviral transcription. However, studies have shown that both naive and memory CD4+ T cells contain integrated viral DNA [34], and that direct infection of resting CD4+ T cells in lymphoid tissue results in productive infection [40]. These findings suggest that HIV latency can also be established in another way: direct infection of resting CD4+ T cells. In this study, we show that all three scenarios can produce latent HIV infection. We further show that HIV latency can be established in activated CD4+ T cells without them first returning to a resting state. Additionally, infecting activated CD4+ T cells is more likely to result in productive infection, while infecting resting CD4+ T cells is more likely to result in latent infection. Finally, HIV latency is more likely to occur in resting lymphoid cell aggregates than in resting CD4+ T cells cultured alone. Using primary CD4+ T cells isolated from the blood of uninfected donors, we demonstrate that infecting resting and activated CD4+ T cells with our HIV Duo-Fluo I virus causes both productive and latent infection in the two populations. In activated CD4+ T cells, HIV latency is established within the first few days of infection and does not require the cell to return to a resting state. We showed this previously [18], as did another group that developed a similar dual-reporter virus [54]. That construct uses a different LTR-independent promoter (CMV) than our EF1α promoter, and it places the LTR-driven eGFP cassette in the Gag region, while ours replaces the Nef open reading frame. Despite these differences, both dual-reporter viruses can detect latent infection events in activated CD4+ T cells early after the initial infection, and these latently infected cells can be reactivated by different stimuli. Additionally, we sorted these latently infected cells and showed that they still express significant amounts of both CD69 and CD25 activation markers; the cells only stop expressing these markers as they are allowed to return to a resting state. As they return to a resting state, latently infected CD4+ T cells also stop expressing the EF1α-driven mCherry marker, suggesting that as these cells return to resting, both promoters become silenced, perhaps by packaging into heterochromatin [55]. This means that HIV latency may be established after activated CD4+ T cells are initially infected, and it is these cells, potentially, that survive and return to a resting memory state, thus significantly contributing to the latent pool. How HIV latency is established in activated CD4+ T cells immediately after infection is still unknown, but it may arise from stochastic viral gene expression [56–59]. Our studies also suggest that activated CD4+ T cells that become productively infected can contribute to the latent pool as they return to a resting state. In our studies, these cells did not return to a completely resting state because so many of the cells died (S6 Fig), a likely consequence of viral cytopathic effects. However, the data clearly indicate that a small population of productively infected cells starts to return to a resting state and as they do, they lose expression of the LTR-driven GFP marker. However, when these cells were then stimulated with αCD3/αCD28, they failed to express GFP, suggesting that they could not be reactivated by CD3/CD28 stimulation, though it is possible that other reactivating agents could work. Finally, it is important to note that these productively infected CD4+ T cells that did eventually shut down LTR-driven GFP expression, did so in a culture dish. It remains to be seen, in vivo, if productively infected CD4+ T cells can survive long enough to return to a resting state and contribute to the latent pool, or if activated CD4+ T cells that become latently infected immediately after infection are the major contributors. Finally, infecting activated CD4+ T cells produces more productively infected cells than latently infected cells, while infecting resting CD4+ T cells produces more latently infected cells. These results reflect that HIV replicates more efficiently in activated CD4+ T cells, but they also show that resting CD4+ T cells can support HIV infection, at least up to the point of viral integration. In resting primary CD4+ T cells, we show that both productive and latent HIV infection can be achieved, though at levels much lower than those seen in activated CD4+ T cells. The infection kinetics in resting CD4+ T cells seem to be slower than in activated cells, since peak infection was not reached until 6 days after infection, while activated cells reached peak infection 4 days after infection (S1 Fig). These results agree with other’s findings [22, 60]. Also in agreement with previous findings, resting CD4+ T cells were made more permissive to HIV infection when exposed to the chemokine CCL19, which increases the ability of resting CD4+ T cells to support latent infection [47]. However, our data demonstrate that CCL19 also increases permissibility to productive infection, although its overall effect on resting CD4+ T cells increases latent infection. Interestingly, the cytokine, IL-7, which increases permissibility of resting CD4+ T cells to productive HIV infection, also increased both productive and latent infection in resting CD4+ T cells in our study. Lastly, infecting untreated resting CD4+ T cells with a Vpx-containing virus significantly increased productive infection but only modestly increased latent infection. Interestingly, infecting resting CD4+ T cells with our HIV Duo-Fluo I virus produced a significant amount of silent infection events, in which expression of both fluorescent proteins was silenced, camouflaging latently infected cells within our uninfected population. In fact, the isolated uninfected population of resting CD4+ T cells contained more silently infected cells than the number of latently infected cells that were identified via the mCherry fluorescent marker after the initial infection. This was true for all untreated and treated resting CD4+ T cells, except CCL19-treated cells, and was highest in IL-7-treated cells and untreated resting CD4+ T cells infected with a Vpx-containing virus. The reasons for this are unclear. Within resting CD4+ T cells, viral integration occurs in regions of the host genome that are unfavorable for viral gene expression [61], and studies also suggest that latently infected cells are more likely to contain provirus in or near heterochromatin [62, 63]. Integration into such regions would be unfavorable not only for LTR-driven gene expression but also for EF1α-driven gene expression. In the presence of Vpx, SAMHD1 cannot inhibit HIV reverse transcription, allowing the virus to bypass one of the major obstacles to replication in resting CD4+ T cells. Therefore, integration of the viral cDNA may occur more readily in these unfavorable heterochromatic regions. Treating cells with IL-7, which signals through the JAK/STAT pathway [64], may produce a similar situation. Lastly, previous studies have reported that resting CD4+ T cells can only be infected by HIV in the context of total lymphoid cell aggregates [41]. However, our results show that infecting untreated resting CD4+ T cells (alone) and untreated resting total lymphoid cells from peripheral blood and lymphoid tissue all produced productive and latent populations. Although, we did find that latent infection is more likely to occur in total resting lymphoid cell aggregates than in resting CD4+ T cells alone. The reasons for this are still unclear, but recent studies have shown that co-culture of resting CD4+ T cells with myeloid dendritic cells [65], or co-culture of resting CD4+ T cells with endothelial cells [66], enhances HIV latency, further proving that the lymphoid environment plays an important role in how HIV latency is established within resting CD4+ T cells. Overall, our studies show that HIV infection can occur in both resting and activated CD4+ T cells, such that infection of resting cells more often results in latent infection and infection of activated cells more often results in productive infection. Based on our data, we now have a better understanding of the contribution that each infected cell type makes to the latent reservoir. Our study underscores why we must consider both resting and activated CD4+ T cells when investigating how HIV latency occurs.

Materials and Methods Virus production Pseudotyped HIV Duo-Fluo I viral stocks were generated by co-transfecting (using the standard calcium phosphate transfection method) HEK293T cells with a plasmid encoding HIV Duo-Fluo I and a plasmid encoding HIV-1 dual-tropic envelope (pSVIII-92HT593.1). We generated a Vpx-containing HIV Duo-Fluo I pseudotyped virus by co-transfecting HEK293T cells with the HIV Duo-Fluo I plasmid, the pSVIII-92HT593.1 plasmid, and a plasmid encoding a Vpr-Vpx fusion protein (pSIV3+, generously donated by Warner Greene). Supernatants were collected after 72 h and filtered through a 0.45 μM membrane to clear cell debris, and were then concentrated by ultracentrifugation (76,755 x g) for 2 h at 4°C. Concentrated virions were resuspended in complete media and stored at -80°C. Virus concentration was estimated by p24 titration (HIV-1 alliance p24 ELISA kit, Perkin-Elmer). Primary cell isolation and cell culture Primary CD4+ T cells and peripheral blood mononuclear cells (PBMCs) were purified from healthy donor blood (Blood Centers of the Pacific, San Francisco, CA, USA and Stanford Blood Center). CD4+ T cells were isolated by negative selection using the RosetteSep Human CD4+ T Cell Enrichment Cocktail (StemCell Technologies). PBMCs were purified by Histopaque-1077 density gradient. Purified resting CD4+ T cells and PBMCs from peripheral blood were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (50 U/ml), and streptomycin (50 mg/ml). Human lymphoid aggregate cultures (HLACs) were purified using tonsillar or splenic tissue from uninfected donors (Cooperative Human Tissue Network) as previously described [67]. CD4+ T cells were isolated from HLACs by negative selection using the EasySep Human CD4+ T Cell Enrichment Kit (StemCell Technologies). HLACs and CD4+ T cells isolated from splenic and tonsillar tissues were cultured in RPMI 1640 supplemented with 20% heat-inactivated FBS, 100 mg/ml gentamicin, 200 mg/ml ampicillin, 1 mM sodium pyruvate, 1% nonessential amino acids (Mediatech, Manassas, VA, USA), 2 mM L-glutamine, and 1% fungizone (Invitrogen, Indianapolis, IN, USA) Cell treatment and infection Purified resting CD4+ T cells were either left untreated or treated for 3 days with 20 ng/ml IL-7 (R&D Systems) or 100 μM CCL19 (R&D Systems). Purified CD4+ T cells isolated from peripheral blood and tonsillar and splenic tissues, as well as PBMCs and HLACs, were stimulated with αCD3/αCD28 activating beads (Life Technologies) at a concentration of 1 bead/cell in the presence of 30 U/ml IL-2 (PeproTech) for 3 days. All cells were spinoculated with either HIV Duo-Fluo I alone or Vpx-containing HIV Duo-Fluo I at a concentration of 100 ng of p24 per 1 × 106 cells for 2 h at 1,200 × g at 37°C. After spinoculation, all cells were returned to culture in the presence of 30 U/ml IL-2, except for CD4+ T cells pre-stimulated with αCD3/αCD28 activating beads, which were placed back in culture with the αCD3/αCD28 activating beads and 30 U/ml IL-2. Flow cytometry and cell sorting Uninfected cells were stained in fluorescence-activated cell sorting (FACS) buffer (phosphate buffered saline supplemented with 2 mM EDTA and 2% FBS) with αCD69-PE and αCD25-APC (eBioscience) and fixed in 1% paraformaldehyde. Infected cells were stained in FACS buffer with αCD69-V450 and αCD25-APC/Cy7 (BD Biosciences) and fixed in 1% paraformaldehyde. Data were collected on a FACS Caliber and a FACS LSRII (BD Biosciences), and analyses were performed with FlowJo software (TreeStar). Untreated and treated CD4+ T cells from Figs 1F and S4 were sorted with a FACS AriaII (BD Biosciences) based on their GFP and mCherry fluorescence at 6 days post-infection, and they were placed back in culture with or without 30 μM Raltegravir (National AIDS Reagent Program). CD4+ T cells stimulated with αCD3/αCD28 activating beads in the presence of 30 U/ml IL-2 from Fig 3 were sorted based on their GFP and mCherry fluorescence at 4 days post-infection. SAMHD1 protein analysis Untreated resting primary CD4+ T cells infected with either HIV-Duo-Fluo I alone or Vpx-containing HIV Duo-Fluo I were lysed 6 days post-infection in radioimmunoprecipitation assay buffer (150 mm NaCl, 1% Nonidet P-40 (vol/vol), 0.5% AB-deoxycholate (vol/vol), 0.1% sodium dodecyl sulfate (SDS) (vol/vol), 50 mm Tris-HCl (pH 8), 1 mm DTT, and EDTA-free Protease Inhibitor (Calbiochem). Cell lysates were used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting analysis. The primary antibodies used were rabbit polyclonal anti-SAMHD1 (Sigma-Aldrich, Cat# SAB2102077) and monoclonal anti-β-actin (A5316, Sigma-Aldrich). HIV integration DNA was prepared after cell sorting of uninfected, productively infected and latently infected cell populations using the DNeasy Kit (Qiagen). Real-time PCR was used to detect total HIV DNA, β-globin, and integrated HIV DNA as previously described [68].

Supporting Information S1 Fig. Resting primary CD4+ T cells reach peak infection levels with HIV Duo-Fluo I 6 days post infection. Infection profiles of untreated or stimulated primary CD4+ T cells over the course of 6 days after infection. Untreated resting CD4+ T cells were infected with either HIV Duo-Fluo I virus alone or the Vpx-containing HIV Duo-Fluo I virus. Stimulated cells were infected with HIV Duo-Fluo I alone. (A) Latent infection (mCherry+) and (B) productive infection (GFP+ and GFP/mCherry double-positive) were analyzed by flow cytometry every 24 hrs following infection. https://doi.org/10.1371/journal.ppat.1004955.s001 (PDF) S2 Fig. Env-deficient HIV Duo-Fluo I does not contain any replication-competent virus. Primary CD4+ T cells were either mock infected or infected with either a replication-competent HIV-GFP (NLENG1, David N. Levy, University of Alabama, Birmingham) or one of three distinct batches of env-deficient HIV Duo-Fluo I (primary infection). Four days post-infection, supernatant was collected from the cultures, cleared of cell debris via centrifugation, and applied to freshly activated primary CD4+ T cells (secondary infection). Secondary infection was monitored for 12 days following infection. (A) Infection profiles for primary and secondary infections of activated primary CD4+ T cells. Data shown are from a single donor but are representative of three separate donors. (B) Quantified values of latent infection and productive infection from primary infections in panel A. (C) Quantified values of latent infection and productive infection from secondary infections in panel A. Data from panels B and C represent the average of three donors. https://doi.org/10.1371/journal.ppat.1004955.s002 (PDF) S3 Fig. Infection of primary CD4+ T cells with HIV Duo-Fluo I containing Vpx leads to SAMHD1 degradation and has no effect on T cell activation. (A) Protein expression levels of SAMHD1 in resting primary CD4+ T cells infected with either HIV Duo-Fluo I alone or HIV Duo-Fluo I containing Vpx at 6 days after infection. (B) Expression of activation markers CD69 and CD25 in untreated resting primary CD4+ T cells infected with either HIV Duo-Fluo I alone or HIV Duo-Fluo I containing Vpx at 6 days after infection. Data shown are from a single donor, but are representative of three separate donors. https://doi.org/10.1371/journal.ppat.1004955.s003 (PDF) S4 Fig. Untreated and treated resting primary CD4+ T cells contain reactivatable pre-integration and post-integration latency. Infection profiles for reactivation of pre-integration latent virus and post-integration provirus used to quantify data in Fig 1F. Data shown are from a single donor, but are representative of three separate donors. https://doi.org/10.1371/journal.ppat.1004955.s004 (TIF) S5 Fig. HIV Duo-Fluo I integration events are found within the sorted productive infection and latent infection populations, but not in the uninfected population. Measure of integration events/cell within the sorted populations of activated primary CD4+ T cells. Data represents the average of three donors. https://doi.org/10.1371/journal.ppat.1004955.s005 (PDF) S6 Fig. Cell-size changes of productive, latent, and uninfected cell populations as they return to a resting state. Productive (green), latent (red) and uninfected (black) primary CD4+ T cell populations were analyzed for cell-size changes via the use of the forward scatter parameter (FSC-A) 1, 5 and 11 days post activation, and compared to the cell-size of the untreated, resting population, and the αCD3/αCD28-treated population at day 4 (Fig 3B). Data shown are from a single donor, but are representative of three separate donors. https://doi.org/10.1371/journal.ppat.1004955.s006 (PDF) S7 Fig. Latently infected primary CD4+ T cells that lose expression of their fluorescent markers are more likely to exhibit a resting phenotype. (A) Expression of activation markers CD69 and CD25 within GFP/mCherry double-negative (1), mCherry single-positive (2) and GFP/mCherry double-positive (3) cells from latently infected primary CD4+ T cells at 11 days after activation (Fig 3C). https://doi.org/10.1371/journal.ppat.1004955.s007 (PDF)

Acknowledgments We thank Gary Howard for editorial assistance, John Carroll and Teresa Roberts for graphics, and Veronica Fonseca for administrative assistance. We also thank Marielle Cavrois and Marianne Gesner for assistance with flow cytometry and cell sorting, and Gilad Doitsh and Kate Monroe for technical assistance.

Author Contributions Conceived and designed the experiments: EV LC. Performed the experiments: LC. Analyzed the data: EV LC. Contributed reagents/materials/analysis tools: VC. Wrote the paper: EV LC VC.