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Epub ahead of print July 1, 2013 - doi:10.1189/jlb.1112566

Brief Conclusive Report

Cocaine exposure enhances permissiveness of quiescent T cells to HIV infection Sohn G. Kim,* James B. Jung,† Dhaval Dixit,† Robert Rovner Jr.,‡ Jerome A. Zack,*,†,§ Gayle C. Baldwin,*,†,§ and Dimitrios N. Vatakis*,§,1 Departments of *Medicine, Division of Hematology-Oncology, †Microbiology, Immunology and Molecular Genetics, and Molecular, Cell and Developmental Biology, §University of California Los Angeles AIDS Institute, David Geffen School of Medicine at University of California Los Angeles, California, USA



RECEIVED NOVEMBER 8, 2012; REVISED JUNE 13, 2013; ACCEPTED JUNE 18, 2013. DOI: 10.1189/jlb.1112566

ABSTRACT In vivo and in vitro exposure to stimulants has been associated with increased levels of HIV infection in PBMCs. Among these lymphocyte subsets, quiescent CD4! T cells make up the majority of circulating T cells in the blood. Others and we have demonstrated that HIV infects this population of cells inefficiently. However, minor changes in their cell state can render them permissive to infection, significantly impacting the viral reservoir. We have hypothesized that stimulants, such as cocaine, may perturb the activation state of quiescent cells enhancing permissiveness to infection. Quiescent T cells isolated from healthy human donors were exposed to cocaine and infected with HIV. Samples were harvested at different time-points to assess the impact of cocaine on their susceptibility to infection at various stages of the HIV life cycle. Our data show that a 3-day exposure to cocaine enhanced infection of quiescent cells, an effect that appears to be mediated by !1R and D4R. Overall, our results indicate that cocaine-mediated effects on quiescent T cells may increase the pool of infection-susceptible T cells. The latter underscores the impact that stimulants have on HIV-seropositive individuals and the challenges posed for treatment. J. Leukoc. Biol. 94: 000 – 000; 2013.

Introduction Quiescent CD4 T cells constitute the majority of circulating T cells in blood. As they are the major targets for HIV infection, factors that influence their infectivity can have a major impact on HIV pathogenesis. We [1, 2] and others [3, 4] have demonstrated that quiescent T cells are refractory to HIV infection. More specifically, viral reverse transcription and integration are marred by inefficiencies severely impacting the kinet-

Abbreviations: 7-AAD"7-amino-actinomycin D, CFAR"Center for AIDS Research, DRD4"dopamine D4, H!L"heavy and light; IP3"inositol trisphosphate, MOI"multiplicity of infection, UCLA"University of California Los Angeles

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ics of infection [2]. However, the aforementioned defects can be overcome as these cells can become permissive to infection if exposed to the appropriate factors, which include, but are not limited to, cytokines, chemokines, or short-term CD3– CD28 activation [5–10]. Moreover, the majority of these factors does not cause full T cell activation. It has been shown that driving cells to the G1b stage of the cell cycle, which is characterized by high levels of cellular RNA synthesis but no DNA replication, is sufficient to render quiescent cells permissive to infection [6]. In addition to immune-related factors, T cells can be targeted by nonimmune effectors, resulting in changes of immune function [11]. Stimulant exposure, such as cocaine, has been shown to affect immune function [12–23]. Regarding HIV infection, in vitro and in vivo studies have suggested that cocaine exposure enhances viral replication [12, 16, 18, 20 – 25]. Cohort studies have well-demonstrated that cocaine abuse results in increased HIV pathogenesis and morbidity [26 –28]. In vitro studies have supported these observations, as exposure of PBMC to cocaine resulted in increased levels of infection [12, 19 –21, 23, 25, 29]. The mechanisms of this phenomenon, however, have not been fully elucidated. Studies have suggested that cocaine-induced enhancement of HIV infection may be mediated by the secretion of cytokines, such as TGF-" and IL-10 [16, 17, 20, 25, 30]. Furthermore, cocaine users have been shown to have elevated levels of neopterin in blood, indicating a state of increased immune activation [24]. Finally, previous studies have shown that the effect of cocaine on lymphocytes could be mediated by its interaction with the !1R [16, 22, 31]. The impact of cocaine on HIV infection has been focused mainly on total PBMC populations. To this date, there have been no studies examining the impact of cocaine exposure on the permissiveness of quiescent T cells to HIV. Here, we focus

1. Correspondence: UCLA AIDS Institute, David Geffen School of Medicine, 615 Charles E. Young Dr. S., BSRB 173, Mail Code 736322, Los Angeles, CA 90095, USA. E-mail: [email protected]; Twitter: https:// twitter.com/vatakisd

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Copyright 2013 by The Society for Leukocyte Biology.

on the effect cocaine has on this major lymphocyte population. Our studies suggest that cocaine exposure renders quiescent T cells permissive to HIV infection by bypassing the block in the early stages of RT, seen previously in quiescent cells. This potentiating effect of cocaine can have a major impact on HIV pathogenesis and may influence the size of the viral reservoir.

caine for 3 days prior to infection. Cocaine remained in culture after infection. Same procedure was followed when using agonists CP-226,269 (DRD4R at 32 nM) and L-687,384 (!1R at 40 #M). For the antagonist studies, cells were pretreated with antagonists for $4 h, followed by exposure to cocaine. The antagonists used were L-745,870 (DRD4R at 0.1 #%) and BD1047 (!1R at 1 #%). DMSO-treated cells were used as solvent controls. Cells were then infected with HIV89.6 by incubation at 37°C, 5% CO2 with mild shaking at a MOI of 1 for 1–2 h. The cells were washed to remove residual free virus and recultured under the appropriate conditions.

MATERIALS AND METHODS

Flow cytometric analysis for markers and cell cycle

Quiescent T cell isolation

Purity of the quiescent cell population was assessed by staining cells with fluorescently conjugated mAb as described previously and acquired on an LSRFortessa (BD Biosciences, San Jose, CA, USA) [2]. Data were analyzed by using FlowJo 9.6 (TreeStar, Ashland, OR, USA). The protocol described above was used to determine the levels of CD25, CD38, CD69, CCR5, CXCR4, and HLA-DR on all cell types. For cell cycle analysis, cells were suspended in a buffer containing 0.03% saponin (Sigma-Aldrich, St. Louis, MO, USA). Fifty microliters of 400 #M 7-AAD (Calbiochem, La Jolla, CA, USA) was added at a final concentration of 20 mM. The cells were incubated at room temperature for 30 min and cooled on ice for at least 5 min, and 3 #l 1.7 mM Pyronin Y (Polysciences, Warrington, PA, USA) was added at a final concentration of 5 #M; the cells were then incubated for an additional 10 min on ice and analyzed. The quadrants were set based on n-butyrate (G1a arrest) and aphidocolin (G1b arrest) treatment of these cells, as described previously [10]. For the expression of the DRD4R, quies-

Human PBMCs from nondrug-using, healthy human donors were incubated with a cocktail of mouse mAb against human lymphocyte markers to remove activated CD4! T cells (CD25, CD38, CD69, HLA-DR) and unwanted cell lineages, such as CD8 T cells, monocytes, B cells, granulocytes (CD123), and NK cells [2]. Labeled cells were removed after incubation with magnetic beads coated with goat antibodies against mouse IgG (Miltenyi Biotec, Auburn, CA, USA) and separated using an autoMACS (Miltenyi Biotec) cell sorter [2].

Quiescent T cell treatment and infection To generate stimulated cells, quiescent cells were cultured for 48 h with plate-bound anti-CD3 (1 #g/ml) and soluble anti-CD28 (50 ng/ml). To assess the effects of cocaine, quiescent cells were treated with 10#8 M co-

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Figure 1. Cocaine treatment of quiescent T cells induces phenotypic changes. Quiescent T cells (Quiescent) were exposed to cocaine (Cocaine) for 3 days or stimulated with anti-CD3/anti-CD28 (CD3/CD28). Cells were then harvested and analyzed by flow cytometry for cell cycle progression and surface marker expression changes. (A) For cell cycle progression, cells were stained with 7-AAD (DNA) and Pyronin Y (RNA), as shown in the upper panels from one representative donor. The increased entry into G1b, following a 3-day cocaine treatment, is statistically significant, as shown in the lower bar graph (n"7; **P&0.01, one-tailed Student’s t-test). Cocaine treatment had no negative effect on cell viability. (B) Cells were also assessed for the expression of CCR5 (n"8; **P&0.01, one-tailed Student’s t-test) and CXCR4 (not significant), as well as (C) T cell activation markers (not significant between Quiescent and Cocaine groups). (continued on next page)

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Cocaine enhances HIV infection of quiescent T cells

Kim et al.

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cent CD4 T cells were first incubated with a DRD4-specific antibody (1:100 dilution, clone 2B9; Millipore, Billerica, MA, USA), followed by an incubation with an Alexa Fluor 647 goat anti-mouse IgG (H!L) secondary antibody, and analyzed as above (1:100 dilution; Invitrogen, San Diego, CA, USA). The !1R was also detected by flow cytometry through intracellular staining of quiescent T cells using an anti-!1R antibody, clone ICR-SIG1R-A (1:100 dilution; Millipore), and the Alexa Fluor 488 goat anti-rat IgG (H!L) secondary antibody and analyzed as above (1:100 dilution; Invitrogen).

DNA real-time PCR Quantitative real-time DNA PCR was performed, as described previously [2], using a primer-probe pair that amplifies full-length HIV reverse transcripts (the long-terminal repeat-gag junction), formed near the completion of the reverse-transcription process. All amplifications were performed on the ABI7700 (Applied Biosystems, Foster City, CA, USA), in parallel with a set of known quantitative standards. Detection and quantitation of integrated viral DNA were assessed by Alu PCR, as described previously [2].

HIV mRNA RT-PCR Real-time multiplex reverse transcription and PCR amplification were performed simultaneously for viral full-length and tat/rev, as well as cellular "2m, RNAs with the Qiagen One Step RT-PCR kit (Qiagen, Chatsworth, CA, USA), using the manufacturer’s protocol. HIV full-length, tat/rev, and "2m primers have been described [2]. All RT-PCR amplifications were performed on the ABI7700 (Applied Biosystems).

Statistical analysis Statistical analyses were conducted by one-tailed Student’s t-test or repeated measures one-way ANOVA, followed by Tuckey’s post hoc testing using GraphPad Prism 6.0. Statistical significance was set at P & 0.05.

RESULTS AND DISCUSSION

Cocaine treatment causes subtle changes in quiescent T cell state To determine if cocaine treatment of quiescent T cells resulted in any phenotypic changes that would suggest increased susceptibility to HIV, we examined the levels of activation markers, such as CD25, CD38, CD69, and HLA-DR, as well as changes in cell cycle progression and HIV coreceptor expression. Quiescent cells were isolated and pretreated with cocaine for 3 days. Samples were then harvested and analyzed for cell cycle progression and relevant surface marker expression by flow cytometry. As shown in Fig. 1A, 3-day exposure of quiescent T cells to cocaine led to increasing levels of cells in the G1b stage of the cell cycle. Cells in this stage are characterized by increased levels of transcription and can become infected when exposed to HIV [32]. Cocaine exposure did not cause any changes in cell viability (Fig. 1A). Furthermore, cocaine treatment resulted in an increased percentage of CCR5-expressing cells, with no effects on the levels of CXCR4 (Fig. 1B), as well as the levels of T cell activation marker expression, such as CD25, CD38, CD69, and HLA-DR (Fig. 1C). Similarly, we did not observe induction of IL-10 or TGF-" expression, as seen by others (data not shown) [20, 33]. Therefore, these data suggest that in vitro cocaine exposure may increase susceptibility of quiescent T cells to HIV infection via alternative stimulation pathways. Such a pattern has been demonstrated elsewhere, as T cell activation and HIV coreceptor expression 4 Journal of Leukocyte Biology

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modulation occurred following exposure of quiescent T cells to neurotransmitters [5, 11]. Furthermore, the lack of TGF-" and IL-10 up-regulation is quite possible, as the secretion of these cytokines was only seen previously in mixed lymphocyte populations that contain macrophages, DCs, and activated T cells that could have released these immune effectors instead. The latter, in addition to the lack of an effect on cell viability, would suggest that the cocaine-induced changes in quiescent T cells are more likely direct.

Cocaine exposure of quiescent cells enhances the kinetics of HIV infection To further examine the effect of cocaine on the infectivity of quiescent T cells by HIV, we purified quiescent T cells from nondrug-using, healthy human donors and treated with cocaine for 3 days. Following drug pretreatment, the cells were infected with HIV-189.6 at a MOI of 1. Untreated quiescent cells and CD3/CD28-stimulated T cells served as negative and positive controls, respectively. Following infection, cells were harvested at different time-points and used in a series of assays to determine the impact of cocaine on the HIV life cycle. To determine the effect of cocaine exposure on HIV reverse transcription, cells were harvested, and total cellular DNA was purified for quantitative real-time PCR analysis. As shown in Fig. 2A, cocaine-treated cells displayed increased levels of fulllength viral cDNA and accelerated kinetics of reverse transcription when compared with quiescent T cells. Interestingly, the rate of reverse transcription was comparable with that of stimulated cells, but the levels of full-length viral cDNA were approximately tenfold lower. Based on our previous studies, the main block to HIV infection in quiescent cells was detected postentry at the early stages of reverse transcription [2]. Based on our data, exposure of quiescent T cells to cocaine has alleviated this early block in reverse transcription, allowing for efficient completion and generation of full-length viral cDNA. As cocaine treatment resulted in increased levels of HIV cDNA, we predicted that there would be a commensurate positive impact on viral integration. Therefore, infected cells were also analyzed for the efficiency and kinetics of HIV integration. To this end, we used quantitative real-time PCR to measure the levels of 2-LTR circles and integrated viral DNA. 2-LTR circles are abortive products of integration and can be used as a measure of integration efficiency. Based on our data, quiescent and cocaine-treated populations had tenfold higher levels of 2-LTR circles compared with stimulated cells, which had very low levels, close to the limit of detection (Fig. 2B). When we examined the levels of viral integration, the 3-day cocaine-treated cells had tenfold higher levels of proviral DNA than quiescent cells. In addition, as expected, based on the reverse transcription data, HIV integration took place 2 days earlier in the cocaine-treated samples compared with that of the quiescent T cells (Fig. 3). Furthermore, while in stimulated cells, integration peaked at Day 1, in cocaine-treated cells, the trend was reversed, as we observed a gradual increase from Days 1 through 5 (Fig. 3). This delayed rate of integration in cocaine-treated cells would suggest that the process of integration could be less efficient in these cells when compared with their stimulated counterparts. Furthermore, this www.jleukbio.org

Kim et al. Cocaine enhances HIV infection of quiescent T cells 100

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Figure 2. Cocaine treatment of quiescent T cells results in increased levels and kinetics of reverse transcription. Quiescent cells were exposed to cocaine for 3 days. Cells were then infected and harvested at different time-points postinfection. Samples were used to assess the kinetics of HIV infection in cocaine-treated cells compared with quiescent as well as prestimulated T cells. (A) Cells (0.3'106) were harvested at different time-points after infection, and DNA was extracted for viral DNA analysis. DNA extraction and real-time PCR analysis were performed. The limit of detection (indicated by a dashed line) was 0.01% cells, based on AZT-treated negative controls (for Day 1, *P&0.05 Cocaine vs. CD3/CD28; for Days 3 and 5, *P&0.05 Quiescent vs. Cocaine, Student’s t-test). (B) Quantitation of 2-LTR circle formation

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delay in integration would explain the higher levels of 2-LTR circles seen in the cocaine-treated group, as linear viral cDNA would circularize if it remains in a nonintegrated form over a prolonged period of time [34]. As the kinetics of viral cDNA synthesis seem to proceed unabated, the potential limitations can be attributed to delayed nuclear transport of the preintegration complex [35], or limiting amounts of cellular host factors, such as lens epithelium-derived growth factor, needed to tether the provirus into the host genome [36]. However, despite any observed shortcomings, cocaine-treated quiescent cells exhibit similar kinetics of infection to that of activated T cells. Furthermore, cocaine exposure leads to increased viral expression. As shown in Fig. 4A, when viral mRNA levels were determined, we observed an increase in multiply spliced viral RNA in cocaine-treated, infected cells when compared with quiescent cells. Additionally, when cells were analyzed for the expression of the viral protein by flow cytometry, we found that cocaine-treated cells expressed high levels of p24 Gag fol-

in cocaine treated, untreated and activated T cells on day after infection using real time PCR. The data shown are the average from three representative donors (out of 5).

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lowing infection, whereas quiescent cells did not express detectable levels of this marker of HIV infection (Fig. 4B). Finally, whereas the delay in viral integration did not impact virus release, the kinetics of virus production was delayed by $1 day. This increased permissiveness to infection was also observed when we repeated our experiments with HIV-1NL4-3 (X4-tropic) and HIV-1NFNSX (R5-tropic; data not shown). Thus, whereas cocaine exposure resulted in up-regulation of CCR5 (Fig. 1B) in quiescent T cells, this would have an impact only on the R5-tropic virus strains, as increased coreceptor expression would facilitate more infection. However, based on our data thus far, the potentiating effects of cocaine on HIV infection extend beyond viral entry. In our previous studies [2], we have shown that X4-tropic HIV enters quiescent cells very effectively, but viral replication is blocked at the early stages of reverse transcription, suggesting that the resistance seen in these cells is mainly a postentry event. Our data suggest that acute exposure to cocaine renders quiescent T cells permissive to HIV infection, primarily by bypassing the ratelimiting step in reverse transcription, seen previously in quiescent T cells [2].

Cocaine mediates its effect on quiescent T cells via the !1R and D4R The capacity for cocaine to interact with !1Rs has been shown to modulate immune function in vivo and in vitro, promoting a TH2 immune response associated in T cells with increased production of IL-10 and TGF-" [31]. Based on microarray data that we have on quiescent T cells from previous studies [34], 6 Journal of Leukocyte Biology

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we found that the !1R as well as DRD4Rs are highly expressed in these cells. These observations were confirmed further by flow cytometric analysis of quiescent CD4 T cells measuring the expression levels of the !1R and DRD4R (Fig. 5A). We hypothesized that an interaction of cocaine with both or either receptors might also contribute to cocaine-mediated effects on quiescent T cells. To test our hypothesis, we treated quiescent T cells with !1 and D4 agonists and antagonists. As in the previous assays, quiescent cells from healthy human donors were isolated and pretreated with cocaine or the receptor agonists. A subset of the cocaine-treated cells was pretreated with the receptor antagonists, 5 h prior to cocaine exposure. The reagents used were BD1047 and L-687,384 !1R antagonist and agonist, respectively, as well as CP-226,269 and L-745,870 D4R agonist and antagonist, respectively. Cocaine and agonist treatment was carried out for 3 days and then assayed to assess how the drug impacts quiescent T cells. Firstly, as shown in Fig. 1B, cocaine caused an up-regulation of CCR5 expression in quiescent CD4! T cells. Thus, we assessed whether either cocaine receptor may be involved in modulating the expression of CCR5. As shown in Fig. 5B, the effect of the !1R on CCR5 expression is minor. On the other hand, the effect elicited by the D4 agonist is quite pronounced, resulting in a significant up-regulation of CCR5. In addition, pretreatment of quiescent cells with the D4 antagonist blocked the cocaine-induced up-regulation of CCR5. Thus, our data suggest that the cocaine-mediated increase in CCR5 expression is via binding to the D4R. Furthermore, when we examined the impact on infection, !1 (L-687,384) and the D4 www.jleukbio.org

Kim et al.

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Figure 5. Cocaine effects are mediated via the DRD4R. (A) Quiescent CD4 T cells from four donors (**P&0.01; ***P&0.001) were stained for antibodies against the !1R and DRD4R (see Materials and Methods) and analyzed by flow cytometry, as shown on the scatter dot plots. The groups labeled Staining control are cells incubated with secondary antibody alone. Histogram panels are from one representative donor. The dashed-line histograms are cells incubated with secondary antibody alone. (B) Quiescent CD4 T cells were treated with cocaine, D4R, or !1R agonists for 3 days. A subset of the cocaine-treatment group was pretreated with DRD4R or !1R antagonists, 4 h prior to cocaine exposure. The reagents used are: CP-226,269 (DRD4R agonist at 32 nM), L-687,384 (!1R agonist at 40 #M), L-745,870 (DRD4R antagonist at 0.1 #%), and BD1047 (!1R antagonist at 1 #%). Cells treated with cocaine or the D4 agonist resulted in increased CCR5 expression. The data are the average from three different donors (left, *P&0.05; **P&0.01). Treated cells were also infected with HIV-189.6 and assayed for p24(Gag) expression. D4 agonisttreated cells mimicked the cocaine-induced permissiveness to infection. The data indicate the fold increase of percent p24-positive cells over cocaine only-treated cells and are the average from four different donors (right, *P&0.05 D4 antagonist vs. Cocaine and D4 Agonist vs. Cocaine).

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(CP-226,269) agonists mimicked the cocaine-induced effect on quiescent cells (Fig. 5B), and the D4 agonist had a more pronounced effect on the treated cells. The potentiating effect of cocaine on quiescent T cells was blocked by antagonists BD1047 and L-745,870; however, the latter was more effective at limiting permissiveness to infection. The above data demonstrate that cocaine impacts quiescent T cells by binding to the !1R and mainly, the DRD4Rs. Whereas our data lend further support on the key role for the !1R [16, 22, 31], use of the DRD4R seems to result in more definitive changes on the cell state of quiescent CD4 T cells. The distinct responses elicited by the two receptors are yet to be explained but can be attributed to different expression levels, subcellular localization of the receptor (surface vs. intracellular), as well as distinct signaling pathways, especially regarding the increased levels of CCR5 expression. Additional studies addressing these and other questions should further assess the role and contribution of each receptor on the effects of cocaine on quiescent T cells. Substance abuse has been linked to increased HIV pathogenesis, disease progression, and mortality. Despite a significant number of epidemiological studies that support the impact, direct and indirect, of stimulants, such as cocaine, on HIV infection [26 –28], the underlying mechanisms remain unclear. In the study presented here, we examined the impact of cocaine on the permissiveness of quiescent T cells to HIV infection. Quiescent T cells are the main subset of circulating T cells in the blood and a major target of HIV infection. Studies have shown that they are refractory to HIV infection [1– 4]. However, a number of groups, including ours, have shown that minimal activation can render these cells permissive to infection [5–10]. This is quite crucial, as several studies have demonstrated that the nondividing or slowly dividing T cell populations are the major component of the long-lasting HIV reservoir [37, 38]. Thus, any factors that can minimally impact infectivity of resting T cells can potentially contribute to the size of the HIV reservoir. To this end, we carried out a series of studies to examine whether exposure of quiescent T cells to cocaine provides sufficient activation to render them permissive to HIV infection. We have shown that cocaine exposure resulted in improved infection kinetics and viral production by bypassing the block in early reverse transcription, seen previously in quiescent T cells. The increased permissiveness caused by cocaine exposure may be mediated by, but not limited to, the !1R and especially, the DRD4R. Our studies suggest that the effect of cocaine on quiescent T cells is direct and not dependent on other bystander immune cells, such as macrophages, which have been shown, following cocaine exposure, to release cytokines that can render quiescent cells permissive to infection [29]. As to the mechanism(s) behind the block to HIV infection, seen in quiescent T cells, this is still a subject of intense investigation. However, based on our data, there are several ways that cocaine exposure may enhance quiescent T cell permissiveness to HIV infection. The DRD4R pathway has been implicated in cytoskeletal rearrangement [39]. T cell cytoskeletal organization plays a crucial role in establishing a produc8 Journal of Leukocyte Biology

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tive infection in resting cells by impacting viral uncoating, reverse transcription, and integration [40]. Moreover, the D4R pathway results in down-regulation of adenylate cyclase, thus inhibiting cAMP and PKA [39]. Whereas the role of cAMP in HIV is still under investigation, it is suggested that up-regulation of the mediator results in decreases of DNA reverse transcription, thus blocking HIV replication [5, 11]. Therefore, decreases in cAMP can have a potentiating effect. The !1R pathway is characterized by increased levels of cytoplasmic Ca ! 2 and activation of mediators, such as IP3 [31]. Whereas these mediators have not been implicated much in the early stages of HIV infection, recent work has suggested that IP3 may be involved in viral release [40]. Therefore, based on our results, a future, careful examination of the DRD4R and the !1R signaling pathways can potentially shed more light on how cocaine exposure alleviates the block seen in quiescent T cells. In conclusion, we have shown that cocaine modulates the permissiveness of quiescent cells to HIV. The potential for cocaine to augment the pool of HIV target cells with a commensurate increase in the viral reservoir has significant implications for HIV-seropositive individuals who abuse or use stimulants such as cocaine.

AUTHORSHIP S.G.K., J.B.J., D.D., R.R., and D.N.V. performed the experiments. D.N.V. designed and analyzed the experiments and prepared the manuscript. G.C.B. and J.A.Z. designed experiments and prepared and critically reviewed the manuscript.

ACKNOWLEDGMENTS This work was supported by U.S. National Institutes of Health grants R21DA031036-01A1 (to D.N.V.), 5P30 AI028697 (UCLA/CFAR to J.A.Z.), R01AI070010 (to J.A.Z.), and R01DA023386 (to G.C.B.). We thank the UCLA/CFAR Virology Core Laboratory for blood products, real-time PCR, and p24 ELISA support. Also, we thank the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research Flow Cytometry Core. DISCLOSURES

The authors declare no conflict of interest.

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KEY WORDS: human immunodeficiency virus ! stimulant abuse ! !1 and D4 receptors ! life cycle ! reservoir

Volume 94, October 2013

Journal of Leukocyte Biology 9