Agents that activate cannabinoid receptor pathways have been tested as treatments for cachexia, nausea or neuropathic pain in HIV-1/AIDS patients. The cannabinoid receptors (CB 1 R and CB 2 R) and the HIV-1 co-receptors, CCR5 and CXCR4, all signal via Gαi-coupled pathways. We hypothesized that drugs targeting cannabinoid receptors modulate chemokine co-receptor function and regulate HIV-1 infectivity. We found that agonism of CB 2 R, but not CB 1 R, reduced infection in primary CD4+ T cells following cell-free and cell-to-cell transmission of CXCR4-tropic virus. As this change in viral permissiveness was most pronounced in unstimulated T cells, we investigated the effect of CB 2 R agonism on to CXCR4-induced signaling following binding of chemokine or virus to the co-receptor. We found that CB 2 R agonism decreased CXCR4-activation mediated G-protein activity and MAPK phosphorylation. Furthermore, CB 2 R agonism altered the cytoskeletal architecture of resting CD4+ T cells by decreasing F-actin levels. Our findings suggest that CB 2 R activation in CD4+ T cells can inhibit actin reorganization and impair productive infection following cell-free or cell-associated viral acquisition of CXCR4-tropic HIV-1 in resting cells. Therefore, the clinical use of CB 2 R agonists in the treatment of AIDS symptoms may also exert beneficial adjunctive antiviral effects against CXCR4-tropic viruses in late stages of HIV-1 infection.

Funding: This work was supported by United States National Institutes of Health grants R01AI074420 and DP1DA028866 awarded to BKC and NIH grants DA008863 and DA019521 awarded to LAD. CMC is further supported by NIH CTSA grant UL1RR029887 awarded to the Mount Sinai School of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2012 Costantino 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.

To test this hypothesis, we examined the effect of cannabinoid receptor activation on HIV viral transmission and productive infection in CD4+ T cells using a GFP-expressing, CXCR4-tropic HIV-1 variant. We found that activation of CB 2 R on CD4+ T cells significantly inhibited viral infection in a CB 2 R -selective and dose-dependent manner. Viral inhibition was more pronounced in resting cells that were activated after infection. We investigated signaling in these cells and found that CB 2 R agonism significantly decreased SDF-1α-induced CXCR4 activation. Furthermore, CB 2 R agonism altered HIV-induced cytoskeletal rearrangement, associated with productive HIV infection in resting cells. We found that CB 2 R was not sufficient to block viral transfer or fusion, but did significantly diminish productive viral infection. We conclude that CB 2 R is a novel modulator of CXCR4-tropic HIV infection in CD4+ T cells.

Although coreceptor signaling is not essential for HIV-1 infection, several recent studies have suggested that chemokine receptor signaling enhances infection of resting CD4+ T cells [19] – [21] . These cells express CXCR4, but not CCR5, whose expression is restricted to a small subset of memory CD4+ T cells [22] . In patients, the emergence of CXCR4-tropic virus usually occurs after years of infection and correlates with more rapid progression to AIDS [23] , [24] , [25] . Viral conversion to CXCR4-tropism increases the number of targets available to the virus [26] . Additionally, as HIV-1 can establish latency in resting T cells [27] , a switch to CXCR4-tropism could enhance the establishment of a latent pools of virus within lymphoid tissues. The increased number of new targets may explain the rapid decline in CD4+ T cell numbers and increased viral load in late-stage AIDS patients with CXCR4-tropic virus [1] , [24] . The late-stage patients who frequently harbor CXCR4-tropic virus are also the most likely to benefit from cannabinoid drug use. It is therefore relevant to study the potential for cannabinoid signaling to modulate CXCR4 activity and alter the course of HIV infection, Interactions between GPCRs like CB 2 R and CXCR4 can cause cross-desensitization, allosteric modulation, dimerization, changes in receptor localization, and alteration of physiological function among GPCR pairings [28] . Given that direct antagonism of chemokine receptor function can block viral infection [19] , [29] , [30] , it is possible that allosteric modulation of CXCR4 through a GPCR partner may also reduce HIV-1 permissiveness. Indeed, oligomerization of the chemokine co-receptors including CXCR4 using conformationally specific monoclonal antibodies can inhibit HIV-1 entry into target cells [31] , [32] . These experiments demonstrate signaling-independent modulation of coreceptor function. Allosteric agents that disrupt HIV-1 infection by modulating chemokine receptor signaling have not yet been identified. Should CB 2 R-induced signaling alter CXCR4 co-receptor function, this would represent first known example of a signaling-dependent GPCR interaction leading to viral inhibition.

Cannabinoid agonists activate the CB 1 R and CB 2 R cannabinoid receptors. Like the HIV chemokine co-receptors CXCR4 and CCR5, CB 1 R and CB 2 R are members of the Gαi-coupled family A GPCRs [10] . CB 2 R is highly expressed on all CD4+ T cells [11] , whereas CB 1 expression is found in activated, memory subsets [12] . CB 1 and CB 2 have been classified as immunosuppressive receptors on CD4+ T cells [13] , although antagonism of CB 1 R and CB 2 R does not enhance immune activation and knock-out mice do not exhibit differences in T cell frequency or increases in autoimmune pathogenesis [14] . The mechanism(s) by which cannabinoid agonists can modulate CD4+ T cell function remain unclear. Activation of CB 2 R has been shown to inhibit inflammatory cytokine production in CD4+ T cells [11] , which may account for the decrease in autoimmune pathogenesis observed in therapeutic trials of cannabinoid agonists in animal models of multiple sclerosis [14] , [15] . CB 2 R may also function as a chemotactic modulator, as CB 2 R activation inhibits CXCR4-induced chemotaxis in transformed lymphocytes [16] . CB 2 R has further been shown to regulate lymphocyte egress from the bone marrow in a role previously attributed largely to CXCR4 [17] , [18] . These findings suggest that CB 2 R may play a role in regulating chemokine receptor signaling, specifically the activity of CXCR4. Such cross-talk between CB 2 R and CXCR4 may have implications for AIDS patients who take cannabinoid-derived agents for therapeutic purposes.

Cannabinoid agonists are currently under investigation for the treatment of AIDS-associated cachexia, nausea, and neuropathic pain [1] – [3] . One such drug, dronabinol (Δ 9 -THC; Marinol®), has won Food and Drug Administration (FDA) approval for treatment of HIV-associated anorexia [4] . Additionally, the prescription of smoked or ingested cannabis (marijuana) for treatment of AIDS-related symptoms has been approved in 14 states [5] . Despite the use of cannabinoids by HIV/AIDS patients, few studies have investigated the impact of such drugs in regard to viral pathogenesis or immune regulation. Early studies conducted in the pre-HAART era suggested a positive correlation between development of opportunistic infection, progression to AIDS, and marijuana use [6] , [7] . Yet recent analysis of HIV/AIDS patients enrolled a randomized, placebo-controlled clinical trial designed to study the outcome of cannabinoid administration have indicated that cannabinoid use does not result in significant immunosuppression [1] . Indeed, both smoked marijuana and dronabinol were reported to increase total CD4+ T cell number [1] and naïve T cell number [8] over a 21-day period. A decrease in viral load was also observed in these patients [1] . Similarly, in SIV infected rhesus macaques, Δ 9 -THC exposure reduced viral load and CD4+ T cell depletion, significantly increasing animal survival over an 11 month period [9] . Despite these findings, the mechanisms by which cannabinoid drugs can influence viral replication or pathogenicity remain unknown.

For GTPγS binding, JWH-133 treated CD4+ T cells were permeabilized with CHAPS and incubated with increasing concentrations of SDF-1α. Incorporation of radiolabelled GTPγS was assayed as described [38] . For kinase phosphorylation and western blotting, primary CD4+ T cells were treated with antagonist and/or agonist in serum-free RPMI for a total of 4 hours prior to treatment with SDF-1α (PeproTech) or NL4.3 HIV at various times indicated. Levels of phosphorylated MAP kinase and total MAP kinase were determined as described [38] .

Cell-free viral fusion was measured using a method described previously [37] . BlaM-VPR was a gift from Michael Miller (Merck Research Laboratories). Viral infections were done with 20 ng of virus at a concentration of 200 ng/ml for 2 hrs at 37°C. Cells were analyzed on an LSRII flow cytometer (Becton Dickinson).

HIV-expressing Jurkat donor cells were generated by transfection using HIV Gag-iCherry, as described previously [34] . To generate infected CD4+ T cell donors, PHA activated CD4+ T cells were spinoculated with either HIV NL-GI or for 90 minutes at 1200×g. After 24–48 hours, approximately 10–30% of donor cells were infected. Donor cells were labeled with 10 µM CellTracker blue (CMF2HC) fluorescent dye (Invitrogen) and then co-cultured with unstimulated and antagonist and/or agonist-treated target cells in a 1∶1 ratio for 3 hours at 37°C, as described previously [33] . Virus transfer was terminated by washing with PBS and treatment with 0.05% trypsin-EDTA (Invitrogen) for 5 minutes. Cells were stained for viability and for CD45RO prior to fixation and acquisition on a LSRII (BD Biosciences). Sorted GFP − Cherry − CD45RO + or CD45RO − targets were seeded into 96-well plates coated with 2.5 µg/ml anti-CD3 in RPMI media containing 1 µg/ml anti-CD28 and 20 U/ml rIL-2 for activation. After 4 days, cells were harvested, fixed, and analyzed for fluorescence.

CD4+ T cells were thawed, resuspended in RPMI medium containing 20 U/ml recombinant IL-2 and stimulated with 1 µg/ml PHA (Sigma) overnight. Cells were cultured for four days and reseeded into 96 well flat-bottom plates (Costar) at a density of 10 5 cells/well prior to treatment and infection. Treated cells were incubated with antagonist or vehicle (DMSO) for one hour at 37°C followed by incubation with agonist or vehicle (DMSO or 0.1% ethanol) for another three hours. Following this treatment, triplicate cultures were infected with 10 ng/well HIV NL-GI. To assess HIV infection, fluorescence was assessed at day 4 post-treatment. Harvested cells were stained for viability and fixed with 4% paraformaldehyde, prior to acquisition on a FACSCalibur (BD Biosciences) and analysis with FloJo software (TreeStar).

HIV NL-GI and Gag-iCherry are NL4-3 based CXCR4-tropic HIV-1 molecular clones that have been described previously [34] . NL-GI expresses green fluorescent protein (GFP) in place of the viral early gene nef, and nef expression is maintained by insertion of an internal ribosome entry site (IRES) [35] . Gag-iCherry carries the GFP variant Cherry inserted internally into Gag between the MA and CA domains. For CCR5-tropic virus, a variant of NL-GI expressing the Env gene from the molecular clone JRFL [36] was used. Virus was produced in HEK293T cells and p24 concentration was calculated by ELISA prior to use.

Anonymous blood donations were obtained from New York Blood Center. Cells were cultured as previously reported [33] . The purified monoclonal antibodies (mAbs) 〈CD2 (OKT3) and 〈CD28 (28.2) were purchased from eBioscience. Phytohemagglutinin (PHA) and carboxyfluorescein succinimidyl ester (CFSE) was purchased from Sigma. Recombinant human IL-2 and CXCR4 antagonist AMD3100 were obtained through the AIDS Research and Reference Reagent Program (NIH). Fluorophore-conjugated CXCR4 (12GS), CCR5 (T21/8), CD25 (BC96), and CD45RO were purchased from Biolegend. The CB2 agonists JWH-133, JWH-150, Ser160, 2-arachidonoylglycerol, and anandamide and the CB2 antagonist AM630 were purchased from Tocris.

Results

CB 2 -specific agonists inhibit HIV-1 infection in primary CD4+ T cells As activation of the cannabinoid receptor CB 2 R has been shown to modify chemokine receptor activity [16] and cannabinoid use in HIV-1 infected individuals is associated with reductions in viral load [1], we hypothesized that CB 2 R agonism may alter the course of HIV-1 infection. Using a GFP-expressing variant of the CXCR4-tropic lab isolate NL4.3 (NL-GI), in which GFP is expressed in place of the viral gene nef [33], we assayed HIV-1 infectivity in primary CD4+ T cells pre-treated with CB 2 R agonist. Purified, blood-derived CD4+ T cells were stimulated with low-dose mitogen for four days prior to a three-hour cannabinoid treatment. To directly test the capacity for CB 2 R activation to inhibit viral infection, we pretreated activated CD4+ T cells with a potent and selective CB 2 R agonist that is approximately 200-fold selective for CB 2 R over CB 1 R (Ki = 3.4 nM) [39] (JWH-133) prior to HIV-1 exposure. Treated cells were then washed and exposed to virus in suspension (10 ng/2×105 cells) for four days. After this time, the frequency of GFP+ infected cells was measured and the frequency of inhibition, as compared to control (DMSO treated) infection, was calculated. When cells were treated with 100 nM of JWH-133 prior to viral exposure, we observed an approximately 40% reduction in HIV-1 infected cells after four days (Figure 1A–B). This inhibition was significantly reduced when CB 2 R activity was selectively blocked using the antagonist AM630, indicating that the antiviral activity of JWH-133 is indeed CB 2 R-specific (Figure 1A–B). JWH-133-mediated blockade of HIV-1 infectivity was dose-dependent, with an EC50 of 7.59±0.1 nM and a plateau of efficacy at approximately 50% inhibition (Figure 1B). Further, the action of this drug was CXCR4-specific, as JWH-133 treatment was not sufficient to inhibit infection with an isogenic virus that carried a CCR5-tropic Env from molecular clone JRFL (Figure 1C). Therefore, CB 2 activation reduces CXCR4-tropic HIV-1 infection in primary CD4+ T cells in a dose-dependent and receptor-specific fashion. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Cannabinoid inhibition of CXCR4-tropic HIV-1 infection is dose-dependent and CB 2 -selective. (A) HIV-1 (GFP) expression 4 days post-infection in primary CD4+ T cells pretreated with 100 nM of the CB2-specific agonist JWH-133 and/or the CB 2 -specific antagonist AM630 in a representative donor. HIV expression (GFP; FL1) is shown on the x-axis, and an empty channel (FL3) is shown on the y-axis. (B) Dose-dependent inhibition of viral infection by JWH-133 in primary CD4+ T cells is blocked by 100 nM of the CB 2 antagonist AM630 (mean±SEM; n = 7 donors; **p = 0.0032, *p<0.03). (C) Inhibition of CXCR4-tropic (NL4-3) but not CCR5-tropic (JRFL) HIV-1 after 4 days in primary CD4+ T cells pretreated with 100 nM of JWH-133 (mean±SEM; n = 5 donors). (D) Inhibition of viral infection in cells pretreated with the CXCR4 antagonist AMD3100 (which is predicted to block viral binding [57]), or 1 µM of cannabinoid agonists ACEA, Hu210, JWH-133, JWH-015 and Ser016 with or without pretreatment with the CB 1 antagonist AM251 or the CB 2 antagonist AM630. (E–F) Dose-dependent inhibition of NL-G1 infection in primary CD4+ T cells pretreated with (F) 2-arachidonoylglycerol (2-AG) or (G) anandamide (AEA) is blocked by 1ìM of the CB 2 antagonist AM630 (mean±SEM; n = 5 donors; *p<0.03). https://doi.org/10.1371/journal.pone.0033961.g001 To further confirm this novel role for CB 2 R, we tested other CB 2 R-selective agonists (JWH-015 and Ser016) to see if treatment was sufficient to reduce viral infectivity. Both of the highly specific CB 2 R agonists we tested proved to be antiviral at concentrations of 1 µM; these were JWH-015 (35.9±11.96% inhibition) and Ser016 (30.7±13.72% inhibition) (mean±SEM, n = 8 donors) (Figure 1D). Treatment with 1 µM of the pan-cannabinoid agonist Hu210 also significantly reduced HIV-1 infection, although to a lesser extent than with CB 2 R-selective agonists (23.9±7.45%, mean±SEM, n = 8 donors) (Figure 1D). The reduction in infection efficiency observed with Hu210 was CB 2 R-specific and was not observed when cells were pretreated with the CB 2 R-selective antagonist AM630 (Figure 1D). Consistent with these findings, 1 µM pretreatment with the CB 1 R selective agonist arachidonyl-2′-chloroethylamide (ACEA) did not significantly reduce viral infection (4.76±3.13%, mean±SEM, n = 5 donors) (Figure 1D). These results indicate that CB 2 R-selective agonists, but not CB 1 R-selective agonists, can inhibit HIV-1 permissiveness. Given that the pan-cannabinoid agonist Hu210 possessed antiviral activity, we predicted that naturally occurring endogenous ligands of the cannabinoid receptors, the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) could reduce infectivity via CB 2 R activation. We found that pretreatment with either 2-AG or AEA significantly inhibited HIV-1 infection in a dose-dependent and CB 2 -specific manner (Figure 1E–F). AEA, which is reported to have higher affinity for CB 2 R than 2-AG (Ki = 371 nM versus 1400 nM, respectively) [40], proved to be a more potent inhibitor of virus, with an EC50 of 8.59±0.09 nM, as compared to 1.82±0.25 µM for 2-AG. The antiviral activity of both of these endocannabinoid agents was abrogated by treatment with 1 µM of the CB 2 R-selective antagonist AM630. These data demonstrate that, like synthetic cannabinoid agonists, the endocannabinoids can activate CB 2 R to inhibit HIV-1 infectivity in CD4+ T cells.

CB 2 activation does not alter CXCR4 expression or T cell activation We next sought to determine the mechanism of HIV-1 inhibition via CB 2 R. Previous reports have indicated that cannabinoid treatment of immune cells can lead to changes in cell surface chemokine receptor expression [41], loss of proliferative capacity [11], and reduction in effector function [42], [43]. We examined whether CB 2 R activation in our infection model led to either a reduction in co-receptor expression or in host fitness, rendering the cells incapable of harboring productive viral infection. To test this possibility, we treated CD4+ T cells with the CB2 agonist JWH-133 with a concentration of drug sufficient to inhibit viral infection (100 nM). We found that this treatment did not lead to significant reduction of CXCR4 cell surface expression (Figure 2A–B) or total CXCR4 protein expression (data not shown). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. CB 2 agonism at concentrations sufficient to inhibit HIV-1 infection does not significantly alter cell-surface CXCR4 expression or inhibit cellular proliferation in primary CD4+ T cells. (A–B) Cell surface expression of CXCR4 in CD4+ T cells pretreated with either DMSO or 100 nM of JWH-133 (A) in a representative donor and (B) in multiple donors (mean±SEM; n = 4 donors). (C–E) Proliferation, as indicated by CFSE dilution; and activation, as measured by CD25 expression; in CD4+ T cells, stimulated with cross-linking anti-CD3 and anti-CD28 mAb, 4 days after pretreatment with 100 nM of JWH-133 (C) in a representative donor and (D–E) in multiple donors (mean±SEM; n = 5 donors). https://doi.org/10.1371/journal.pone.0033961.g002 Likewise, pretreatment with up to 1 µM of JWH-133 prior to TCR-mediated activation did not reduce T cell activation, as measured by an increase in CD25 expression, or proliferation, as indicated by CFSE dilution after stimulation with anti-CD3 and anti-CD28 antibodies (Figure 2C–E). Concentrations of JWH-133 ranging from 1 µM and below did not lead to a change in cell viability as compared to DMSO treated controls (data not shown). Higher concentrations of JWH-133 or Hu210 (10 µM and above) did lead to apoptosis and cell death, consistent with published observations [11]. Our findings indicate that low doses of the CB 2 R agonist JWH-133 are sufficient to inhibit viral infection without significant disruption of CD4+ T cell co-receptor expression, CD4 expression, CD25 upregulation, or proliferation. Further, CB 2 R agonism did not alter the ability of the CD4+ T cell to support viral infection, as JWH-133 treated cells are readily infected by the CCR5-tropic JRFL virus (Figure 1C). Taken together, these data suggest that CB 2 R activation is altering a pathway specifically required for CXCR4-tropic infection.

The antiviral activity of CB2 agonist JWH-133 is enhanced in unstimulated CD4+ T cells Despite intense study, the importance of GPCR-mediated signaling during CXCR4-tropic HIV-1 infection remains unclear. Recently, several lines of study have demonstrated a role for chemokine receptor signaling in resting CD4+ T cells [44]. Activation of Gαi-coupled chemokine receptors enhances infectivity in resting CD4+ T cells that are stimulated with gp120 or chemokine agonist after exposure to HIV-1 [19], [20]. Conversely, inhibition of GPCR function by pertussis toxin inhibits viral infection [19]. This has been described as a model for latent infection in resting cells [19], [45]. We tested to see if the CB 2 agonist JWH-133 differentially inhibited HIV-1 infection in resting versus activated cells. Cells were activated with mitogen four days before (activated) (Figure 3A) or four days after (resting) (Figure 3B) treatment with 100 nM JWH-133 or 1 µM pertussis toxin and exposure to virus. We found that JWH-133 and pertussis toxin partially inhibited viral replication in both activated and resting cultures (Figure 3). Consistent with previous reports [19], inhibition by pertussis toxin was augmented in resting cultures as compared to activated cultures (59.38±29.5% versus 41.16±20.6%, respectively, *p<0.05) (mean±SEM, n = 4). Similarly, we found that the efficacy of JWH-133–mediated viral inhibition was significantly increased in resting, as compared to activated, cultures (63.34±31.7% versus 36.31±18.2%, respectively, *p<0.05 (mean±SEM, n = 4) (Figure 3C). These data are consistent with the premise that alteration of GPCR signaling predominantly alters viral infection in resting cells. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Inhibition of HIV-1 infection by CB 2 activation is enhanced in CD4+ T cells infected prior to activation. (A–C) Infection, as measured by GFP expression, in primary CD4+ T cells activated with mitogen (A) for 4 days prior or (B) 4 days after treatment with pertussis toxin or 100 nM of the CB2 agonist JWH-133 (A, B) in a representative donor and (C) in multiple donors (mean±SEM; n = 4 donors; *p<0.05). https://doi.org/10.1371/journal.pone.0033961.g003

The CB 2 R agonist JWH-133 decreases F-actin in CD4+ T cells Down-regulation of CXCR4 signaling with pertussis toxin has been shown to decrease actin dynamics, disrupting the remodeling of the cortical actin barrier required for HIV-1 infection [19]. Given the capacity for CB 2 R agonism to inhibit upstream CXCR4-mediated signaling events, we hypothesized that CB 2 R could suppress CXCR4-induced actin polymerization. To test for this possibility, we assayed the ability of JWH-133 to inhibit SDF-1α mediated actin polymerization, as visualized by phalloidin binding. While a significant difference in the rate of SDF-1α induced F-actin accumulation was not detected (data not shown), we did find that JWH-133-treatment led to a significant decrease in phalloidin binding as compared to untreated control cells (Figure 6A–B) (at time 0, 46.2±17.46 versus 64.36±24.34 MFI, respectively; after 30 minutes of SDF-1α treatment, 23.36±11.68 versus 31.3±15.65 MFI, respectively) (mean±SEM, n = 7, **p = 0.0021, *p = 0.04). Although CB 2 R agonism was found to disrupt SDF-1α induced G protein binding and signaling (Figure 4 and 5), this decrease did not translate into acute disruption of the rate of actin polymerization following SDF-1α treatment. Therefore it is possible that CB 2 R exerts its effect at the level of F-actin formation as treatment with JWH-133 reduces the level of F-actin in the steady state. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. Alteration of actin organization in primary CD4+ T cells pretreated with CB 2 agonist. (A–B) Total F-actin, as measured by phalloidin binding, following 3 hours of pretreatment with 100 nM of the CB2 agonist JWH-133 in (A) a representative donor and (B) multiple donors (mean±SEM; n = 7 donors; **p = 0.0021). (C) Normalized change in phalloidin binding as a result of HIV-1-mediated CXCR4 activation in CD4+ T cells pretreated with DMSO or JWH-133 (mean±SEM; n = 4 donors; *p<0.05). (D) Mean phalloidin binding in DMSO or JWH-133 pretreated CD4+ T cells treated for 1 minute with wild type (WT) or Env null (ÄEnv) HIV-1 (mean±SEM; n = 4 donors; *p<0.05). (D–E) Cofilin phosphorylation following HIV-1 or VSV-pseudotyped viral exposure in CD4+ T cells pretreated with DMSO or JWH-133 in (D) a representative donor and (E) in multiple donors (mean±SEM; n = 3 donors; *p<0.05). (F) Cofilin phosphorylation and total cofilin following HIV-1 exposure in CD4+ T cells treated with JWH-133. https://doi.org/10.1371/journal.pone.0033961.g006 During HIV-1 infection, the virus itself acts as an agonist to stimulate CXCR4 and induce actin remodeling in resting cells [46], [47]. We tested to see whether changes in actin reorganization caused by viral binding were altered by CB 2 R agonist pretreatment. To do so, we pretreated CD4+ T cells with JWH-133, incubated these cells with HIV-1 viral particles, and then measured changes in phalloidin binding to F-actin over time. We found that HIV-1 induced transient upregulation in phalloidin binding activity peaking at approximately 1 minute after addition in both control and JWH-133 treated cells (Figure 6C). Like with SDF-1α treatment, the rate of increase in phalloidin binding was not significantly altered in CB 2 R agonist pretreated cells. Consistent with our previous observations, we found that that basal phalloidin binding was significantly reduced in JWH-133 treated cells. The reduction in F-actin formation in JWH-133 treated cells was consistent over time despite the transient upregulation of phalloidin stain induced by virus (Figure 6C). Taken together, these data suggest that reduction of F-actin induced by CB 2 R agonism results in the reduction of total F-actin over time, despite addition of CXCR4 agonists such as SDF-1α or HIV-1 viral particles. The rate of actin polymerization remained constant after acute CXCR4 activation, but the total amount of F-actin induced was significantly lower in cells pretreated with CB 2 R agonist. Indeed, the amount of F-actin induced by wild type virus in JWH-133 pretreated cells was similar to that induced by Env-null viral particles in control treated cells (at 1 minute, 107.5±53.7 versus 95.9±47.9) (mean±SEM, n = 4) (Figure 6D). Therefore, JWH-133 treatment reduces F-actin concentration to background levels, that is, the same level as non-specific induction of actin by Env-null virus. This basal reduction in actin polymerization via CB 2 R may reduce actin rearrangement sufficiently to block viral infection.

CB 2 R agonism decreases HIV-induced cofilin activation HIV-triggered actin rearrangement is regulated in part by the severing protein cofilin, which dissociates and facilitates depolymerization of actin filaments thereby promoting actin dynamics [45]. In the inactive state, cofilin is phosphorylated; agonism of Gαi-coupled chemokine receptors initiates cofilin de-phosphorylation and activity [20]. Induction of cofilin de-phosphorylation increases HIV infection [19], we therefore hypothesized that JWH-133 treatment, which decreases HIV infection, would lead to increased cofilin phosphorylation. To identify changes in cofilin regulation, we measured phosphorylated cofilin (p-cofilin) expression in CD4+ T cells pretreated with JWH-133 and exposed to HIV. We detected a significant increase of p-cofilin over time in JWH-133 treated cells, as compared to control treated cells (216.42±124.95 versus 146.83±84.77, respectively, p-cofilin density normalized to actin at 120 minutes) (mean±SEM, n = 3, *p<0.05) (Figure 6E–F). This data suggests that CB 2 R agonism not only blocks HIV-induced cofilin de-phosphorylation, but also enhances p-cofilin. Indeed, CB 2 R-induced p-cofilin was also observed in cells exposed to a control, VSV-envelope pseudotyped virus (183.11±91.56 JWH-133 versus 138.32±69.16 control, p-cofilin density normalized to actin at 120 minutes) (mean±SEM, n = 4, *p<0.05) (data not shown). We did not observe changes in total cofilin levels in cells treated with HIV and JWH-133 (Figure 6G). Taken together, our data indicate that CB 2 R agonist pretreatment leads to accumulation of p-cofilin as well as the inhibition of cofilin dephosphorylation, i.e. activation in the presence of HIV; findings consistent with a known requirement for cofilin activation in HIV infection [19]. The increase of inactive cofilin in JHW-133 treated cells is a likely mechanism for the reduction of actin dynamism and subsequent inhibition of viral infection in these cells.

CB 2 R agonism inhibits productive infection, but not viral fusion Alteration of actin dynamics has been shown to inhibit productive viral infection but not viral binding or fusion [46]. To confirm that CB 2 R-mediated changes in the actin cytoskeleton did not inhibit viral fusion, we assessed levels of fusion using a β-lactamase (BlaM)-Vpr fusion assay [37]. NL4.3 virions that incorporated BlaM-VPR were used to infect JWH-133 and control treated cells loaded with the β-lactamase substrate CCF2-AM. Upon viral membrane fusion, BlaM-Vpr is released into the cytoplasm where it is able to cleave CCF2-AM. Infection of primary, resting CD4+ T cells induced fusion in approximately 4% of cells (Figure 7A). We found that blockade of HIV binding with the CXCR4 antagonist AMD3100 significantly reduced viral fusion and CCF2-AM cleavage, but pretreatment with JWH-133 had no effect (Figure 7 A–B). This data indicates that CB 2 R antagonism of CXCR4 function does not block the early stages of viral infection binding and fusion. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 7. CB 2 agonism inhibits productive infection of CXCR4-tropic HIV-1, but not cell-associated transfer or viral fusion. (A–B) Viral fusion, as indicated by â-lactamase cleavage, in primary CD4+ T cells pretreated with either JWH-133 or AMD3100 in (A) a representative donor and (B) in multiple donors (mean±SEM; n = 4 donors). (C) Transfer of HIV (Gag-iCherry) from infected Jurkat donors to memory (CD45RO+) or naive (CD45RO-) primary CD4+ T cells pretreated with 100 nM of the CB2 agonist JWH-133 or the CXCR4 antagonist AMD3100, in a representative donor. (D) Quantification of viral HIV (Gag-iCherry) transfer from Jurkat donors after 3 hours in CD45RO+ and CD45RO- cells pretreated with JWH-133 or AMD3100 (mean±SEM; n = 4 donors). (E–G) Productive infection following HIV transfer from primary CD4+ donors infected with HIV (GFP). Pretreated CD4+ targets were cocultured with infected CD4+ donor for 3 hours, sorted by CD45RO expression, and then activated. HIV (GFP) expression is shown in (E) CD45RO- naive or (F) CD45RO+ memory CD4+ T cells after 4 days of activation with IL-2 and anti-CD3/CD28 or anti-CD3 alone, respectively. (G) Quantification of productive infection post-activation in sorted CD45RO- or CD45RO+ CD4+ T cells pretreated with JWH-133 or AMD3100 (mean±SEM; n = 4 donors; **p = 0.0099; *p<0.05). https://doi.org/10.1371/journal.pone.0033961.g007