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. Author manuscript; available in PMC: 2016 Sep 1.
Published in final edited form as: Virology. 2015 May 15;483:185–202. doi: 10.1016/j.virol.2015.03.036

Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR

Geetaram Sahu 1, Kalamo Farley 1, Nazira El-Hage 2, Benjamas Aiamkitsumrit 1, Ryan Fassnacht 1, Fatah Kashanchi 3, Alex Ochem 4, Gary L Simon 1, Jonathan Karn 5, Kurt F Hauser 2, Mudit Tyagi 1,6,#
PMCID: PMC4516702  NIHMSID: NIHMS679748  PMID: 25980739

Abstract

Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-κB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-κB at 276th serine residue. These modifications enhance the interaction of NF-κB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication.

Keywords: HIV-1, Cocaine, NF-κgreen, MSK1, RSK1, H3, p65, chromatin, transcription, elongation

INTRODUCTION

Cocaine is one of the most widely abused drugs in the United States and remains a significant cofactor for human immunodeficiency virus type 1 (HIV-1) infection and transmission. In addition to increasing the severity of the infection (Arthur, 2008; Ioannidis et al., 2000; Skalka, 2013), cocaine impairs the normal function of the cells within the brain and promotes HIV-1 replication in the central nervous system (CNS) (Nath et al., 2002; Nath et al., 2001). The long-lived CNS glial cells, microglia, macrophages and astrocytes are the main targets and reservoirs of HIV-1 infection in the brain (Nath et al., 2002; Nath et al., 2008). Previous studies reported that HIV-1 infected individuals who abuse cocaine experience a more severe and rapid onset of neuro-acquired immunodeficiency syndrome (neuroAIDS) than non-abusing individuals (Anthony et al., 1991; Chiasson et al., 1990; Ioannidis et al., 2000; Nath et al., 2001; Parikh et al., 2014). It has been well established that cocaine modulates several signaling pathways primarily MAPK pathway, PI3K/Akt pathway and PKC signaling pathways (Estaquier et al., 2013; Ioannidis et al., 2000; Liu et al., 2000; Nath et al., 2000a; Parikh et al., 2014; Skalka, 2013), which result in the activation of downstream protein factors, including transcription factors. These downstream transcription factors in addition to modifying the expression of cellular genes, modulate the expression of integrated HIV-1 proviruses and ultimately enhance HIV-1 replication, transmission and HIV-1-associated neurological disorders (HAND) (Hauser et al., 2006; Nath et al., 2000b; Nath et al., 2002). However, the mechanism underlying the complex interplay between drugs of abuse and HIV-1 replication is still uncertain.

Similar to the transcription of most cellular genes, HIV-1 transcription progresses through two major phases, initiation and elongation, in order to generate full viral genomic transcripts (Estaquier et al., 2013; Kilareski et al., 2009; Kobor and Greenblatt, 2002; Krogan et al., 2002; Liu et al., 2000; Nath et al., 2000a; Turk and Stoka, 2007). Transcriptional initiation efficiently starts when transcription factors such as specificity protein 1 (Sp1), nuclear factor (NF)-κB and nuclear factor of activated T cells (NFAT) proteins bind to the HIV-1 Long Terminal Repeat (LTR) (Karn, 2011; Kinoshita et al., 1997; Nabel and Baltimore, 1987; Perkins et al., 1997a). These factors subsequently promote the recruitment of histone modifying enzymes which establish transcriptionally active chromatin structures at the HIV-1 LTR and facilitates the recruitment of other components of transcription machineries at LTR promoter (Kilareski et al., 2009; Kobor and Greenblatt, 2002; Krogan et al., 2002; Peterlin and Price, 2006; Turk and Stoka, 2007). RNA polymerase II (RNAP II) initiates the elongation phase of HIV-1 transcription, but it halts just after generating incomplete shorter transcripts due to the binding of inhibitory complexes, which primarily include the negative elongation factor (NELF) and the 5,6-dicholoro-1-β-D-ribofuranoxylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) at the HIV-1 LTR (Jadlowsky et al., 2014; Zhang et al., 2007). To overcome this inhibition, HIV-1 has a specialized protein, trans-activator of transcription (Tat), that directly interacts with elongation factor and recruits the host protein complex, positive transcriptional elongation factor b (P-TEFb) to the HIV-1 LTR (Rockstroh et al., 2011; Volberding and Deeks, 2010; Wei et al., 1998). P-TEFb consists of an enzymatic component, cyclin-dependent kinase 9 (CDK9) and cofactor cyclin T (T1 or T2) (Lee et al., 2006). P-TEFb is required for the generation of full-length HIV transcripts as the lack of P-TEFb in quiescent T cells has shown to be one of the major factors that restrict HIV-1 transcription during latency (Budhiraja et al., 2013; Tyagi et al., 2010). The recruitment of P-TEFb at the HIV-1 LTR, primarily facilitated by the Tat protein of HIV-1 (Peterlin and Price, 2006; Wei et al., 1998), enhances the processivity of RNAP II by hyper-phosphorylating the C-terminal domain (CTD) of RNAP II (Adelman and Lis, 2012). Moreover, P-TEFb also phosphorylates the different subunits of inhibitory complexes leading to either their removal from the LTR or their conversion into positive factors (Bourgeois et al., 2002; Fujinaga et al., 2004; Isel and Karn, 1999; Kim et al., 2002; Parada and Roeder, 1996). These events relieve all the restrictions, allowing the recruitment of a super elongation complex at HIV-1 LTR, resulting in a productive elongation phase (He et al., 2011; Liu et al., 2012; Sobhian et al., 2010).

Cocaine activates NF-κB, an important transcriptional activator (Ang et al., 2001; Dhillon et al., 2007; Hou et al., 1996; Yao et al., 2010). The NF-κB family consists of five related proteins, p65 (Rel A), Rel B, c-Rel, p50/p105 (NF-κB) and p52/p100 (NF-κB2) and they can form homo- or heterodimers with each other (Hayden and Ghosh, 2004). Of these, one of the best-characterized NF-κB heterodimers is comprised of Rel A (p65) and p50, which widely expressed and heavily involved in NF-KB-regulated transactivation (Nath et al., 2001; Schmitz and Baeuerle, 1991). In its inactive state, the NF-κB dimer is bound to the inhibitory IκB in the cellular cytoplasm (Hayden and Ghosh, 2004; Sobhian et al., 2010). Upon stimulation, serine residues 32 and 36 (S32 and S36) of IκB are phosphorylated by the IκB kinase (IKK) complex, which is composed of IKKα, IKKβ and IKKγ (Bonizzi et al., 2004; Bonizzi and Karin, 2004; Brown et al., 1995; Hayden and Ghosh, 2004). This leads to the ubiquitination of IκB at lysine residues 21 and 22 (K21 and K22) and leads to the 26S proteasome degradation and the subsequent activation and nuclear translocation of NF-κB (Brown et al., 1995; Kerr et al., 1991). During and following nuclear translocation, the p65 subunit gets phosphorylated at selective serine residues each of which play a different role in the transcriptional activation of NF-κB (Naumann and Scheidereit, 1994). Phosphorylation of p65 at serine 536 (S536) promotes its nuclear translocation and is primarily catalyzed by IKKβ and ribosomal S6 kinase-1 (RSK1) (Bohuslav et al., 2004; Brami-Cherrier et al., 2005; Chandrakesan et al., 2010). The phosphorylation of p65 at serine 276 (S276) enhances its functional activity (Hu et al., 2004) which supports the association of NF-κB with chromatin remodeling enzymes such as histone acetyltransferase (HATs) (Perkins et al., 1997a; Vermeulen et al., 2002; Vermeulen et al., 2003; Yao et al., 2010; Zhong et al., 2002).

Mitogen-and stress-activated protein kinase 1 and 2 (MSK1 and MSK2) are two protein isoforms of MSK that are expressed predominantly in the nucleus and are involved in cellular proliferation and survival (Dunn et al., 2005; Vermeulen et al., 2003). Both MSK proteins require a series of phosphorylation steps to become catalytically proficient, prior to phosphorylating or activating other proteins in downstream cascades (Dunn et al., 2005; Vermeulen et al., 2003). MSK1 has been identified as one of the kinases that catalyze the phosphorylate histone H3 at its serine residue 10 (Thomson et al., 1999). Besides histones, MSK proteins phosphorylate various protein targets including histone proteins, cyclic AMP-responsive element binding protein (CREB), ATF1, NF-κB, as well as genes involved in transcriptional activation such as c-fos and c-jun (Dunn et al., 2005; Panneer et al., 2014; Soloaga et al., 2003; Vermeulen et al., 2003). In the context of cocaine, previous studies in a mouse model have shown a role for MSK1 in the activation of the immediate early gene, c-fos, and the extracellular signal-regulated kinase (ERK) signaling pathway in the striatum following cocaine exposure (Ang et al., 2001; Brami-Cherrier et al., 2005; Dhillon et al., 2007; Hou et al., 1996; Nowak and Corces, 2004; Yao et al., 2010). However, its role in human cells and how MSK1 influences HIV-1 transcription have never been investigated. In this study, for the first time, we established the important role of MSK1 during HIV-1 transcription, by performing a comprehensive investigation using myeloid cell lines and primary cells. We found that MSK1 promotes both the initiation and elongation phases of HIV-1 transcription by catalyzing the phosphorylation of p65 at serine 276, p-p65 (S276) and histone H3 at serine residue 10, p-H3S10, respectively. Phosphorylation of p65 subunit of NF-κB at 276 enhances the interaction and recruitment of HATs, such as p300 at gene promoters and the induced euchromatin structures further promotes the access to transcriptional machinery at promoter and augment the initiation phase of HIV transcription. In parallel MSK1 induced p-H3S10 epigenetic modification, besides facilitating the establishment of transcriptionally active chromatin structures promotes the recruitment of P-TEFb to the HIV LTR {Ivaldi, 2007 #3345;Kizaki, 2009 #5052;Itzen, 2014 #5053}. Consequently, cocaine is able to accelerate HIV-1 transcription by promoting both initiation and elongation of viral transcripts via activating NF-κB and MSK1.

MATERIALS AND METHODS

Transfection and VSV-G pseudotyped virus generation

Human Embryonic Kidney 293 cells (HEK 293 or 293T) cells were cultured with RPMI-1640 media supplemented with 2.05mM L-glutamine (Hyclone, ThermoScientific), 10% fetal bovine serum (Gemini), and 1U/mL penicillin/streptavidin. Cells were grown to 70–80% confluency and washed with Opti-MEM® GlutaMAX reduced serum media (Gibco) prior to transfection. Transfection with Lipofectamine 3000 (Invitrogen) was carried out according to the manufacturer’s protocol. Briefly, 35 µL of Lipofectamine 3000 reagent was diluted in 500 µL Opti-MEM. In a separate tube, 18 µg of plasmid DNA mixture (4µg pMD.G, 3 µg pCMVΔ 8.9.1, 3µg pMDL-g/p-RRE, 1µg pRSV-Rev, and 7 µg of either pNL4-3-ΔE-EGFP or pHR’-P-Luc, in order to generate a NL4-3-ΔE-EGFP and a HR’-P-Luc pseudotyped viruses, respectively) (Bonacini et al., 1999; Butler et al., 2001; Dull et al., 1998; Jadlowsky et al., 2014; Naldini et al., 1996; Zhang et al., 2004) and 35 µL of P3000 reagent were diluted in 500µL Opti-MEM. The two separated dilutions were mixed and allowed to incubate at room temperature for 10 minutes to form the lipid-DNA complex, which were then added to the cells. Three to five hours following the addition of the transfection cocktail, RPMI culture medium was added to the cells. The virus containing cell supernatant was collected at 48 hours and 72 hours post transfection. The successful generation of replication incompetent pseudotyped virus expressing EGFP reporter gene under the control of the HIV-1 LTR was confirmed by fluorescent microscopy or FACS analysis.

Monocyte-derived macrophages (MDM) isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by density centrifugation. Briefly, the blood samples were spun at 400xg for 10 minutes and the plasma layer removed. The cells were then diluted with Hank’s balanced salt saline (HBSS) and layered onto Ficoll- Hypacque (1.077 g/ml; GE Healthcare) and spun at 2000 rpm for 30 minutes with no braking. The PBMC layer was collected, washed once with HBSS and plated in T75 cell culture flasks in RPMI for 3 – 4 hours at 37°C. The non-adherent cells were aspirated, and the remaining adherent monocytes were washed once with RPMI and incubated with media supplemented with 100ng/ml M-CSF (Biolegend) for 7 days to allow for differentiation to macrophages.

Viral Titer

The viral titer was assessed by infecting defined number of Jurkat cells with the different dilutions of concentrated Vesicular Stomatitis Virus g protein (VSV-G) pseudotyped lentiviral particles. The viral titer was assessed by calculating the number of GFP positive cells obtained in higher dilutions of virus. The cell lines were infected with about one multiplicity of infection (MOI) of viral particles; however for primary cells we used two MOI of virus.

Cocaine Treatment

The cocaine-HCl (referred to as “cocaine” hereafter) was obtained from National Institute on Drug Abuse (NIDA). The concentration of cocaine to be utilized was first optimized by a dose-response experiment of cocaine concentration and HIV-1 transcription using THP-1 cells. MDM and human THP-1 monocytic cells were treated with 5 µM cocaine for 10 to 60 minutes in our acute model of cocaine treatment. In the chronic model of cocaine treatment the cells were treated with 5 µM cocaine for 72 hours, twice a day. To assess the effects of cocaine on HIV-1 gene expression, MDM and U937 cell were pre-incubated with 1–10 µM of cocaine for 15 hours and then treated with the HIV-1NL4-3-ΔE-EGFP pseudotyped virus. Cocaine was added daily for 7 days to MDM cells and 2 days to U937 cell and GFP expression levels were then determined by FACS analysis.

Cytoplasm and Nuclear extract preparation

5 × 106 HIV-1 infected cells were plated in 6-well plates overnight and incubated with 5 µM cocaine for 30 minutes to 72 hours as indicated in each experiment. The cells were collected and washed with ice-cold PBS. The samples were then lysed by incubating on ice for 10 minutes with 0.5% NP-40 supplemented with 10 mM HEPES-KOH pH 7.9, 60 mM KCL, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and a protease inhibitor cocktail (Roche), followed by brief vortexing and centrifuged for 10 minutes at 4°C. The cytoplasmic protein extract was collected and the remaining nuclear pellet was washed with 500 µL of ice-cold PBS. The purified nuclei were then lysed by 6 freeze-thaw cycles in a buffer comprising of 250 mM Tris pH7.8, 60 mM KCL, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and a protease inhibitor cocktail. The nuclear protein extract was collected by centrifugation, and solubilized with 5x SDS-PAGE loading buffer. For optimal histone protein isolation, the nuclear extracts were sonicated by three 10-second pulses. The sonicated nuclear extract samples were subsequently collected by centrifugation at 13,000 rpm for 2 minutes and solubilized with the 5X SDS-PAGE loading buffer.

Western Immunoblotting

The cytoplasm and nuclear protein extracts were run on 4–15% Tris-glycine gels (Bio-Rad) and transferred to nitrocellulose membranes (GE). The proteins of interest were detected by incubating with primary antibodies such as NFkB-P65(C-20)(Cat.no:SC-372), IKKα (Cat.no:SC-7218), IKKβ (Cat.no:SC-7329), RSK1(Cat.no:SC-130870), MSK1(Cat.no:SC-25417), H3-Histone(Cat.no:SC-8654), SPT5(Cat.no:SC-28678), GAPDH(Cat.no:SC-25778), Lamin A/C(Cat.no:SC-7292), Phospho-RSK1(Thr348)(Cat.no:SC-101770), Phospho-p65(Ser 276) (Cat.no: SC-101749) were purchased from Santa Cruz Biotechnology. Antibodies against p65 (phospho-S536; Cat no-3033), phospho-MSK1 (Thr 581) (Cat no-9595), Phospho-IKKα (Ser 176/180)(Cat no-2697) and phospho-Histone-H3-Serine 10(Cat no-12201) were purchased from Cell Signaling. Antibodies against Phospho-IKKβ (Tyr 199) (Cat no-PA5–35838) were purchased from life technologies. The appropriate secondary antibodies were applied after 3 washes with 0.05% Tween 20 in phosphate or Tris buffered saline (PBST/TBST). The blots were incubated with substrate ECL (Santa Cruz) prior to exposure to X-ray film (Fuji). GAPDH, Lamin A/C, Histone H3 and SPT5 were used as loading controls for cytoplasmic and nuclear protein fractions. Densitometric analysis on the western blots was performed using NIH-Image J software.

Chromatin Immunoprecipitation (ChIP) assay

ChIP analysis was performed as previously described (Tyagi et al., 2010). Briefly, (1×108) THP-1 cells were infected by the virus carrying pNL4-3-ΔE-EGFP. In this assay we used 30–40% of infected cells per antibody. The infected THP-1 cells were stimulated with 5µM cocaine for 1hour or 6 hours (as indicated in each experiment) in petri plates. DNA and proteins were cross-linked with 0.5% formaldehyde followed by cell lysis in the buffer previously described. The remaining nuclei were lysed with 1% SDS buffer (supplemented with 10 mM EDTA, 50 mM Tris HCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and a protease inhibitor cocktail). For each immunoprecipitation reaction nuclear extract of 5 to 8 million cells was used. The immunoprecipitation was performed with antibodies specific to transcription factors required for HIV transcription including RNAPII(Cat no:SC-9001, Santa Cruz Biotech), HDAC3 (Cat no:SC-11417, Santa Cruz Biotech), p-p65(Ser 276) (Cat no:SC-101749, Santa Cruz Biotech), p-MSK1(Thr 581)(Cat.no:9595,Cell Signaling ), P300 (Cat no: SC-584, Santa Cruz Biotech), c-Jun (Cat no: SC-1694, Santa Cruz Biotech), Cyclin T1(Cat no:SC-8127, Santa cruz Biotech). The chromatin structure at HIV-1 LTR was analyzed by utilizing antibodies specific to selective epigenetic modifications such as, Ac-H3(Cat no: 07-352,Millipore), H3K9me3 (Cat no: 07-442, Millipore), H3K27me3(Cat no:07-449, Millipore), H4K12-Ac(Cat no: 04119, Millipore) and phospho-Histone-H3-Serine10(Cat no: 12201, Cell signaling). The samples were then reverse cross-linked and the immunoprecipitated DNA was analyzed by q-RT-PCR.

Quantitative Real Time PCR

Each ChIP DNA sample was analyzed by quantitative real-time PCR to determine the amount of sample immunoprecipitated by the antibody sets. Primers sets to the promoter region −116 to +4 (5’-AGCTTGCTACAAGGGACTTTCC-3’ and 5’-ACCCAGTACAGGCAAAAAGCAG-3’), and to the nucleosome 1 region +30 to +134 (5’-CTGGGAGCTCTCTGGCTAACTA-3’ and 5’-TTACCAGAGTCACACAACAGACG-3’) were used to examine the proviral genome. The quantitative data analysis was normalized with the appropriate IgG control and GAPDH.

Luciferase Assay

2×106 THP-1-pHR’-P-Luc-IRES-GFP cells were plated in 12-well plates with RPMI supplemented with 10% FBS, penicillin and streptavidin. The cells were incubated with 5 µM cocaine for 48 hours prior to harvesting and washed twice with PBS. Luciferase levels in the cells were assessed using a Luciferase Assay System kit (Promega). Briefly, the cells were lysed for 30 minutes at room temperature with passive lysis buffer and centrifuged at 14,000rpm for 2 minutes. 10 µl of each sample was added to individual wells, followed by 50 µl of luciferase substrate/assay buffer. Each sample was tested in triplicate. Luminescence was read in a Veritas Microplate Luminometer (Turner Biosystems).

Flow cytometry (FACs) analysis

FACS analyses were performed on cells infected with VSV-G pseudotyped HIV-1 virus carrying the GFP gene under control of the HIV-1 LTR promoter. The analyses were done with a FACS Calibur (BD Biosciences), using FlowJo software (Treestar Inc.) to collect and analyze the data.

Statistical Analysis

All analyses were performed using GraphPad Prism software. Data were analyzed by T test or One-way Anova. Data were expressed as means ± SEM and a p-value < 0.05 was considered statistically significant.

RESULTS

Cocaine enhances HIV-1 gene expression

THP-1 cells were infected with a HR’P-Luc pseudotyped virus which is an HIV-1 based lentiviral vector expressing luciferase reporter gene under the control of HIV-1 LTR promoter (Dull et al., 1998; Pearson et al., 2008; Tyagi and Karn, 2007; Zufferey et al., 1998). The cells were treated with different concentrations of cocaine, 2.5 µM, 5 µM and 10 µM for 48 hours. These cells were then lysed and the level of the reporter protein expression was determined by the luciferase assay. It was shown that cocaine treatment induced HIV gene expression in a dose-dependent manner (Figure 1A). Specifically, incubation with 5 µM of cocaine led to an approximate 2-fold increase in HIV-1 transcription (Figure 1A). Following incubation, we determined if the effect of cocaine treatment on viral transcription observed in THP-1 cells held true in other monocytic cell-types. 5×105 primary monocyte derived macrophages (MDM) or U937 cells were plated in 12-well plates, followed by pre-incubation with 1, 5 or 10 µM cocaine for 15 hours prior to infection with the HIV-1NL4-3-ΔE-EGFP VSV-G pseudotyped virus. We observed that after seven days of daily cocaine treatment, there is a dose-dependent increase in the GFP expression, indicating an increase in HIV-1 gene expression within the infected MDM cells (Figure 1 B-E). Similarly, cocaine treatment increased the expression of GFP after only two days infection in a dose-dependent manner in U937 cells (Figure 1 F-I). Although the 10 µM dose of cocaine produced the strongest response in the cell lines tested, the 5 µM concentration dose was chosen for all subsequent experiments because it fell well within physiological range and could still significantly stimulate the cells {Nair, 2000 #3636}. In addition, there was no decrease in cell viability following cocaine treatment as determined by trypan blue (data not shown).

Figure 1. Cocaine accelerates HIV-1 transcription in a dose-dependent manner in THP-1, U937 and Monocyte-derived Macrophages (MDMs).

Figure 1

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(A) THP-1-pHR’-P-Luc cells were treated with 2.5 – 10µM of cocaine for 48 hours. The cells were lysed and the luciferase assay was performed on the cell lysates. In all treated samples luciferase levels were calculated as a percentage of the untreated group. Statistical analysis was done in 3 experiments * p<0.05, ** p<0.01 vs. unstimulated control. MDM and U937 cells were pre-incubated with 1, 5 or 10 µM cocaine for 15 hours then treated with the HIV-1NL4-3-ΔE-EGFP VSVG-pseudotyped virus. The primary MDM cells were further exposed to cocaine once daily for 7 days, and GFP expression within (B) untreated and (C) 1µM, (D) 5 µM and (E) 10 µM cocaine-treated MDM was analyzed by FACS. GFP expression within the (F) untreated and (G) 1µM, (H) 5 µM and (I) 10 µM cocaine-treated U937 cells was assessed two days after cocaine treatment. Statistical analysis was done from 3 separate experiments; * p<0.05, ** p<0.01 vs. unstimulated control.

Cocaine enhances the nuclear translocation of NF-κB in HIV-1 infected cells

The activation of HIV-1 gene expression involves the interaction of several transcription factors with specific DNA binding sites within HIV-1 LTR. NF-κB is one of the nuclear proteins that play an important role in the expression of several cellular genes, and its binding sites are found within HIV-1 LTR (Kilareski et al., 2009; Nabel and Baltimore, 1987). Therefore, the activation of NF-κB upon cocaine treatment was assessed in our system. THP-1 cells were infected with replication incompetent HIV-1 (NL4-3-ΔE-EGFP) overnight. The following day, the NL4-3-ΔE-EGFP virus infected-cells were treated with 5 µM cocaine for different time intervals (30 min to 6 hour) in our acute cocaine intake model. The translocation of NF-κB, from cytoplasm to nucleus was evaluated by western blotting using cytoplasmic and nuclear fractions.

The nuclear levels of the p65 subunit of NF-κB significantly increased following cocaine treatment with more than 2 fold at 30 minutes and peaking at around 3 hours (Figure 2B and Figure S1). Corresponding decrease in the cytoplasmic levels of p65 was quite evident (Figure 2A and Figure S1), illustrating the nuclear translocation of cytoplasmic p65 protein following cocaine treatment. Nuclear translocation of p65 was even more pronounced in case of TNF-α treatment, which is one of the strongest and specific activators of NF-κB. Although cocaine is not as potent as TNF-α in inducing the translocation of NF-κB, cocaine nevertheless is a highly efficient activator of NF-κB. Similarly, the nuclear translocation of NF-κB was also observed in our chronic model of cocaine treatment although not as profound as in acute model. In the chronic model of cocaine treatment cells were treated repeatedly for 3 days with 5 µM of cocaine every 12 hours in order to mimic chronic/repeated cocaine intake conditions (Figure 2C and 2D). In the chronic model p65 levels peak at 48 hours. Longer treatment (72 hours) did not enhance p65 levels further. Together, these results suggested that cocaine intake promotes HIV-1 gene expression by enhancing both the nuclear translocation and functional activity of NF-κB (detailed in the following sections).

Figure 2. Cocaine exposure significantly enhances the nuclear translocation of NF-κB in both acute and chronic models of cocaine treatment.

Figure 2

Figure 2

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HIV-1 infected THP-1 cells were treated either acutely (A and B) or chronically (C and D) with 5µM cocaine. For acute exposure, cells were treated once with 5µM cocaine for indicated time intervals; whereas for chronic treatment cells were treated twice a day for up to 3 days. Subsequently, the nuclear translocation kinetics of NF-κB from cytoplasm was assessed by probing for p65 subunit. For cytoplasmic fractions (A and C) GAPDH was used as loading control; while Lamin A/C was used as loading control for nuclear fractions (B and D). As positive control for NF-κB activation, cells were treated with TNF-α (10 ng/ml). Densitometric analysis was done on all immunoblots and statistical analysis was done from 5 experiments; * p<0.05, ** p<0.01 vs. unstimulated control.

Cocaine activates NF-κB by enhancing its phosphorylation at serine residue 536

It has been well established that the phosphorylation of p65 subunit of NF-kB at serine residue 536 (S536) promotes and marks the nuclear translocation of NF-κB due to its uncoupling from the cytoplasmic inhibitory IκB complex {Bohuslav, 2004 #6304;Chandrakesan, 2010 #6305;Brami-Cherrier, 2005 #3546;Hayden, 2012 #4827}. Therefore, we assessed the p65 subunit phosphorylation at S536 following cocaine treatment to determine if this was part of the molecular mechanism of NF-κB activation by cocaine in HIV-infected cells. The NL4-3-ΔE-EGFP virus infected THP-1 cells were treated with 5 µM cocaine acutely and chronically. We found cocaine treatment greatly enhanced the phosphorylation of S536 of p65, P-p65(S536) (more than 4 fold within 30 minute) and remain higher even after 6 hours (Figure 3B). However, cytoplasmic levels of P-p65(S536) did not show significant change following cocaine exposure (Figure 3A). Stimulation with TNF-α was used as a positive control and resulted in a substantial increase in p65 phosphorylation at serine 536 (more than 6 folds, Figure 3B). Interestingly, analogous to the nuclear translocation of p65 (Figure 2D), we found a similar pattern of S536 phosphorylation during chronic cocaine treatment (Figure 3D), with peak phosphorylation at 48 hours. Overall, the results so far have shown that cocaine enhances HIV gene expression by activating NF-κB.

Figure 3. Cocaine enhances phosphorylation of the p65 subunit of NF-κB at S536.

Figure 3

Figure 3

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Phosphorylation of cytoplasmic and nuclear NF-κB at serine 536 was assessed by immunoblotting following acute (A and B) or chronic (C and D) cocaine exposures to HIV-infected THP-1 cells for indicated time periods. GAPDH and Lamin A/C were used as loading controls for cytoplasmic and nuclear extracts, respectively. Cells treated with 10 ng/ml TNF-α were used as positive control. Densitometry analysis was done for all immunoblots. Densitometry analysis was done on all immunoblots and statistical analysis was done from 5 experiments; * p<0.05, ** p<0.01 vs. unstimulated control.

Cocaine enhances nuclear translocation of NF-κB by activating IKKβ and RSK1

It is established that in addition to inhibitory kappa B (IκB) phosphorylation, the ribosomal S6 kinase-1 (RSK1) and IκB kinase (IKK) complexes catalyze the phosphorylation of p65 at S536, and facilitate the nuclear translocation of NF-κB {Bohuslav, 2004 #6304;Chandrakesan, 2010 #6305;Brami-Cherrier, 2005 #3546;Hayden, 2012 #4827;Hu, 2004 #2704;Naumann, 1994 #4831}. Therefore, the cell extracts from cocaine treated NL4-3-ΔE-EGFP virus infected THP-1 cells were analyzed for the functionally active (phosphorylated) forms of IKKβ (Tyr 199) and RSK1 (Thr 359) both in acute and chronic cocaine treatment models. Following acute cocaine treatment, we found a sizeable increase in the phosphorylation level of IKKβ (Figure 4A) at 30 min, whereas total IKKβ protein level significantly increases from 30 min to 3 hours and then returns to basal level at 6hours. In our chronic model, we observed that, phosphorylated forms of IKKβ (Tyr 199) increases for up to 48 hours of cocaine treatment and then return to basal level at 72 hour; we found no change in the level of total IKKβ in the chronic cocaine treatment model (Figure 4B).

Figure 4. Cocaine promotes the nuclear translocation of NF-κB by activating IKKβ and RSK1.

Figure 4

Figure 4

Figure 4

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The cell lysates of the HIV-1-infected THP-1 cells were examined for the stimulation of the total and phosphorylated forms of IKKβ and RSK1 at tyrosine 199 and threonine 359, respectively. Total and the catalytically active, phosphorylated, forms of enzymes were analyzed by immunoblotting following acute (A and C) or chronic (B and D) cocaine exposures to HIV-infected THP-1 cells for time periods as indicated. GAPDH was used as loading control for cytoplasmic fraction and Lamin A/C for nuclear fraction. Densitometry analysis was done on all immunoblots and statistical analysis was done from 5 experiments; * p<0.05, ** p<0.01 vs. unstimulated control.

In the case of RSK1, there was a significant increase in the phosphorylation level of RSK1 at threonine residue position 359 (T359) in both acute and chronic cocaine treatment (Figure 4C and 4D), showing the induction of catalytically active forms of RSK1. In contrast, the total RSK1 levels were essentially constant. These results firmly establish the role of these enzymes in catalyzing the phosphorylation of p65 at S536 following cocaine administration.

Cocaine enhances NF-κB functional activity by promoting its phosphorylation through MSK1 activation

The phosphorylation of p65 at S276, primarily catalyzed by MSK1, has been shown to enhance the functional activity of NF-κB by facilitating its interaction with histone acetyl transferases (HATs) (Perkins et al., 1997b; Vermeulen et al., 2003; Zhong et al., 2002). In mouse striatum cocaine was reported to activate MSK1 (Brami-Cherrier et al., 2005). We therefore hypothesized that cocaine-activated MSK1 could lead to the phosphorylation of p65 at S276 in human myeloid cells. The NL4-3-ΔE-EGFP virus infected THP-1 cells were acutely treated with 5 µM cocaine. Cytoplasmic and nuclear extracts were subsequently examined by western immunoblotting using an antibody against the phosphorylated form of p65 at Ser 276, P-p65 (S276). We found that cocaine treatment substantially enhanced the phosphorylation of p65 at S276 in the nucleus (Figure 5B). However in the cytoplasm, we did not detect the phosphorylation of p65 at S276 (Figure 5A). TNF-α treated cells were used as a positive control. In our chronic model of cocaine treatment, the levels of P-p65 (S276) peaked at 48 hours, however further cocaine treatment resulted in its reduction; akin to the total p65 levels (Figure 2D). Similar to the acute treatment, we did not detect P-p65 (S276) in the cytoplasmic fraction of chronically treated cells (Figure 5C).

Figure 5. Phosphorylation of the p65 subunit at serine 276 is significantly increased during both acute and chronic cocaine treatments.

Figure 5

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HIV-1 infected THP-1 cells were treated with 5µM cocaine acutely (A and B) or chronically (C and D) for different time intervals. NF-κB phosphorylated at serine 276 (p-p65 (Ser 276) was assessed by western immunoblotting. GAPDH is used as a loading control for cytoplasmic extract (A and C) and Lamin A/C used as a loading control for nuclear extract (B and D). Cells treated with 10 ng/ml TNF-α were used as positive control. Densitometry analysis was done on all immunoblots and statistical analysis was done from 5 experiments; * p<0.05, ** p<0.01 vs. unstimulated control.

In parallel experiments, NL4-3-ΔE-EGFP virus infected THP-1 cells were treated with 5 µM cocaine and the activation of MSK1 was assessed by evaluating the phosphorylation of MSK1 at threonine residue 581 (T581), a critical modification that indicates the functionally active form of MSK1 (McCoy et al., 2005). As anticipated, analogous to the phosphorylation of p65 (S276) (Figure 5B and 5D), we noticed markedly enhanced phosphorylation of MSK1 at T581 in nucleus, following cocaine exposure (Figure 6B and 6D). Notably, a rapid activation of MSK1 upon acute cocaine exposure was observed (Figure 6B). During chronic cocaine treatment we also found sizable MSK1 phosphorylation. Interestingly we detected a corresponding loss of unphosphorylated form of MSK1, especially during chronic treatment (Figure 6D).

Figure 6. Cocaine profoundly activates MSK-1 during both acute and chronic cocaine exposures.

Figure 6

Figure 6

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We characterized the impact of cocaine in inducing catalytically active form of MSK1, besides total MSK1 levels, after treating HIV-1 infected THP-1 cells with 5µM cocaine either acutely (A and B) or chronically (C and D). The cytoplasmic and nuclear fractions of the cells were probed with antibody specific for phospho-MSK1 at threonine 581 (T581) and total MSK1 antibody. GAPDH was used as loading control for cytoplasmic fraction and Lamin A/C for nuclear fraction. Densitometry analysis was done on all immunoblots and statistical analysis was done from 5 experiments; * p<0.05, ** p<0.01 vs. unstimulated control.

The lack of phospho-p65 (S276) in the cytoplasmic fraction (Figure 5A and 5C) was further confirmed by the absence of the functionally active form of MSK1 (Figure 6A and 6C) Thus, direct correlation between phospho-MSK1 (T581) and phospho-p65 (S276) levels suggests that MSK1 is the primary kinases that catalyzes the phosphorylation of the p65 subunit, specifically at S276 following cocaine exposure, thereby enhancing the functional activity of NF-κB.

Cocaine activated MSK1 promotes histone H3 phosphorylation

The functional role of the phosphorylation at the serine residue 10 of histone H3 (H3S10) during transcription was highlighted by the recent discovery that this event is crucial for recruiting P-TEFb to the gene promoters via the bromodomain protein 4 (Brd4) (Itzen et al., 2014; Kizaki et al., 2009). Accordingly, the temporal changes in the phosphorylation of H3S10 (p-H3S10) were investigated in the cocaine treated NL4-3-ΔE-EGFP virus-infected THP-1 cells by western blotting. In parallel to the observed MSK1 activation kinetics, a significant increase in the level of p-H3S10 was observed just after 30 minutes of cocaine treatment (Figure 7A). Furthermore, similar to the levels of MSK1 activation, the higher levels of p-H3S10 persisted for up to 72 hours in the chronic treatment model (Figure 7B). In order to confirm the direct role of MSK1 in catalyzing p-H3S10 phosphorylation, we analyzed the impact of the MSK1 inhibitor, RO31-8220 (Santa Cruz), in restricting MSK1 activity and corresponding H3S10 phosphorylation. The potent inhibition of cocaine-induced MSK1 activation was observed following 1 hour pre-incubation with 5 µM RO31-8220 (Figure 7C). This inhibition also coincided with a significant decrease in H3S10 phosphorylation induced by cocaine (Figure 7D). These results confirm that cocaine activated MSK1 plays a major role in H3S10 phosphorylation following cocaine treatment.

Figure 7. Cocaine augments the phosphorylation of H3S10 via MSK1 activation.

Figure 7

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HIV-1- infected THP-1 cells were treated with acutely and chronically with 5 µM cocaine (A and B), lysed and the nuclear fraction sonicated and analyzed for the phosphorylated form of H3S10 by immunoblotting. Total Histone H3 was used as a loading control. In panel C and D, cells were pre-treated with MSK1 inhibitor RO31 (3 µM and 5 µM) for 1 hour, before treating them with cocaine (5 µM) for 30 min. Nuclear fractions were assessed for p-MSK1 (C) and p-H3S10 (D) before and after treating cells with MSK1 inhibitor RO31 (5µM). Densitometry analysis was done on all immunoblots and statistical analysis was done from 4 experiments; * p<0.05, ** p<0.01 vs. unstimulated control.

Cocaine activated IKKα too promotes histone H3 phosphorylation

Besides MSK1, several other enzymes are known to catalyze the phosphorylation of H3S10. IKKα is a primary example of such an enzyme. In order to determine whether cocaine activates IKKα, we treated NL4-3-ΔE-EGFP virus-infected THP-1 cells acutely and chronically with cocaine. Western blots were performed using antibody specific to the phosphorylated form of IKKα, P-IKKα (S176/180). The nuclear levels of P-IKKα (S176/180), which indicates the catalytically active form peaked at 30 minutes (Figure 8B) and showed a clear relation to the H3S10 phosphorylation (Figure 7A and Figure 8B). During chronic treatment p-IKKα (S176/180) remained elevated after 72 hours post treatment (Figure 8D). Similar to p-MSK1 (Figure 6A and 6C), the catalytically active phosphorylated form of IKKα is exclusively present in the nuclear fraction, where its substrate, p-H3S10 resides, suggesting a clear role of IKKα in catalyzing phosphorylation of H3S10 (Figure 8A and 8C). Moreover, cocaine also enhanced the overall levels of IKKα in the nucleus (Figure 8B and 8D). However, cytoplasmic levels of IKKα were unaffected upon cocaine exposure (Figure 8A and 8C).

Figure 8. Cocaine activates IKKα in both in acute and chronic cocaine model.

Figure 8

Figure 8

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The cytoplasmic and nuclear protein fraction of HIV-1-infected THP-1 cells treated either acutely (A and B) or chronically (C and D) with 5µM cocaine were probed with antibodies against unphosphorylated and phosphorylated catalytically active forms of IKKα. Cytoplasmic (A and C) and nuclear fractions (B and D) were analyzed. GAPDH was used as a loading control for cytoplasmic fraction and Lamin A/C for nuclear fraction. Densitometry analysis was done on all western immunoblots. Densitometry analysis was done on all immunoblots and statistical analysis was done from 3 experiments; * p<0.05, ** p<0.01 vs. unstimulated control.

In order to determine the role of IKKα in catalyzing p-H3S10 phosphorylation during cocaine exposure, we analyzed the impact of the IKKα inhibitor, BAY11-7082 (Santa Cruz) on restricting IKKα activity and analyzed the impact on IKKα-induced H3S10 phosphorylation. The robust inhibition of cocaine-induced IKKα activation by BAY11-7082 was quite evident (Figure 9A). However, strong inhibition of IKKα did not translate into proportional p-H3S10 inhibition (Figure 9B). These results confirmed that IKKα surely contributes to overall cocaine induced p-H3S10 phosphorylation, but plays a subordinate role to MSK1 in catalyzing H3S10 phosphorylation following cocaine treatment.

Figure 9. IKKα modestly support cocaine induced overall H3S10 phosphorylation.

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HIV-1 infected THP-1 cells were pre-treated with the IKKα inhibitor BAY-11 (3 µM and 5 µM) for one hour prior to incubation with 5µM cocaine for 30 minutes. The nuclear levels of IKKα (A) and p-H3S10 (B) were determined by immunoblotting. SPT5 was used as loading control for IKKα and Histone 3 for phosphorylated H3S10. Statistical analysis was done from 4 experiments, * p<0.05 and ** p<0.001 vs. unstimulated control or RO-31 treated samples.

Cocaine promotes HIV-1 transcription by enhancing the recruitment of both initiation and elongation factors in addition to establishing transcriptionally active chromatin structures at HIV-1 LTR

Chromatin modification is a crucial step to initiate transcription of most eukaryotic genes (Kouzarides, 2007; Nowak and Corces, 2004). Although the proteins that induce chromatin reorganization play critical roles during overall transcription, their primary effect occurs during the initiation phase of transcription (Gray et al., 1999; Thorne et al., 2009). Therefore, in order to determine the factors involved in HIV-1 transcription following cocaine treatment, we analyzed their recruitment and resultant epigenetic modifications at the HIV-1 LTR by performing chromatin immunoprecipitation (ChIP) assays.

THP-1 cells were infected with HIV-1NL4-3-ΔE-EGFP and treated with cocaine for either 1 hour or 6 hours followed by ChIP assays. The immunoprecipitated DNA was analyzed for the recruitment of different transcription and epigenetic factors and the changes in epigenetic modifications at both the promoter and nucleosome-1 (Nuc-1) regions using primer sets specific for the indicated regions as detailed in materials and methods. As demonstrated above, via activating MSK1, cocaine enhances the functional activity of NF-κB by promoting the phosphorylation of p65 at S276. The recruitment of p-p65 (S276) was analyzed and we found more than 2-fold enhanced recruitment of p-p65 within 1 hour and remained significantly elevated even after 6 hours post-cocaine treatment at promoter (Figure 10A and 10C). In agreement with the established findings that the phosphorylation of p65 at S276 enhances the interaction of NF-κB with histone acetyl transferases (HATs), we found higher recruitment of P300 at the HIV-1 LTR after cocaine exposure (Figure 10A and 10B). Concomitantly, we found a rapid loss of histone deacetylase 3 (HDAC3) from the LTR upon cocaine treatment; the higher ratio of HATs to HDACs translated into enhanced acetylation of core histones H3 (H3-Ac) and H4 (H4K12-Ac) at the HIV-1 LTR (Figure 10A and 10B). The H3-Ac and H4K12-Ac are euchromatic marks, which represent the establishment of transcriptionally active open chromatin structures at the HIV-1 LTR allowing access to transcription machinery (Ishida et al., 2006; Nakayama et al., 2001; Nakayama and Takami, 2001; Xu et al., 2003). This is evidenced by the elevated presence of RNA polymerase II (RNAP II) following cocaine treatment representing enhanced ongoing HIV-1 transcription (Figure 10 A-D). Upon cocaine treatment, a rapid, concurrent loss of several heterochromatic marks from the LTR, including trimethylated histone H3 at lysine positions 9 (H3K9-me3) and lysine position 27 (H3K27-me3) (Figure 10C and 10 D) further validate the establishment of transcriptionally active chromatin structures at LTR.

Figure 10. Cocaine activated NF-kB enhances HIV-1 gene expression by recruiting P300 at the HIV-1 LTR, which acetylate the core histones and establish transcriptionally active chromatin structures at the HIV-1 LTR.

Figure 10

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HIV infected THP-1 cells were treated with 5 mM cocaine for either 1 hour or 6 hours. The chromatin immunoprecipitation (ChIP) analysis was performed to evaluate the reorganization of chromatin structure at HIV-1 LTR following cocaine treatment in order to measure the changes in selective epigenetic modifications. We also assessed the recruitment of different transcription factors and epigenetic enzymes at LTR. (A and C) Primer sets directed to the Promoter region (-116 to +4 with respect to transcription start site); (B and D) Nucleosome 1 (+30 to +134 with respect to transcription start site) of HIV-1 LTR. The depicted ChIP assay results were reproduced more than 7 times.

We demonstrated earlier that MSK1 catalyzes the phosphorylation of histone H3 at the serine residue 10 (P-H3S10) (Figure 7) which suggests the recruitment of MSK1 at HIV LTR. The enhanced recruitment of MSK1 at HIV LTR following cocaine treatment was observed (Figure 10A and 10B). The enrichment of MSK1 at promoter was greater than on Nuc-1 region (Figure 10A and 10B), and accordingly the enhanced recruitment of MSK1 translated into higher levels of p-H3S10 to the HIV-1 LTR following cocaine treatment (Figure 10C and 10D). Similar to our western blotting analysis, levels of H3S10 phosphorylation remained significantly upregulated even after 6 hours of cocaine exposure (Figure 7, 10C and 10D). Besides contributing to the establishment of transcriptionally active chromatin structures, H3S10 phosphorylation promotes the recruitment of P-TEFb, which is indicated by the recruitment of cyclin T1 to the HIV-1 LTR following cocaine treatment (Figure 10C and 10D). It is noteworthy that the presence of p-H3S10 and H4K12-Ac marks newly reorganized chromatin structures (Strahl and Allis, 2000). The accumulation of p-H3S10 and H4K12-Ac epigenetic marks following cocaine treatment further validates our hypothesis that cocaine enhances HIV-1 transcription by modifying chromatin structures at the LTR. Overall, these results confirm that cocaine exposure converts transcriptionally repressive chromatin (heterochromatin) structures into transcriptionally active chromatin (euchromatin) structures at the HIV-1 LTR.

Considering that the resolution of the ChIP assays range between 500 and 700 base pairs (bp), some signal overlap between the promoter and Nuc-1 regions of the LTR was expected. Accordingly, we found some overlap of signal; nevertheless, as anticipated, p65 is largely restricted to its binding sites, which exist near the promoter region (Figure 10 A-D).

Previous studies established that AP-1 is activated efficiently by cocaine treatment (Nestler, 2012). Therefore, as a positive control, we assessed the recruitment of the c-Jun subunit of AP-1 at HIV-1 LTR (Figure 10A and 10B). Possibly resulting from cocaine degradation in the cell culture milieu and/or cocaine receptor internalization/desensitization after 6 hours, the levels of all the factors at the HIV-1 LTR decreased. Interestingly, however, despite the reductions in the recruitment of most of the transcription factors at the LTR, histone modifications (acetylation and phosphorylation) and the recruitment of AP-1 to its downstream binding sites were still increasing (Figure 10A and 10B).

Thus, cocaine appears to facilitate HIV-1 transcription by promoting the recruitment of transcription factors that facilitate both the initiation and the elongation phases of HIV-1 transcription. This eventually results in the removal of heterochromatin structures and leads to the establishment of euchromatin structures at LTR, promoting HIV-1 transcription.

Cocaine promotes HIV transcription by activating NF-kB and MSK1 in primary monocyte-derived macrophages (MDM)

In figure 1 we demonstrated that cocaine enhances HIV-1 gene expression in primary monocyte derived macrophages (MDM) in a dose dependent manner (Figure 1B–E). In order to further establish the role of cocaine in physiologically more relevant primary MDM cells, the MDMs were treated with either 1 or 5 µM cocaine for 30 minutes. The cytoplasmic and nuclear fractions were assessed to examine the degree of activation of different factors and post-translational modification. As expected, similar to the results observed in the THP-1 cell line, western blot analysis of MDM showed a dose-dependent increase in the nuclear levels of total NF-κB, p-p65 (S536) and p-p65 S276), following cocaine exposure (Figure 11A, 11B, 11C and S2). Additionally, we also found increased expression of MSK1 phosphorylated at threonine 581 (T581) (Figure 11D) as well as phosphorylation of histone H3 at serine 10 (Figure 11E).

Figure 11. NF-κB is dose-dependently activated in primary monocyte-derived macrophages (MDM) by cocaine treatment.

Figure 11

Figure 11

Figure 11

Figure 11

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MDM isolated from three healthy donors were treated with 1 or 5 µM cocaine for 30 minutes. The cells were lysed, cytoplasmic and the nuclear fractions were probed for (A) p65 (B) p-p65 (S536) (C) p-p65 (S276, (D) p-MSK1 (T581) and (E) p-H3S10 by immunoblotting. GAPDH and Lamin A/C were used as loading controls for cytoplasmic and nuclear extract. Densitometry analysis was done on all western immunoblots. Densitometry analysis was done on all immunoblots and statistical analysis was done using 3 experiments; * p<0.05 and ** p<0.01 vs. unstimulated control.

DISCUSSION

In this study the molecular mechanisms by which cocaine enhances HIV-1 gene expression during acute and chronic cocaine exposure have been explored, and the key steps/pathways through which cocaine enhances HIV-I transcription have been elucidated. We have shown that cocaine promotes HIV-1 transcription by activating both NF-κB and MSK1. More importantly, results from primary MDM cells confirmed the activation of NF-κB and MSK1 following cocaine treatment. In both cell types, we found NF-κB and MSK1 activation rose sharply following cocaine exposure. When we examined HIV-1 gene expression in infected primary MDM and U937 cells by Flowcytometric (FACS) analysis, corresponding increase in GFP expression following cocaine treatment was found. These results strongly support the concept that cocaine-induced activation of NF-κB and MSK1 drives HIV-1 transcription and replication within monocytes/macrophages lineages.

The importance of NF-κB in inducing HIV-1 transcription (Nabel and Baltimore, 1987) and activation of NF-κB by cocaine have been demonstrated by several previous studies (Ang et al., 2001; Dhillon et al., 2007; Hou et al., 1996; Yao et al., 2010). However, the underlying molecular mechanisms through which cocaine modulates NF-κB activation were not well defined. In this study we have demonstrated that besides stimulating IKKβ, cocaine profoundly activates ribosomal S6 kinase-1 (RSK1); both enzymes besides inducing the phosphorylation of IκB, catalyze the phosphorylation of the p65 at serine residue 536, a marker of nuclear translocation or activation of NF-kB {Bohuslav, 2004 #6304;Chandrakesan, 2010 #6305;Brami-Cherrier, 2005 #3546;Hayden, 2012 #4827}. Furthermore, we found that MSK1 is specifically activated by cocaine, as TNF-α was unable to activate MSK1. The findings in our study have shown that cocaine-activated MSK1 plays a pivotal role in HIV-1 gene expression by facilitating both the initiation and elongation phase of HIV-1 transcription. These findings provide, for the first time, a comprehensive analysis of the important role of MSK1 in supporting HIV-1 transcription. In addition to inducing the levels of MSK1, cocaine stimulates its functional activity by enhancing its phosphorylation at threonine residue 581 (T581), which is a marker of the catalytically active form of MSK1 (Arthur, 2008; Markou and Lazou, 2002; Zhong et al., 1998). Cocaine activated MSK1 subsequently phosphorylates the p65 subunit of NF-κB selectively at serine residue 276 (S276). This phosphorylation event is known to augment the interaction of p65 with histone acetyl transferases (HATs), such as P300 and consequently enhances the transcriptional ability of NF-κB (Perkins et al., 1997b; Vermeulen et al., 2003; Zhong et al., 2002). As result, we found enhanced recruitment of histone acetyltransferase, such as p300, to the HIV-1 LTR. HATs subsequently catalyze the acetylation of core histones and, as a result, higher amounts of hyperacetylated forms of histone H3 and H4 (H3-Ac and H4K12-Ac) were found at the HIV-1 LTR. Concurrently cocaine treatment triggers the removal of heterochromatin marks, including reduction in the trimethylation of histone H3 at K9 (H3K9-Me3) and K27 (H3K27-Me3) from the HIV-1 promoter. These epigenetic changes have resulted in the establishment of transcriptionally active, open chromatin structures at the HIV-1 LTR that further promotes the access of transcription machineries at the promoter. This concept is highly supported by our results showing several fold enrichment of RNAP II at the LTR upon cocaine treatment. All of these events facilitate the initiation of HIV-1 transcription.

In addition to phosphorylating the p65 subunit of NF-κB at serine 276, MSK1 also catalyzes the phosphorylation of histone H3 at serine 10 (p-H3S10), and both of these events correlate well with the kinetics of MSK1 activation following cocaine treatment. Furthermore, we found enhanced recruitment of MSK1 at HIV LTR after cocaine treatment. Accordingly, inhibiting MSK1 activity by a specific inhibitor caused a significant reduction in cocaine-induced H3S10 phosphorylation, further establishing the direct role of MSK1 in catalyzing these modifications. Along with MSK1 several other enzymes are known to catalyze the phosphorylation of H3S10, which we plan to investigate in detail in our future studies, IKKα is one of them. We found efficient activation of IKKα upon cocaine treatment; however our inhibition studies clearly confirm the predominant role of MSK1 in catalyzing H3S10 phosphorylation during cocaine exposure. P-H3S10 is a euchromatic mark, which contributes to the establishment of transcriptionally active chromatin structure (Soloaga et al., 2003; Wei et al., 1998). Hence, we found higher levels of p-H3S10 in the nucleus and its enrichment at HIV-1 LTR after cocaine treatment, again demonstrating the establishment of transcriptionally active chromatin structures at HIV-1 LTR following cocaine treatment.

Besides facilitating the establishment of transcriptionally active chromatin structures, p-H3S10 promotes the recruitment of P-TEFb to the promoter sites, which in turn leads to the enhanced processivity of RNAP II (Ivaldi et al., 2007). Accordingly, we found higher recruitment of P-TEFb to the HIV-1 LTR following cocaine treatment. The role of P-TEFb in facilitating the elongation phase of HIV-1 transcription is well established (Bourgeois et al., 2002; Fujinaga et al., 2004; Ivanov et al., 2000; Karn, 2011; Kim et al., 2002; Parada and Roeder, 1996; Peterlin and Price, 2006; Wei et al., 1998). Hence, our results demonstrate that cocaine enhances HIV-1 gene expression by inducing both the initiation and elongation phases of HIV-1 transcription by activating NF-κB and MSK1.

Despite the use of highly active antiretroviral therapy (HAART), the prevalence of HIV-associated neurocognitive disorder or HAND in drug abusing populations is significantly high (Ferris et al., 2008; Nath et al., 2002; Nath et al., 2001). HIV-1 can be found in the CNS within days of infection, where it can be present either in a transcriptionally active or impaired form (Kaul et al., 2001; Nath et al., 2002; Nath et al., 2001). Within the brain, HIV-1 is harbored primarily in myeloid cells and these cell types act as the main HIV-1 reservoirs in the CNS (Nath et al., 2002; Nath et al., 2008). Several viral proteins, including Tat, Vpr and gp120 are known to contribute to HAND. In addition, cocaine abuse further accelerates the susceptibility of the brain to HIV-1 and enhances the severity of neurocognitive dysfunction in drug abusing HIV-1-infected populations (Anthony et al., 1991; Chiasson et al., 1990; Dhillon et al., 2007; Goodkin et al., 1997; Kim et al., 2013; Nath et al., 2001). Thus, consumption of cocaine remains a significant cofactor for HIV-1 infection, transmission, and consequent neurodegeneration, even in the era of HAART (Alcabes and Friedland, 1995; Bagasra and Pomerantz, 1993; Dhillon et al., 2007; Nath et al., 2002; Peterson et al., 1991; Roth et al., 2002). Since the CNS is the common target for both cocaine and HIV-1, the CNS paradoxically provides an exclusive opportunity for cocaine and HIV-1 to interact, and in some instances cooperate synergistically to diminish CNS function.

Our results, in myeloid cells, provide compelling evidence that cocaine enhances HIV-1 transcription by promoting both initiation and elongation phase during either acute or chronic cocaine intake conditions. Another striking finding of our study is that both acute and chronic cocaine treatment activates NF-κB and MSK1. However, upon chronic cocaine treatment NF-κB activation is delayed and comparatively less profound. MSK1 subsequently promotes the HIV-1 transcriptional initiation by augmenting the functional activity of NF-κB through P-p65 (S276), which enhances the interaction of NF-κB with HATs such as P300. In parallel MSK1 also induces the transcriptional elongation by promoting the recruitment of P-TEFb at LTR via P-H3S10.

Collectively, our data supports the previous observations and suggests that cocaine exposure promotes neurodegeneration not only by inducing the overall replication of HIV-1 (Goodkin et al., 1997; Nath et al., 2002; Nath et al., 2001), but also by enhancing the cellular levels of viral proteins such as Tat, Vpr and gp120, which are well established viral contributors to neurodegeneration during HIV-1 infection (Kaul and Lipton, 2006; King et al., 2006; Kruman et al., 1998; Mocchetti et al., 2007; Nath et al., 1999). Moreover, NF-κB is known to induce the expression of several pro-inflammatory genes transcription (Tak and Firestein, 2001) and the involvement of several pro-inflammatory cytokines has already been implicated in neurodegeneration during HIV-1 infection (Kaul et al., 2005). Hence, it is quite plausible that besides elevated levels of HIV-1 and its proteins; the up-regulation of several pro-inflammatory cytokines contributes to neurodegeneration via neuroinflammation, during cocaine intake.

We have demonstrated the important role of MSK1 in HIV-1 transcription. Recently, we have also found that MSK1 is a common target of several pathways that lead to activate HIV-1 transcription, via different stimuli (work in progress). Additional studies are required to investigate if cocaine also activates MSK1 in other cell types, especially T cells and, potentially, may have an impact on replication and latency (work in progress). Moreover, targeting critical components of these cocaine-induced cascades could prove beneficial in combination with suppressive HAART, especially for cocaine abusing HIV-1 patients.

In conclusion, utilizing both myeloid cell lines and primary human macrophages (MDM), we have demonstrated that cocaine promotes HIV-1 replication by enhancing HIV-1 transcription through the activation of NF-κB and MSK1. The specific epigenetic changes caused by the cocaine activation of these proteins, leads to the development of a transcriptionally active open chromatin structures around the HIV-1 promoter. Our findings further suggest a previously unknown role for MSK1 in enhancing HIV-1 transcription and replication, more importantly during chronic cocaine usage. MSK1 prolonged activation leads to extended NF-κB and H3S10 phosphorylation, two of the key events which promote both the initiation and elongation phases of HIV-1 gene transcription. We have uncovered the underlying molecular mechanisms through which cocaine induces HIV-1 gene expression and promotes neurodegeneration in cocaine using HIV patients.

Conclusions

Cocaine promotes HIV replication by enhancing both initiation and elongation phases of HIV transcription by activating NF-κB and MSK1.

Supplementary Material

Acknowledgments

We thank the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, US National Institutes of Health; M. Gately (Hoffmann La Roche) for human recombinant interleukin 2; from Drs. H. Zhang, Y. Zhou and R. Siliciano (Johns Hopkins University) for pNL4-3-ΔE-EGFP (Cat# 11100) (Zhang et al., 2004). The cocaine-HCl was kindly provided by National Institute of Drug Abuse. We are also thankful to the Flow Cytometry core facility of George Washington University. The research is funded by the NIDA/NIH (5R21DA033924-02 and 5R03DA033900-02 to MT, and K02 DA027374 and R01 DA033200 to KF. This work is also supported by grants of the District of Columbia Developmental Center for AIDS Research (DC D-CFAR), an NIH-funded program 5P30AI087714-02 and startup funds from the George Washington University to MT. This work is also supported partially by the AmFAR (the Foundation for AIDS Research) and Case Western Reserve University C-FAR grants to MT The content is solely the responsibility of the authors and does not necessarily represent the official views of National Center for Research Resources or the US National Institutes of Health.

Footnotes

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Competing Interests

The authors declare that they have no financial competing interests.

Authors‘ contributions

Research designed: MT; Research performed: MT, GS, KF, RF and NH; Data analysed: MT, GS, KF, AO, RF and KH; Manuscript written: MT, KF, GS, AO, JK, FK, KH and GLS. All authors read and approved the final manuscript.

REFERENCES

  1. Adelman K, Lis JT. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nature reviews. Genetics. 2012;13:720–731. doi: 10.1038/nrg3293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alcabes P, Friedland G. Injection drug use and human immunodeficiency virus infection. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 1995;20:1467–1479. doi: 10.1093/clinids/20.6.1467. [DOI] [PubMed] [Google Scholar]
  3. Ang E, Chen J, Zagouras P, Magna H, Holland J, Schaeffer E, Nestler EJ. Induction of nuclear factor-kappaB in nucleus accumbens by chronic cocaine administration. J Neurochem. 2001;79:221–224. doi: 10.1046/j.1471-4159.2001.00563.x. [DOI] [PubMed] [Google Scholar]
  4. Anthony JC, Vlahov D, Nelson KE, Cohn S, Astemborski J, Solomon L. New evidence on intravenous cocaine use and the risk of infection with human immunodeficiency virus type 1. American journal of epidemiology. 1991;134:1175–1189. doi: 10.1093/oxfordjournals.aje.a116021. [DOI] [PubMed] [Google Scholar]
  5. Arthur JS. MSK activation and physiological roles. Front Biosci. 2008;13:5866–5879. doi: 10.2741/3122. [DOI] [PubMed] [Google Scholar]
  6. Bagasra O, Pomerantz RJ. Human immunodeficiency virus type 1 replication in peripheral blood mononuclear cells in the presence of cocaine. J Infect Dis. 1993;168:1157–1164. doi: 10.1093/infdis/168.5.1157. [DOI] [PubMed] [Google Scholar]
  7. Bohuslav J, Chen LF, Kwon H, Mu Y, Greene WC. p53 induces NF-kappaB activation by an IkappaB kinase-independent mechanism involving phosphorylation of p65 by ribosomal S6 kinase 1. J Biol Chem. 2004;279:26115–26125. doi: 10.1074/jbc.M313509200. [DOI] [PubMed] [Google Scholar]
  8. Bonacini M, Govindarajan S, Blatt LM, Schmid P, Conrad A, Lindsay KL. Patients co-infected with human immunodeficiency virus and hepatitis C virus demonstrate higher levels of hepatic HCV RNA. Journal of viral hepatitis. 1999;6:203–208. doi: 10.1046/j.1365-2893.1999.00153.x. [DOI] [PubMed] [Google Scholar]
  9. Bonizzi G, Bebien M, Otero DC, Johnson-Vroom KE, Cao Y, Vu D, Jegga AG, Aronow BJ, Ghosh G, Rickert RC, Karin M. Activation of IKKalpha target genes depends on recognition of specific kappaB binding sites by RelB:p52 dimers. EMBO J. 2004;23:4202–4210. doi: 10.1038/sj.emboj.7600391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 2004;25:280–288. doi: 10.1016/j.it.2004.03.008. [DOI] [PubMed] [Google Scholar]
  11. Bourgeois CF, Kim YK, Churcher MJ, West MJ, Karn J. Spt5 cooperates with Tat by preventing premature RNA release at terminator sequences. Mol. Cell. Biol. 2002;22:1079–1093. doi: 10.1128/MCB.22.4.1079-1093.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Brami-Cherrier K, Valjent E, Herve D, Darragh J, Corvol JC, Pages C, Arthur SJ, Girault JA, Caboche J. Parsing molecular and behavioral effects of cocaine in mitogen- and stress-activated protein kinase-1-deficient mice. J Neurosci. 2005;25:11444–11454. doi: 10.1523/JNEUROSCI.1711-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Brown K, Gerstberger S, Carlson L, Franzoso G, Siebenlist U. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science. 1995;267:1485–1488. doi: 10.1126/science.7878466. [DOI] [PubMed] [Google Scholar]
  14. Budhiraja S, Famiglietti M, Bosque A, Planelles V, Rice AP. Cyclin T1 and CDK9 T-loop phosphorylation are downregulated during establishment of HIV-1 latency in primary resting memory CD4+ T cells. J Virol. 2013;87:1211–1220. doi: 10.1128/JVI.02413-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Butler SL, Hansen MS, Bushman FD. A quantitative assay for HIV DNA integration in vivo. Nature medicine. 2001;7:631–634. doi: 10.1038/87979. [DOI] [PubMed] [Google Scholar]
  16. Chandrakesan P, Ahmed I, Anwar T, Wang Y, Sarkar S, Singh P, Peleg S, Umar S. Novel changes in NF-{kappa}B activity during progression and regression phases of hyperplasia: role of MEK, ERK, and p38. J Biol Chem. 2010;285:33485–33498. doi: 10.1074/jbc.M110.129353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Chiasson MA, Stoneburner RL, Lifson AR, Hildebrandt DS, Ewing WE, Schultz S, Jaffe HW. Risk factors for human immunodeficiency virus type 1 (HIV-1) infection in patients at a sexually transmitted disease clinic in New York City. American journal of epidemiology. 1990;131:208–220. doi: 10.1093/oxfordjournals.aje.a115491. [DOI] [PubMed] [Google Scholar]
  18. Dhillon NK, Williams R, Peng F, Tsai YJ, Dhillon S, Nicolay B, Gadgil M, Kumar A, Buch SJ. Cocaine-mediated enhancement of virus replication in macrophages: implications for human immunodeficiency virus-associated dementia. J Neurovirol. 2007;13:483–495. doi: 10.1080/13550280701528684. [DOI] [PubMed] [Google Scholar]
  19. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 1998;72:8463–8471. doi: 10.1128/jvi.72.11.8463-8471.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Dunn KL, Espino PS, Drobic B, He S, Davie JR. The Ras-MAPK signal transduction pathway, cancer and chromatin remodeling. Biochemistry and cell biology Biochimie et biologie cellulaire. 2005;83:1–14. doi: 10.1139/o04-121. [DOI] [PubMed] [Google Scholar]
  21. Estaquier J, Zaunders J, Laforge M. HIV integrase and the swan song of the CD4 T cells? Retrovirology. 2013;10:149. doi: 10.1186/1742-4690-10-149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ferris MJ, Mactutus CF, Booze RM. Neurotoxic profiles of HIV, psychostimulant drugs of abuse, and their concerted effect on the brain: current status of dopamine system vulnerability in NeuroAIDS. Neuroscience and biobehavioral reviews. 2008;32:883–909. doi: 10.1016/j.neubiorev.2008.01.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fujinaga K, Irwin D, Huang Y, Taube R, Kurosu T, Peterlin BM. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol. Cell. Biol. 2004;24:787–795. doi: 10.1128/MCB.24.2.787-795.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Goodkin K, Wilkie FL, Concha M, Asthana D, Shapshak P, Douyon R, Fujimura RK, LoPiccolo C. Subtle neuropsychological impairment and minor cognitive-motor disorder in HIV-1 infection. Neuroradiological, neurophysiological, neuroimmunological, and virological correlates. Neuroimaging Clin N Am. 1997;7:561–579. [PubMed] [Google Scholar]
  25. Gray SG, Eriksson T, Ekstrom TJ. Methylation, gene expression and the chromatin connection in cancer (review) International journal of molecular medicine. 1999;4:333–350. doi: 10.3892/ijmm.4.4.333. [DOI] [PubMed] [Google Scholar]
  26. Hauser KF, El-Hage N, Buch S, Nath A, Tyor WR, Bruce-Keller AJ, Knapp PE. Impact of opiate-HIV-1 interactions on neurotoxic signaling. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology. 2006;1:98–105. doi: 10.1007/s11481-005-9000-4. [DOI] [PubMed] [Google Scholar]
  27. Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev. 2004;18:2195–2224. doi: 10.1101/gad.1228704. [DOI] [PubMed] [Google Scholar]
  28. He N, Chan CK, Sobhian B, Chou S, Xue Y, Liu M, Alber T, Benkirane M, Zhou Q. Human Polymerase-Associated Factor complex (PAFc) connects the Super Elongation Complex (SEC) to RNA polymerase II on chromatin. Proc Natl Acad Sci U S A. 2011;108:E636–E645. doi: 10.1073/pnas.1107107108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hou YN, Vlaskovska M, Cebers G, Kasakov L, Liljequist S, Terenius L. A mu-receptor opioid agonist induces AP-1 and NF-kappa B transcription factor activity in primary cultures of rat cortical neurons. Neuroscience letters. 1996;212:159–162. doi: 10.1016/0304-3940(96)12799-3. [DOI] [PubMed] [Google Scholar]
  30. Hu J, Nakano H, Sakurai H, Colburn NH. Insufficient p65 phosphorylation at S536 specifically contributes to the lack of NF-kappaB activation and transformation in resistant JB6 cells. Carcinogenesis. 2004;25:1991–2003. doi: 10.1093/carcin/bgh198. [DOI] [PubMed] [Google Scholar]
  31. Ioannidis JPA, Havlir DV, Tebas P, Hirsch MS, Collier AC, Richman DD. Dynamics of HIV-1 viral load rebound among patients with previous suppression of viral replication. Aids. 2000;14:1481–1488. doi: 10.1097/00002030-200007280-00003. [DOI] [PubMed] [Google Scholar]
  32. Isel C, Karn J. Direct evidence that HIV-1 Tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation. J Mol Biol. 1999;290:929–941. doi: 10.1006/jmbi.1999.2933. [DOI] [PubMed] [Google Scholar]
  33. Ishida T, Hamano A, Koiwa T, Watanabe T. 5’ long terminal repeat (LTR)-selective methylation of latently infected HIV-1 provirus that is demethylated by reactivation signals. Retrovirology. 2006;3:69. doi: 10.1186/1742-4690-3-69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Itzen F, Greifenberg AK, Bosken CA, Geyer M. Brd4 activates P-TEFb for RNA polymerase II CTD phosphorylation. Nucleic Acids Res. 2014;42:7577–7590. doi: 10.1093/nar/gku449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ivaldi MS, Karam CS, Corces VG. Phosphorylation of histone H3 at Ser10 facilitates RNA polymerase II release from promoter-proximal pausing in Drosophila. Genes Dev. 2007;21:2818–2831. doi: 10.1101/gad.1604007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ivanov D, Kwak YT, Guo J, Gaynor RB. Domains in the SPT5 protein that modulate its transcriptional regulatory properties. Mol. Cell. Biol. 2000;20:2970–2983. doi: 10.1128/mcb.20.9.2970-2983.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Jadlowsky JK, Wong JY, Graham AC, Dobrowolski C, Devor RL, Adams MD, Fujinaga K, Karn J. Negative elongation factor is required for the maintenance of proviral latency but does not induce promoter-proximal pausing of RNA polymerase II on the HIV long terminal repeat. Mol Cell Biol. 2014;34:1911–1928. doi: 10.1128/MCB.01013-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Karn J. The molecular biology of HIV latency: breaking and restoring the Tat-dependent transcriptional circuit. Current opinion in HIV and AIDS. 2011;6:4–11. doi: 10.1097/COH.0b013e328340ffbb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kaul M, Garden GA, Lipton SA. Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature. 2001;410:988–994. doi: 10.1038/35073667. [DOI] [PubMed] [Google Scholar]
  40. Kaul M, Lipton SA. Mechanisms of neuronal injury and death in HIV-1 associated dementia. Current HIV research. 2006;4:307–318. doi: 10.2174/157016206777709384. [DOI] [PubMed] [Google Scholar]
  41. Kaul M, Zheng J, Okamoto S, Gendelman HE, Lipton SA. HIV-1 infection and AIDS: consequences for the central nervous system. Cell death and differentiation 12 Suppl. 2005;1:878–892. doi: 10.1038/sj.cdd.4401623. [DOI] [PubMed] [Google Scholar]
  42. Kerr LD, Inoue J, Davis N, Link E, Baeuerle PA, Bose HR, Jr, Verma IM. The rel-associated pp40 protein prevents DNA binding of Rel and NF-kappa B: relationship with I kappa B beta and regulation by phosphorylation. Genes Dev. 1991;5:1464–1476. doi: 10.1101/gad.5.8.1464. [DOI] [PubMed] [Google Scholar]
  43. Kilareski EM, Shah S, Nonnemacher MR, Wigdahl B. Regulation of HIV-1 transcription in cells of the monocyte-macrophage lineage. Retrovirology. 2009;6:118. doi: 10.1186/1742-4690-6-118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Kim SG, Jung JB, Dixit D, Rovner R, Jr, Zack JA, Baldwin GC, Vatakis DN. Cocaine exposure enhances permissiveness of quiescent T cells to HIV infection. Journal of leukocyte biology. 2013;94:835–843. doi: 10.1189/jlb.1112566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Kim YK, Bourgeois CF, Isel C, Churcher MJ, Karn J. Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation. Mol. Cell. Biol. 2002;22:4622–4637. doi: 10.1128/MCB.22.13.4622-4637.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. King JE, Eugenin EA, Buckner CM, Berman JW. HIV tat and neurotoxicity. Microbes and infection / Institut Pasteur. 2006;8:1347–1357. doi: 10.1016/j.micinf.2005.11.014. [DOI] [PubMed] [Google Scholar]
  47. Kinoshita S, Su L, Amano M, Timmerman LA, Kaneshima H, Nolan GP. The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells. Immunity. 1997;6:235–244. doi: 10.1016/s1074-7613(00)80326-x. [DOI] [PubMed] [Google Scholar]
  48. Kizaki T, Shirato K, Sakurai T, Ogasawara JE, Ohishi S, Matsuoka T, Izawa T, Imaizumi K, Haga S, Ohno H. Beta2-adrenergic receptor regulate Toll-like receptor 4-induced late-phase NF-kappaB activation. Molecular immunology. 2009;46:1195–1203. doi: 10.1016/j.molimm.2008.11.005. [DOI] [PubMed] [Google Scholar]
  49. Kobor MS, Greenblatt J. Regulation of transcription elongation by phosphorylation. Biochim. Biophys. Acta. 2002;1577:261–275. doi: 10.1016/s0167-4781(02)00457-8. [DOI] [PubMed] [Google Scholar]
  50. Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693–705. doi: 10.1016/j.cell.2007.02.005. [DOI] [PubMed] [Google Scholar]
  51. Krogan NJ, Kim M, Ahn SH, Zhong G, Kobor MS, Cagney G, Emili A, Shilatifard A, Buratowski S, Greenblatt JF. RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol. Cell. Biol. 2002;22:6979–6992. doi: 10.1128/MCB.22.20.6979-6992.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Kruman II, Nath A, Mattson MP. HIV-1 protein Tat induces apoptosis of hippocampal neurons by a mechanism involving caspase activation, calcium overload, and oxidative stress. Experimental Neurology. 1998;154:276–288. doi: 10.1006/exnr.1998.6958. [DOI] [PubMed] [Google Scholar]
  53. Lee PK, Kieffer TL, Siliciano RF, Nettles RE. HIV-1 viral load blips are of limited clinical significance. The Journal of antimicrobial chemotherapy. 2006;57:803–805. doi: 10.1093/jac/dkl092. [DOI] [PubMed] [Google Scholar]
  54. Liu M, Hsu J, Chan C, Li Z, Zhou Q. The ubiquitin ligase Siah1 controls ELL2 stability and formation of super elongation complexes to modulate gene transcription. Molecular cell. 2012;46:325–334. doi: 10.1016/j.molcel.2012.03.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Liu Y, Jones M, Hingtgen CM, Bu G, Laribee N, Tanzi RE, Moir RD, Nath A, He JJ. Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands. Nat. Med. 2000;6:1380–1387. doi: 10.1038/82199. [DOI] [PubMed] [Google Scholar]
  56. Markou T, Lazou A. Phosphorylation and activation of mitogen- and stress-activated protein kinase-1 in adult rat cardiac myocytes by G-protein-coupled receptor agonists requires both extracellular-signal-regulated kinase and p38 mitogen-activated protein kinase. Biochem J. 2002;365:757–763. doi: 10.1042/BJ20011828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. McCoy CE, Campbell DG, Deak M, Bloomberg GB, Arthur JSC. MSK1 activity is controlled by multiple phosphorylation sites. Biochem J. 2005;387:507–517. doi: 10.1042/BJ20041501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Mocchetti I, Nosheny RL, Tanda G, Ren K, Meyer EM. Brain-derived neurotrophic factor prevents human immunodeficiency virus type 1 protein gp120 neurotoxicity in the rat nigrostriatal system. Annals of the New York Academy of Sciences. 2007;1122:144–154. doi: 10.1196/annals.1403.010. [DOI] [PubMed] [Google Scholar]
  59. Nabel G, Baltimore DA. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature. 1987;326:711–713. doi: 10.1038/326711a0. [DOI] [PubMed] [Google Scholar]
  60. Nakayama J, Rice JC, Strahl BD, Allis CD, Grewal SI. Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science. 2001;292:110–113. doi: 10.1126/science.1060118. [DOI] [PubMed] [Google Scholar]
  61. Nakayama T, Takami Y. Participation of histones and histone-modifying enzymes in cell functions through alterations in chromatin structure. Journal of biochemistry. 2001;129:491–499. doi: 10.1093/oxfordjournals.jbchem.a002882. [DOI] [PubMed] [Google Scholar]
  62. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996;272:263–267. doi: 10.1126/science.272.5259.263. [DOI] [PubMed] [Google Scholar]
  63. Nath A, Anderson C, Jones M, Maragos W, Booze R, Mactutus C, Bell J, Hauser KF, Mattson M. Neurotoxicity and dysfunction of dopaminergic systems associated with AIDS dementia. J Psychopharmacol. 2000a;14:222–227. doi: 10.1177/026988110001400305. [DOI] [PubMed] [Google Scholar]
  64. Nath A, Conant K, Chen P, Scott C, Major EO. Transient exposure to HIV-1 Tat protein results in cytokine production in macrophages and astrocytes. J. Biol. Chem. 1999;274:17098–17102. doi: 10.1074/jbc.274.24.17098. [DOI] [PubMed] [Google Scholar]
  65. Nath A, Hauser KF, Wojna V, Booze RM, Maragos W, Prendergast M, Cass W, Turchan JT. Molecular basis for interactions of HIV and drugs of abuse. J Acquir Immune Defic Syndr. 2002;2(31 Suppl.):S62–S69. doi: 10.1097/00126334-200210012-00006. [DOI] [PubMed] [Google Scholar]
  66. Nath A, Maragos WF, Avison MJ, Schmitt FA, Berger JR. Acceleration of HIV dementia with methamphetamine and cocaine. J Neurovirol. 2001;7:66–71. doi: 10.1080/135502801300069737. [DOI] [PubMed] [Google Scholar]
  67. Nath A, Schiess N, Venkatesan A, Rumbaugh J, Sacktor N, McArthur J. Evolution of HIV dementia with HIV infection. Int Rev Psychiatry. 2008;20:25–31. doi: 10.1080/09540260701861930. [DOI] [PubMed] [Google Scholar]
  68. Naumann M, Scheidereit C. Activation of NF-kappa B in vivo is regulated by multiple phosphorylations. EMBO J. 1994;13:4597–4607. doi: 10.1002/j.1460-2075.1994.tb06781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Nestler EJ. Transcriptional mechanisms of drug addiction. Clinical psychopharmacology and neuroscience : the official scientific journal of the Korean College of Neuropsychopharmacology. 2012;10:136–143. doi: 10.9758/cpn.2012.10.3.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Nowak SJ, Corces VG. Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet. 2004;20:214–220. doi: 10.1016/j.tig.2004.02.007. [DOI] [PubMed] [Google Scholar]
  71. Panneer N, Lontok E, Branson BM, Teo CG, Dan C, Parker M, Stekler JD, DeMaria A, Jr, Miller V. HIV and HCV infection in the United States: Whom and How to Test. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2014 doi: 10.1093/cid/ciu396. [DOI] [PubMed] [Google Scholar]
  72. Parada CA, Roeder RG. Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxy-terminal domain. Nature. 1996;384:375–378. doi: 10.1038/384375a0. [DOI] [PubMed] [Google Scholar]
  73. Parikh N, Dampier W, Feng R, Passic SR, Zhong W, Frantz B, Blakey B, Aiamkitsumrit B, Pirrone V, Nonnemacher MR, Jacobson JM, Wigdahl B. Cocaine Alters Cytokine Profiles in HIV-1-Infected African American Individuals in the DrexelMed HIV/AIDS Genetic Analysis Cohort. Journal of acquired immune deficiency syndromes. 2014 doi: 10.1097/QAI.0000000000000163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Pearson R, Kim YK, Hokello J, Lassen K, Friedman J, Tyagi M, Karn J. Epigenetic silencing of human immunodeficiency virus (HIV) transcription by formation of restrictive chromatin structures at the viral long terminal repeat drives the progressive entry of HIV into latency. J. Virol. 2008;82:12291–12303. doi: 10.1128/JVI.01383-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Perkins ND, Felzien LK, Betts JC, Leung K, Beach DH, Nabel GJ. Regulation of NF-kappaB by cyclin-dependent kinases associated with the p300 coactivator. Science. 1997a;275:523–527. doi: 10.1126/science.275.5299.523. [DOI] [PubMed] [Google Scholar]
  76. Perkins ND, Felzien LK, Betts JC, Leung K, Beach DH, Nabel GJ. Regulation of NF-kB by cyclin-dependent kinases associated with the p300 coactivator. Science. 1997b;275:523–527. doi: 10.1126/science.275.5299.523. [DOI] [PubMed] [Google Scholar]
  77. Peterlin BM, Price DH. Controlling the elongation phase of transcription with P-TEFb. Mol. Cell. 2006;23:297–305. doi: 10.1016/j.molcel.2006.06.014. [DOI] [PubMed] [Google Scholar]
  78. Peterson PK, Gekker G, Chao CC, Schut R, Molitor TW, Balfour HH., Jr Cocaine potentiates HIV-1 replication in human peripheral blood mononuclear cell cocultures. Involvement of transforming growth factor-beta. Journal of immunology. 1991;146:81–84. [PubMed] [Google Scholar]
  79. Rockstroh JK, Lennox JL, Dejesus E, Saag MS, Lazzarin A, Wan H, Walker ML, Xu X, Zhao J, Teppler H, Dinubile MJ, Rodgers AJ, Nguyen BY, Leavitt R, Sklar P, Investigators S. Long-term treatment with raltegravir or efavirenz combined with tenofovir/emtricitabine for treatment-naive human immunodeficiency virus-1-infected patients: 156-week results from STARTMRK. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2011;53:807–816. doi: 10.1093/cid/cir510. [DOI] [PubMed] [Google Scholar]
  80. Roth MD, Tashkin DP, Choi R, Jamieson BD, Zack JA, Baldwin GC. Cocaine enhances human immunodeficiency virus replication in a model of severe combined immunodeficient mice implanted with human peripheral blood leukocytes. J Infect Dis. 2002;185:701–705. doi: 10.1086/339012. [DOI] [PubMed] [Google Scholar]
  81. Schmitz ML, Baeuerle PA. The p65 subunit is responsible for the strong transcription activating potential of NF-kappa B. EMBO J. 1991;10:3805–3817. doi: 10.1002/j.1460-2075.1991.tb04950.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Skalka AM. HIV: Integration triggers death. Nature. 2013;498:305–306. doi: 10.1038/nature12254. [DOI] [PubMed] [Google Scholar]
  83. Sobhian B, Laguette N, Yatim A, Nakamura M, Levy Y, Kiernan R, Benkirane M. HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP. Molecular cell. 2010;38:439–451. doi: 10.1016/j.molcel.2010.04.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Soloaga A, Thomson S, Wiggin GR, Rampersaud N, Dyson MH, Hazzalin CA, Mahadevan LC, Arthur JS. MSK2 and MSK1 mediate the mitogen- and stress-induced phosphorylation of histone H3 and HMG-14. EMBO J. 2003;22:2788–2797. doi: 10.1093/emboj/cdg273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Strahl BD, Allis CD. The language of covalent histone modifications. Nature. 2000;403:41–45. doi: 10.1038/47412. [DOI] [PubMed] [Google Scholar]
  86. Tak PP, Firestein GS. NF-kappaB: a key role in inflammatory diseases. J Clin Invest. 2001;107:7–11. doi: 10.1172/JCI11830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Thorne JL, Campbell MJ, Turner BM. Transcription factors, chromatin and cancer. The international journal of biochemistry & cell biology. 2009;41:164–175. doi: 10.1016/j.biocel.2008.08.029. [DOI] [PubMed] [Google Scholar]
  88. Turk B, Stoka V. Protease signalling in cell death: caspases versus cysteine cathepsins. FEBS letters. 2007;581:2761–2767. doi: 10.1016/j.febslet.2007.05.038. [DOI] [PubMed] [Google Scholar]
  89. Tyagi M, Karn J. CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency. EMBO J. 2007;26:4985–4995. doi: 10.1038/sj.emboj.7601928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  90. Tyagi M, Pearson RJ, Karn J. Establishment of HIV latency in primary CD4+ cells is due to epigenetic transcriptional silencing and P-TEFb restriction. J Virol. 2010;84:6425–6437. doi: 10.1128/JVI.01519-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Vermeulen L, De Wilde G, Notebaert S, Vanden Berghe W, Haegeman G. Regulation of the transcriptional activity of the nuclear factor-kappaB p65 subunit. Biochem. Pharmacol. 2002;64:963–970. doi: 10.1016/s0006-2952(02)01161-9. [DOI] [PubMed] [Google Scholar]
  92. Vermeulen L, De Wilde G, Van Damme P, Vanden Berghe W, Haegeman G. Transcriptional activation of the NF-kappaB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1) EMBO J. 2003;22:1313–1324. doi: 10.1093/emboj/cdg139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  93. Volberding PA, Deeks SG. Antiretroviral therapy and management of HIV infection. Lancet. 2010;376:49–62. doi: 10.1016/S0140-6736(10)60676-9. [DOI] [PubMed] [Google Scholar]
  94. Wei P, Garber ME, Fang SM, Fischer WH, Jones KA. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell. 1998;92:451–462. doi: 10.1016/s0092-8674(00)80939-3. [DOI] [PubMed] [Google Scholar]
  95. Xu W, Cho H, Evans RM. Acetylation and methylation in nuclear receptor gene activation. Methods in enzymology. 2003;364:205–223. doi: 10.1016/s0076-6879(03)64012-7. [DOI] [PubMed] [Google Scholar]
  96. Yao HH, Yang YJ, Kim KJ, Bethel-Brown C, Gong N, Funa K, Gendelman HE, Su TP, Wang JQ, Buch S. Molecular mechanisms involving sigma receptor-mediated induction of MCP-1: implication for increased monocyte transmigration. Blood. 2010;115:4951–4962. doi: 10.1182/blood-2010-01-266221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  97. Zhang H, Zhou Y, Alcock C, Kiefer T, Monie D, Siliciano J, Li Q, Pham P, Cofrancesco J, Persaud D, Siliciano RF. Novel single-cell-level phenotypic assay for residual drug susceptibility and reduced replication capacity of drug-resistant human immunodeficiency virus type 1. J. Virol. 2004;78:1718–1729. doi: 10.1128/JVI.78.4.1718-1729.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  98. Zhang Z, Klatt A, Gilmour DS, Henderson AJ. Negative elongation factor NELF represses human immunodeficiency virus transcription by pausing the RNA polymerase II complex. J. Biol. Chem. 2007;282:16981–16988. doi: 10.1074/jbc.M610688200. [DOI] [PubMed] [Google Scholar]
  99. Zhong H, May MJ, Jimi E, Ghosh S. The phosphorylation status of nuclear NF-kB determines its association with CBP/p300 or HDAC-1. Mol. Cell. 2002;9:625–636. doi: 10.1016/s1097-2765(02)00477-x. [DOI] [PubMed] [Google Scholar]
  100. Zhong H, Voll RE, Ghosh S. Phosphorylation of NF-kB p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol. Cell. 1998;1:661–671. doi: 10.1016/s1097-2765(00)80066-0. [DOI] [PubMed] [Google Scholar]
  101. Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 1998;72:9873–9880. doi: 10.1128/jvi.72.12.9873-9880.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]

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