The effects of cocaine on HIV transcription

Abstract

Illicit drug users are a high risk population for infection with the Human Immunodeficiency Virus (HIV). A strong correlation exists between prohibited drugs use and an increase rate of HIV transmission. Cocaine stands out as one of the most frequently abused illicit drugs and its use is correlated with HIV infection and disease progression. The central nervous system (CNS) is a common target for both drugs of abuse and HIV, and cocaine intake further accelerates neuronal injury in HIV patients. Although the high incidence of HIV infection in illicit drug abusers is primarily due to high risk activities such as needle sharing and unprotected sex, several studies have demonstrated that cocaine enhances the rate of HIV gene expression and replication by activating various signal transduction pathways and downstream transcription factors. In order to generate mature HIV genomic transcript, HIV gene expression has to pass through both the initiation and elongation phases of transcription, which requires discrete transcription factors. In this review, we will provide a detailed analysis of the molecular mechanisms that regulate HIV transcription and discuss how cocaine modulates those mechanisms to upregulate HIV transcription and eventually HIV replication.

HIV infection and neurological disorders

According to a recent estimate, approximately 40 million people worldwide are infected with the human immunodeficiency virus (HIV) [1, 2]. Anti-HIV therapy, commonly known as either highly active antiretroviral therapy (HAART) or combined antiretroviral therapy (cART), has been proven to be extremely successful in controlling viral replication and prolonging the life span of HIV infected individuals. Unfortunately, HAART is not able to fully restore the health of HIV patients, even in those with a sustained undetectable viral load. Nearly one third of all HIV infected patients develop neuropathies, collectively known as HIV-associated neurocognitive disorders (HAND) [3, 4]. Neurocognitive impairment, even in milder forms, impacts on general health, adds financial burdens and leads to a deterioration in quality of life [5, 6]. Numerous mechanisms have been proposed to explain these neurocognitive disorders, including neurotoxicity and glial cell activation by viral proteins such as Gp120 and Tat, immune activation, oxidative stress, BBB deterioration, immune cell infiltration, and increased proinflammatory cytokines [7–10]. The suboptimal penetration of HAART in the brain permits sustained low levels of viral replication and brain adaptive HIV evolution, which eventually contributes to the occasional systemic viral blips [11–14]. Moreover, recently we have proposed that owing to the lack of any transcriptional inhibitor, HAART regimens are unable to restrict HIV transcription [15]. Consequently, viral proteins are produced unchecked even in the presence of effective HAART [16]. The immune response against HIV virions and its proteins further contributes to the persistent immune activation and the deterioration of the central nervous system (CNS) in HIV patients [6, 17–26].

CNS is the common target for cocaine and HIV

Injection and non-injection illicit drug use and abuse contribute significantly to HIV infection and transmission [2, 4, 25]. In the United States, HIV infection in drug addicts accounts for one-third of new cases of HIV [2, 27]. Cocaine, one of the most frequent drugs of abuse has been implicated as a major contributing factor for HIV infection, transmission and AIDS progression [28–30]. Both cocaine and HIV target the CNS. Besides promoting HIV replication, cocaine primes the cells to become more susceptible for HIV infection [31–33]. Within a few days of infection, HIV establishes a reservoir in the CNS by infecting different types of brain cells including resident microglial cells and astrocytes, along with visiting macrophages, and lymphocytes [31, 32, 34–37]. Due to suboptimal presence of HAART in brain, it has been proposed that residual viral replication and the presence of viral proteins (e.g. Tat and gp120); the CNS impair the functioning of different kinds of brain cells, including neurons, which results in the overall deterioration of the immune and nervous system [6, 17–26, 38]. Cocaine further accelerates this deleterious process by promoting HIV replication and enhancing HIV gene expression. Thus, cocaine intake significantly augments HIV-associated neurotoxicity (neuro-AIDS) in HIV patients, which is clinically recognized as HAND [4, 6, 39–41]. Despite the success of HAART in controlling circulating HIV, HAND remains a significant co-morbidity responsible for deterioration in quality of life and enhanced mortality of HIV-infected patients [4, 6, 42]. Poor adherence to HAART treatment by cocaine users further contributes to HIV disease progression [43–45]. It is noteworthy that cocaine using HIV patients, despite adherence to HAART, frequently have higher viral loads and exhibit faster viral rebound following HAART interruption, further indicating that besides promoting HIV replication, cocaine enhances the susceptibility of cells to HIV [46–48]. Thus, illicit drug using HIV patients are more prone to the HIV associated comorbidities. Furthermore, the direct correlation of cocaine use with enhanced HIV replications has been well documented by several investigations using HIV infected humanized mouse models [32, 33, 49]. Hence, drug addiction extraordinarily contributes to the health and financial burden on HIV patients and the society as a whole [5, 6].

HIV transcription

HIV replication primarily relies on efficient transcription to generate full genomic HIV transcripts. HIV performs its transcription mainly by using the host cell transcription machinery, with the help of its own master transactivator protein, transactivator of transcription (Tat). In order to generate complete HIV genomic transcripts, transcription requires successful progression through both the initiation and elongation phases (Figure 1). In a recent publication, we have revealed some of the underlying molecular mechanisms that cocaine utilizes to promote both the initiation and elongation phases of HIV transcription in order to enhance the overall rate of HIV gene expression ( [15] Figure 2).

Figure 1. Different phases of HIV transcription.

A), The binding of NF-κB and NF-AT to the enhancer region of HIV LTR strongly upregulates the initiation phase of HIV transcription. The histone acetyl transferases (HATs) recruited by these factors, catalyze the acetylation of core histones and establish the transcriptionally active chromatin (euchromatin) structures. The establishment of euchromatin structures at and around LTR promoter promotes efficient access of LTR to transcription machinery. Transcriptional initiation is characterized by the phosphorylation of serine residues at position 5 (S5) in the heptapeptide (YSPTSPS)₅₂ repeats of the C-terminal domain (CTD) of RNAPII by TFIIH/Cdk7. B), HIV transcription halts after passing through the TAR elements due to the binding of negative transcriptional elongation factors, mainly NELF and DSIF. C), Tat protein of HIV brings the super elongation complex (SEC), containing P-TEFb at HIV LTR along with it. The CDK9 subunit of P-TEFb subsequently catalyzes the phosphorylation of negative elongation factors DSIF and NELF, which leads to either their dissociation from LTR or reverse their function from negative to positive elongation factor. CDK9 also catalyzes the phosphorylation of CTD of RNAPII, primarily at serine 2 residues. This event makes RNAPII highly processive or transcription elongation proficient that result in the generation of complete and properly processed HIV transcripts.

Figure 2. Schematic diagram showing cocaine accelerates HIV transcription by promoting both initiation and elongation phases of HIV transcription.

Cocaine treatment results in the activation and recruitment of NF-κB and MSK1 at HIV LTR. MSK1 subsequently catalyze the phosphorylation of NF-κB at its serine residue 276. Phosphorylation of NF-κB at 276^th serine residue enhances the functional capability of NF-κB by augmenting the interaction of NF-κB with HATs. HATs such as p300 in turn catalyze the acetylation of core histones (mainly H3 and H4) at HIV LTR and facilitate the establishment of transcriptionally active chromatin structures, which boost the access of transcription machinery to HIV LTR and thus facilitate the initiation phase of HIV transcription. MSK1 in addition to phosphorylating NF-κB catalyzes the phosphorylation of histone H3 at serine 10 (H3S10). Phosphorylated histone H3 at 10^th residue contributes to the establishment of transcriptionally active chromatin structure at HIV LTR. Moreover, phosphorylated H3S10 also facilitates the recruitment of P-TEFb at HIV LTR through a mechanism that is not yet fully defined; usually with the help of BRD proteins which bind specifically to acetylated histones through their bromo domain. P-TEFb, an established elongation factor, subsequently catalyzes the phosphorylation of several proteins, including RNA Polymerase II (RNAP II) and negative elongation factors. These modifications enhance the processivity of RNAP II and nullify the impact of negative elongation factors, and thus promote the elongation phase of HIV transcription.

HIV transcriptional initiation

The initiation phase of HIV transcription involves the binding of transcription factors such as SP1, TATA-box-binding protein (TBP), and TBP-associated factors (TAFs) which are recruited to the core long terminal repeats (LTR) promoter, an essential component sufficient to sustain basal transcription. HIV LTR core promoter consists of TATA box, initiator sequence and three SP1 binding sites [50–54]. However, efficient initiation of HIV transcription occurs only after the binding of transcriptional activators, primarily NF-kB, NF-AT, AP-1 and STAT5 to the enhancer sequences of HIV LTR [55–58]. These factors, after binding to HIV LTR, promote efficient HIV transcription mainly by recruiting histone acetyl transferases (HATs) and through cooperation with SP1 proteins [59–63] (Figure 1).

Cocaine and HIV transcriptional initiation

The role of cocaine in promoting HIV replication is well established. However very little is known about the involved molecular mechanisms that cocaine utilizes to enhance HIV gene expression. Recently, we have investigated some of the molecular mechanisms that are modulated by cocaine in order to enhance HIV transcription [15].

NF-κB plays a critical role during HIV transcription

The NF-κB/Rel proteins are transcription factors that induce expression of several cellular genes. Many of these genes regulate host immunity and inflammatory responses [64, 65]. Transcription factor NF-κB consists of either homo- or heterodimers of five related proteins, p65 (Rel A), Rel B, c-Rel, p50/p105 (NF-κB) and p52/p100 (NF-κB2) [66]. Of these, one of the best-characterized and functionally most active NF-κB protein is the heterodimer which comprises of Rel A (p65) and p50, which is widely expressed and heavily involved in NF-κB -regulated transactivation [6, 67]. In its inactive state, the NF-κB complexes are sequestered in the cytoplasm through their interaction with inhibitory IκB proteins [66, 68]. However, upon cell activation through a wide array of stimuli serine residues 32 and 36 of IκB become phosphorylated by several kinases, including IκB kinase (IKK) complex, which is composed of IKKα, IKKβ and IKKγ [66, 69–71] and Ribosomal S6 kinase1 (RSK1). This event induces the ubiquitination of IκB at lysine residues 21 and 22 and leads to the 26S proteasome degradation. Due to the dissociation of IκB, the nuclear localization signal (NLS) of NF-κB proteins become exposed and consequently NF-κB translocates into the nucleus [69, 72]. Once in the nucleus, NF-κB binds to the cognate binding sites at the promoter and enhancer regions of the genes and activates their transcription including the HIV LTR enhancer region (Figure 1). The HIV enhancer region is one of the best studied elements which usually contains two functional NF-kB binding motifs [55, 59].

Cocaine accelerates HIV transcriptional initiation by both activating and enhancing the functional activity of NF-kB

Several groups including ours have demonstrated that cocaine promotes both the activation of NF-κB and HIV replication [15, 73–76]. To extend those studies further, using monocytic cell lines (THP1 and U937) and primary monocytes derived macrophages (MDMs) [15], we found that during both acute (one time) and chronic (multiple time for 3 days) treatment, cocaine efficiently activates NF-κB. Interestingly, cocaine not only promotes the activation (nuclear translocation) of NF-κB, but also enhances the functional activity of NF-κB by augmenting the ability of NF-κB to interact with histone acetyltransferases (HATs) (Detailed below and Figure 2). Cocaine induced NF-κB activation is primarily mediated via RSK1 activation, instead of IKKβ, a kinase specifically activated by TNF-α, which is one of the strongest and most specific activator of NF-κB [15, 64, 77]. RSK1 is a downstream kinase of the extracellular signal-regulated kinase (ERK) pathway, one of the main pathways through which cocaine exerts many of its effects [78–80]. Cocaine activated RSK1 primarily phosphorylates IκBβ at Ser19 and Ser23 (our unpublished data), instead of IκBα, which is mainly phosphorylated by IKKβ [81]. NF-κB activation in addition to activating the transcription of other genes, promotes the transcription of its own inhibitor IκBα. The newly formed IκBα interacts with NF-κB and takes it to the cytoplasm and thus negatively regulates expression of NF-κB dependent genes. Interestingly, unlike IκBα, the IκBβ promoter does not have NF-κB binding sites. Consequently, cocaine induced NF-κB activation does not directly lead to the synthesis of its inhibitor. Hence, RSK1 induced NF-κB activation lasts longer and eventually contributes to the persistence of a cocaine effect (our unpublished data and [82]).

In addition to the activation of NF-κB, cocaine exposure enhances the functional activity of NF-κB primarily by activating mitogen- and stress-activated kinase 1 (MSK1). MSK1 is another kinase that becomes activated upon the stimulation of MAPK/extracellular-signal regulated kinase (ERK) cascade [83, 84]. MSK1 subsequently phosphorylates the p65 subunit of NF-κB at serine residue 276 (p65S276). This post-translational modification boosts the interaction of NF-κB with histone acetyltransferases (HATs) [15, 85–87]. Enhanced interaction translates into higher recruitment of HATs at HIV LTR through NF-κB binding to its consensus binding sequences at HIV LTR. HATs in turn acetylate the core histones, which reduce the electrostatic interactions between the histones and DNA. Less interaction between DNA and histones results in more relaxed/open chromatin structures near the LTR promoter. These relaxed transcriptionally active chromatin structures consequently further promote access to all the remaining components of the transcription machinery (Holo-transcription machinery) and even augment the overall flow of transcription machinery at the HIV LTR promoter. These events eventually enhance the rate of transcriptional initiation from the LTR promoter [15].

HIV transcriptional elongation

The efficiency of the elongation phase of HIV transcription is predominantly dependent on the HIV protein Tat. However, prior to sufficient Tat protein generation, the elongation phase of HIV transcription proceeds very slowly. The Tat-independent inefficient phase of HIV transcription is primarily attributed to the presence of two elongation inhibitory factors; negative elongation factor (NELF) and DRB sensitivity-inducing factor (DSIF) at HIV LTR (for recent reviews refer to [88–93]). In addition to restricting HIV transcription, these negative elongation factors ubiquitously inhibit the transcription of numerous cellular genes. Moreover, in brain resident macrophages, microglia, HIV transcription is further repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF) interacting protein 2 (CTIP2), an inhibitor of P-TEFb predominantly expressed in microglial cells [94–96]. In addition to using the cellular machinery to relieve the restriction posed by NELF and DSIF, HIV utilizes its specialized protein, Tat to facilitate this function. In the absence of Tat, most of the HIV transcripts halt at approximately 60 nucleotides due to the presence of NELF and DSIF along with non-processive or elongation defective RNA polymerase II (RNAP II) at HIV LTR. Once Tat is synthesized and accumulated in the cell beyond a certain threshold, Tat positively feedbacks the entire system. To perform its functions Tat requires binding to an RNA stem loop structure called the trans-activation response (TAR) element which is present at the 5′ extremity of all HIV transcripts. Along with it Tat brings positive transcription elongation factor b (P-TEFb) protein, which mediates most of the transcriptional functions of Tat [97–99]. The cyclin dependent kinase 9 (CDK9) subunit of P-TEFb subsequently hyper-phosphorylates the C-terminal domain (CTD) of RNA polymerase II (RNAPII) and converts the pausing RNAPII into a processive or elongation proficient polymerase [100, 101]. Besides RNAPII, P-TEFb also phosphorylates inhibitory factors DSIF and NELF. These modifications either dissociate the negative factors or convert them into a positive transcription factor [102–104]. P-TEFb recruitment at LTR is also essential to reactivate latent provirus in primary T cells [105, 106]. Besides recruiting P-TEFb, Tat also brings additional elongation factors, such as ELL2, AFF4, ENL and AF9; together they form a Super Elongation Complex (SEC), at HIV-1 LTR (Figure 1) [68, 107, 108]. ELL2 and the scaffold protein AFF4 have been shown to be the critical components of the SEC, as their knockdown or removal strongly inhibits Tat-dependent LTR-driven reporter gene expression [68, 109]. The binding of AFF4 in the presence of Tat modulates the Tat–TAR recognition motif of Cyclin T1 and increases the affinity of Tat-P-TEFb complex for TAR several folds [110, 111]. The enhanced rate of HIV transcription results in generating more Tat protein and higher Tat levels further accelerate HIV transcription. Hence, HIV transcription enters into the fast Tat-dependent phase, that eventually accelerates HIV transcription several hundred fold [97, 112]. HIV transcription thus differs from normal transcription of cellular genes as it is auto-regulated by Tat protein. These events subsequently relieve all restrictions to HIV transcription, and consequently efficient HIV transcription enhances the rate of generation of full-length mature transcripts. This includes production of unspliced HIV genomic transcripts which get packaged and lead to the generation of new viral particles, indicated as an enhanced rate of HIV replication[113, 114].

Cocaine and HIV transcriptional elongation

It is well known that cocaine augments the rate of HIV replication and that cocaine is able to induce the generation of full-length HIV genomic transcripts. As aforementioned, in order to generate the full genomic transcript, HIV transcription has to pass through both the initiation and elongation phase of transcription. Thus, in addition to NF-κB activation, this implies that cocaine activates P-TEFb, an essential factor required for the elongation phase of HIV transcription (Figure 1).

Cocaine promotes HIV transcriptional elongation primarily by activating MSK1

Cocaine activates MSK1 which, besides catalyzing the phosphorylation of the p65 subunit of NF-κB at serine 276, also catalyzes the phosphorylation of histone H3 at serine 10 (P-H3S10) at the HIV LTR promoter [15]. Notably, cocaine also enhances the phosphorylation of p65 and of histone H3 locally at HIV LTR by augmenting the recruitment of MSK1 at LTR [15]. Comparable observations have also been reported for cellular promoters following cocaine exposure [115]. Although several other enzymes besides MSK1 are known to catalyze H3S10 phosphorylation, the predominant role of MSK1 during cocaine exposure has been well established [116–118]. P-H3S10 is a euchromatic mark which facilitates the establishment of transcriptionally active chromatin structure [115, 119]. Accordingly, MSK1 induced H3S10 phosphorylation promotes the establishment of transcriptionally active chromatin structures at HIV-1 LTR following cocaine treatment [15].

In addition to facilitating the establishment of transcriptionally active chromatin structures, P-H3S10 promotes the recruitment of P-TEFb to HIV LTR analogous to other gene promoters [15, 120, 121]. As noted above, P-TEFb plays an essential role in supporting the elongation phase of HIV-1 transcription by catalyzing several above mentioned phosphorylation events (detailed in HIV transcriptional elongation section, Figures 1 and 2) [99–104, 119, 122]. The resultant, complete unspliced HIV transcripts are then packaged and lead to the generation of new viral particles which are represented as enhanced HIV replication. Thus, cocaine boosts HIV-1 gene expression by inducing both the initiation and elongation phases of HIV-1 transcription.

Role of epigenetics in controlling HIV transcription and latency

Like other retroviruses, HIV integrates itself into the host cell genome, preferentially within the intronic regions of actively transcribing genes. This inclination is due to the selective binding preference of lens epithelium-derived growth factor (LEDGF), a protein that plays an important role during HIV integration. LEDGF shows specificity towards transcriptionally active open chromatin structures [123–128]. Analogous to cellular genes, the expression of integrated HIV genome is facilitated by the formation of transcriptionally active chromatin structures around the LTR promoter [129].

The chromatin structures are characterized by their fundamental subunits, nucleosomes. A nucleosome comprises of an octamer, a pair of four core histones (H3, H4, H2A and H2B), which are wrapped around by 147 base pairs of DNA. These core histones undergo various kinds of post-translational modifications such as acetylation, methylation (mono-, di-, or tri-methylation), sumoylation, phosphorylation, ubiquitinylation, etc. [130]. These modifications are called epigenetic modifications, as they can be passed through to the next generation. These epigenetic modifications eventually define the specific nature of the chromatin structures. Chromatin structures, especially in the vicinity of the promoter region of a gene, regulate its expression. Thus, the type and amount of epigenetic modifications harbored play a decisive role in defining specific chromatin structure and subsequent regulation of gene expression [131–133]. The open or relaxed chromatin structure, which promotes access to the transcription machinery at the promoter region of a gene, is called transcriptionally active or euchromatin structure. On the other hand, the closed or compact chromatin structure, which inhibits the access of transcription machinery to the promoter region of a gene is called transcriptionally repressive or heterochromatin structure [130, 132].

The HIV LTR promoter is precisely flanked by two well-placed nucleosomes, independent of the site of integration in the cellular genome The nucleosome-0 (Nuc-0) is located upstream of the LTR promoter and nucleosome-1 (Nuc-1) is assembled downstream from the LTR promoter [129, 134], Figure 1. The epigenetic modifications of these two nucleosomes (Nuc-0 and Nuc-1) play major role in defining the overall chromatin structure near LTR promoter and consequently controlling the initiation of HIV transcription [129, 135, 136].

The chromatin structures at HIV LTR are remodeled mainly by two kinds of protein complexes. One of these protein complexes modifies chromatin structures by inducing various post-translational epigenetic modifications at the N-terminal tails of histones. It is noteworthy that the binding of the basal transcription factors to the chromatin-free promoter region of LTR is not hindered by the surrounding higher order epigenetic nucleosomal structures. The major chromatin reorganization at HIV LTR begins after the binding of factors, such as NF-κB to the enhancer region of the HIV LTR. NF-κB subsequently recruits histone acetyltransferases (HATs), HATs in turn greatly induce remodeling of the surrounding chromatin structures at HIV LTR. Moreover, Tat also brings HATs containing complexes at HIV LTR. Together recruited HATs successively promote the establishment of transcriptionally active chromatin structures at HIV LTR, which, in turn, support Tat-mediated induction of HIV transcription [137–140]. The second group of chromatin-modifying complexes uses energy to change the structures of nucleosomes in order to opening-up or relax nucleosomal structure. The SWI/SNF is one of such complex that changes the location and reorganization of nucleosomes via an ATP dependent mechanism. The SWI/SNF remodeling complexes facilitates the opening of the nucleosomal structures and promotes access of the LTR promoter to transcription factors (for detail, please refer to [141–149]. These events eventually accelerate the overall rate of HIV transcription.

In addition to the post-translational modification of nucleosomal histones, other epigenetic modifications, such as DNA methylation at the CpG islands flanking the transcription start site have also been implicated in regulating the HIV gene expression [150–152]. Even the role of certain noncoding RNA, primarily microRNA (miRNA) has been implicated in HIV transcription and latency [153, 154]. The miRNAs are single-stranded small RNA molecules that bind to specific complementary sequences on the target mRNA and usually inhibit its translation. However miRNA binding occasionally also results in the degradation of specific mRNA [155]. The vital role of viral and cellular miRNAs has been demonstrated in regulating HIV gene expression and latency. In particular, cellular miR-28, miR-125b, miR-150, miR-223 and miR-382, which are enriched in metabolically silent, resting CD4+ T lymphocytes, suppress HIV translation by targeting its mRNA [156–158]. A number of recent papers provide further details of miRNA-mediated regulation of HIV latency [159–168]. Cocaine has been reported to up- and downregulate multiple miRNAs, such as upregulation of miR-181a and downregulation of miR-124 and let-7d [169, 170]. Accordingly, cocaine has been shown to enhance HIV-1 replication in CD4+ T cells by down-regulating miR-125b [36]. Therefore, innovative methodologies designed to manipulate the action of involved miRNAs could be proved useful in regulating HIV gene expression, latency and modulating impact of drugs of abuse.

It is worthy to note that most of the enzymes that catalyze epigenetic modifications do no bind directly to DNA. As a result, the epigenetic enzymes must be recruited to HIV LTR by a variety of DNA binding proteins. HIV transcriptional repressors such as CBF-1, YY1/LSF1, P50 homodimer, AP4, CTIP2, and thyroid hormone receptor recruit chromatin modifying enzymes to the LTR along with several other proteins as multiprotein complexes, which subsequently induce transcriptionally repressive, heterochromatin structures, at HIV LTR [95, 171–175]. We have demonstrated that CBF-1-induced repressive chromatin structures play an important role in restricting HIV transcription and promote HIV latency in primary CD4+ T cells [105]. The role of repressive epigenetic modifications in restricting HIV transcription during latency is quite evident due to the fact that their removal or inhibition leads to the reactivation of latent proviruses [90, 91, 144, 176–178].

It has been well established that epigenetic modifications play an important role in regulating HIV transcription. However, the precise nature of the underlying molecular mechanisms that regulate these specific epigenetic changes at HIV LTR are still not fully defined. Understanding the precise role of epigenetic modifications and the mechanisms involved could be of therapeutic importance in reactivating latent proviruses which are mainly silent due to the lack of productive HIV transcription. Thus, there is an enormous potential for drugs that could manipulate chromatin structures at HIV LTR and regulate HIV gene expression. Different drugs of abuse including cocaine also induce chromatin structure reorganization at numerous cellular genes and HIV LTR (detailed in following section). However, a huge body of work is still required to precisely characterize and define the vital role of epigenetic modifications induced by cocaine. Thus, there is pressing need to examine the molecular mechanisms involved during interactions between the virus and drugs of abuse at the gene expression level to determine how they may be affecting HIV replication.

Impact of cocaine induced epigenetic changes on HIV transcription

The important role for cocaine induced epigenetic modifications in altering the expression of several genes in the central nervous system has been clearly demonstrated [179–181]. Different types and levels of epigenetic modifications of the nucleosomal core histones eventually define the nature of chromatin structure at and around gene promoters. Transcriptionally active, open/euchromatin structures facilitates transcription, whereas transcriptionally repressive, closed/heterochromatin structures suppress transcription by inhibiting the access of transcription machinery to the genetic elements responsible for transcription. Moreover, the long lasting effects of different stimuli on the brain, including cocaine induced plasticity which can persist even after its removal, can be better explained by the concept of relatively stable specific epigenetic modifications and resulting persistent long term expression of selected CNS genes even after removal of stimuli. Epigenetic dysregulations and the resultant changes in the responsible genes have been implicated in several neuronal dysfunctions, including Huntington’s disease, Friedreich ataxia and Rett syndrome [118, 182]. These observations further strengthen the concept that cocaine induced epigenetic modifications may be involved in the exaggeration of neurodegeneration seen in HIV-infected drug abusers. The better understanding of these mechanisms will reveal new drug targets and open up new avenue for better pharmaceutical interventions in drug addict HIV patients.

Cocaine induced Histone modifications at HIV LTR

Interestingly, the mode of cocaine exposure (acute or chronic) also regulates the activation and duration of gene expression by inducing a selective set of epigenetic modifications [181, 183–188]. Genome wide ChIP analysis following cocaine exposure results in both subtle and persistent induction of genes due to selective histone H3 and H4 acetylation [181, 184, 189]. Interestingly, on a number of genes enhanced acetylation of histone H3 was found to be associated with chronic cocaine treatment, while histone H4 hyper-acetylation was found to occur specifically during acute cocaine treatment. However, a lot of genes did not follow this pattern. These facts further validate that histone code for gene regulation is a complex phenomenon. Histone acetylation, although a vital epigenetic modification, still not sufficient to dictate the expression of a gene and contribution of different epigenetic modifications on a particular gene defines its eventual expression [181, 185, 186, 188–190]. Another important finding from these analyses was that cocaine usually promotes gene expression by inducing both phosphorylation and acetylation of core histones. However, cocaine has also been found to suppress a small subset of genes by inducing histone H3 methylation at lysine 9 [130, 191–194]. To further complicate this scenario, recent studies have confirmed that cocaine specifically inhibits class II HDACs, mainly HDAC-4 and -5, but activates Class III HDACs, SIRT1 and SIRT2 [179, 183, 184]. Moreover, cocaine also promotes gene expression by downregulating histone metyltransferses, including G9a and GLP (G9a-like protein). These enzymes catalyze the methylation of histone H3 at lysine 9, a transcriptionally repressive heterochromatic mark [195]. These observations suggest that besides histones, the post-translational modification of several non-histone proteins by these enzymes is responsible for deciding the expression of cellular genes following cocaine exposure.

In the context of HIV infection, we have investigated the role of cocaine induced epigenetic modifications on HIV gene expression [15]. Our results demonstrate that cocaine treatment greatly enhances the recruitment of histone acetyltransferase (HAT), p300, at HIV LTR and leads to the dissociation of histone deacetylase, HDAC1 and HDAC3, from LTR. These events result in hyper-acetylation of core histones both H3 and H4 [15]. Thus, we found that cocaine induced epigenetic modifications at HIV LTR do not follow any strict core histone acetylation pattern. In addition to histone acetylation, cocaine treatment induces phosphorylation of histone H3 at serine 10 (p-H3S10). Acetylation and phosphorylation of core histones partially neutralizes the positive ionic charge of histones and thus both of these epigenetic modifications contribute to the establishment of relaxed transcription-promoting euchromatin structures at HIV LTR. Interestingly, upon cocaine treatment, we also observed the loss of heterochromatic epigenetic modifications such as trimethylation of histone H3 at lysine 9 (H3K9me³) and lysine 27(H3K27me³) from HIV LTR. As indicated in Figure 2, our results demonstrated that cocaine exposure converts the transcriptionally repressive heterochromatin structures into transcriptionally active euchromatic structures at HIV LTR [15]. Accordingly, we found higher recruitment of RNA polymerase II at HIV LTR; further validating the fact that the euchromatin structure promotes the access to transcription machinery at gene promoters and facilitates HIV transcription.

Therapeutic implications to counter cocaine impact

Development of HAART regimens that penetrate better blood brain barrier (BBB)

Current HAART regimens may result in the establishment of several anatomical sanctuary sites such as the CNS because of poor tissue penetration. In these sanctuary sites, a perpetual low level of viral replication has been documented by several studies [196, 197]. Eradication of HIV from sanctuary sites holds the key to HIV cure. Cocaine use further burdens HIV patients by accelerating ongoing HIV replication, primarily in the CNS of HIV patients [6, 198–201]. Hence, better penetrating HAART regimens into HIV sanctuary sites, including the CNS will improve cognitive performance, as has been demonstrated during HAART intensification with better BBB crossing HAART regimens [202, 203].

Inclusion of drugs in HAART regimens that restrict HIV transcription

While HAART efficiently restrict the HIV replication and infection of new cells, it is unable to prevent the transcription of viral proteins. The viral proteins that are produced induce several side effects, especially chronic inflammation, by entering and interacting with cells and cellular proteins, respectively. The CNS is particularly sensitive to the inflammation induced by viral proteins, such as GP120, Tat and Nef. The produced inflammatory cytokines and cytotoxic products secreted by brain cells, especially microglia and perivascular macrophages and to a lesser extent astrocytes subsequently induce neuropathy [7, 204]. Therefore, it implies that cocaine-mediated enhanced HIV transcription elevate the levels of viral proteins even in the presence of HAART regimens and contribute to chronic brain inflammation and deterioration of CNS functioning [15]. Although some HAART agents have shown clear benefit in protecting against neurocognitive impairments, yet higher penetration of HAART drugs may be associated with neurotoxicity [205–208]. Hence, there is an urgent need to perform systematic studies to settle these confounding results.

Conclusion and perspectives

Studies of the effects of cocaine on HIV gene expression are still in their infancy. Our work was the first to describe the salient underlying molecular mechanisms that cocaine utilizes in order to enhance HIV transcription. The transcription factors discussed above are just a few of many that affect HIV gene expression. The main goal of future research is to obtain a comprehensive view of the transcription factors induced by cocaine and their effect on the initiation and elongation phase of expression of relevant genes, especially those that affect HIV gene expression and brain function.

We found that cocaine induces the chromatin remodeling at HIV LTR via MSK1 and histone H3 phosphorylation. Similar epigenetic modifications at various gene promoters, especially in brain cells have been implicated in some of the long-lasting behavioral consequences of cocaine. In order to better understand the cocaine-induced epigenetic modifications at HIV LTR, it will be necessary to involve high throughput approaches, such as ChIP-Seq, to identify numerous other post-translational modifications in both histone and non-histone proteins.

There remains a huge body of work necessary to precisely characterize and define the vital role of epigenetic modifications induced by cocaine. There is a pressing need to examine the molecular mechanisms that are involved during the interactions between the virus and drugs of abuse at the level of gene expression in order to determine how they may be affecting HIV replication. A better understanding of these mechanisms may reveal new drug targets and open up new avenues for better pharmaceutical interventions in HIV-infected drug abusers.