Cocaine Enhances HIV-1 Replication in CD4+ T Cells by Down-Regulating MiR-125b

Dash CCV (2012) Cocaine Enhances HIV-1 Replication in CD4+ T Cells by Down-Regulating MiR-125b. PLoS ONE 7(12): e51387. doi:10.1371/journal.pone.0051387 Cocaine Enhances HIV-1 Replication in CD4+ T Cells by Down-Regulating MiR-125b Chinmay K. Mantri 0 Jui Pandhare Dash 0 Jyoti Velamarti Mantri 0 Chandravanu C. V. Dash 0 K.T. Jeang, National Institute of Health, United States of America 0 The Laboratory of Retrovirology and Epigenetics, Center For AIDS Health Disparities Research, Vanderbilt-Meharry Center for AIDS Research, Department of Biochemistry and Cancer Biology, Meharry Medical College , Nashville, Tennessee , United States of America The main objective of this study was to examine effects of cocaine on HIV-1 replication in primary CD4+ T cells. Cocaine a commonly used drug among HIV-1 positive individuals serves as a cofactor for HIV-1 infection and progression to acquired immunodeficiency syndrome (AIDS). Accumulating evidence suggest that cocaine increases HIV-1 replication in cell cultures, peripheral blood mononuclear cells (PBMCs) and animal models. Intriguingly, there are no studies on cocaineinduced alterations in HIV-1 replication in primary CD4+ T cells that serve as the main targets for HIV-1 replication in vivo. In this report, we demonstrate cocaine-induced enhancement of HIV-1 replication in primary CD4+ T cells isolated from human PBMCs. To decipher a potential mechanism, we examined whether cocaine targets the innate antiviral immunity of CD4+ T cells mediated by cellular microRNAs (miRNAs). This is because recently a network of anti-HIV miRNAs in CD4+ T cells is highlighted to suppress viral replication. Our genome wide miRNA expression analysis indicated downregulation of several anti-HIV miRNAs (miR-28, miR-125b, miR-150, miR-223, and miR-382) in cocaine treated CD4+ T cells. However, our real-time quantitative PCR analysis revealed significant downregulation of miR-125b only. Our results illustrated that miR-125b knockdown enhances HIV-1 replication, whereas overexpression of miR-125b decreases HIV-1 replication in these cells. Therefore, we believe miR-125b is a key player for the cocaine induced enhancement of HIV-1 replication in CD4+ T cells. Since, miR-125b targets the 39 UTR regions of HIV-1 transcripts and inhibits viral protein translation, our data suggest modulation of post entry steps of HIV-1 by cocaine. Given that a plethora of studies suggest that cocaine regulates HIV entry, our results implicate a potentially novel mechanism by which cocaine can increase viral replication in CD4+ T cells. - Funding: This work is partly supported by grants (DA024558, DA30896, DA033892) from National Institute on Drug Abuse/National Institutes of Health, the Vanderbilt CTSA grant UL1 RR024975-01 from NCRR/NIH, and the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH and U54 MD007593 from NIMHD/NIH to CD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare that no competing interests exist. Illicit drug use remains the second most frequent mode of acquisition of HIV and drugs such as amphetamines, cocaine, marijuana, and opiates serve as cofactors for susceptibility to HIV infection and disease progression [18]. Cocaine, a commonly used drug among HIV-1 positive individuals, serves as a cofactor for HIV-1 infection and progression to acquired immunodeficiency syndrome (AIDS) [913]. Epidemiological studies suggest that HIV positive cocaine users have lower CD4+ T cell counts, increased risk of diseases progression and AIDS-related death [11 13]. In vitro cocaine increases HIV-1 replication in human peripheral blood mononuclear cells (PBMCs) and enhances viral load in humanized mice [1416]. Although, cocaine is known to have immunomodulatory function [1719], the underlying mechanism by which cocaine increases HIV-1 replication remains unclear. It has been proposed that cocaine increases HIV-1 infection/replication by inhibiting HIV-1 protective chemokines and/or upregulating the HIV-1 entry co-receptor [2021]. For example, members of the b-chemokine family that bind to CCR5 such as regulated upon-activation T expressed and secreted (RANTES), macrophage inflammatory protein 1a (MIP-1a), and MIP-1b have been demonstrated to inhibit entry of certain HIV-1 strains [22]. In addition, proteomics analysis of cocaine treated PBMCs isolated from HIV-positive donors suggests that cocaine differentially regulates expression of several key host proteins that may influence HIV-1 replication [23]. Since these studies were conducted using cell culture models or the mixed cell populations of PBMCs, there are no reports on primary CD4+ T cells. Given that CD4+ T cells are the main targets for HIV-1 infection and replication in vivo [24], it is critical to evaluate whether cocaine enhances HIV-1 replication in primary CD4+ T cells. MicroRNAs (miRNAs) are noncoding single-stranded small RNAs that bind to the 39 untranslated region (UTR) of target mRNA and post-transcriptionally regulate gene expression via degradation of specific mRNAs and/or repression of their translation [25]. Therefore, cellular miRNAs play critical roles for the normal function and diseased state of cells [25]. MiR-125 is a highly conserved miRNA from nematode to humans and has recently emerged as a key regulator of cell survival and differentiation [26]. In addition miR-125b has been shown to be dysregulated in multiple malignancies [26]. miR-125b is the ortholog of lin-4 in Caenorhaditis elegans and is transcribed from two loci located on chromosomes 11q23 (hsa-miR-125b-1) and 21q21 (hsa-miR-125b-2) [27]. miR-125b has been reported to target many key proteins that regulate apoptosis, innate immunity, inflammation and hematopoietic differentiation [26]. Recently, miR-125b has been demonstrated to regulate a network of genes in CD4+ T cells that are critical for its differentiation [28]. Intriguingly, miR-125b also belongs to the network of cellular antiHIV miRNAs that suppress viral replication [2930]. These antiHIV miRNAs target the 39 UTR regions of HIV-1 transcripts and inhibit HIV-1 replication. It has been proposed that miR-125b and other anti-HIV-1 miRNAs may be responsible for inducing latency in nave CD4+ T cells [29]. Very recently, it has also been suggested that downregulation of miR-125b in the PBMCs of HIV-1 infected individuals may lead to viremia [31]. Since CD4+ T cells serve as primary targets for HIV-1 infection and replication [24], in this study we have examined whether cocaine enhances HIV-1 replication in CD4+ T cells. Using primary CD4+ T cells isolated from human PBMCs, we illustrate cocaine-induced increase in HIV-1 replication in these cells. In an attempt to decipher the mechanism by which cocaine enhances HIV-1 replication, we examined whether cocaine targets the antiHIV-1 miRNAs in CD4+ T cells. The rationa (...truncated)