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Abbreviations: BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); CD26, cluster of differentiation 4; CD4+, cluster of differentiation 4; CNS, central nervous system; CRISPR, clustered regularly interspaced short palindromic repeats; CSF, cerebrospinal fluid; CXCR4, C-X-C chemokine receptor type 4; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; gp120, envelope glycoprotein 120; HCl, hydrogen chloride; HIV-1, human immunodeficiency virus-1; kDa, kilodalton; LRP1, low density lipoprotein receptor-related protein 1; LTR, long terminal repeat; MERS-CoV, middle east respiratory syndrome coronavirus; MW, molecular weight; NAADP, nicotinic acid adenine dinucleotide phosphate; P2X4, purinergic P2X4 receptors; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PVDF, polyvinylidene difluoride; RIPA, radioimmunoprecipitation assay buffer; SD, standard deviation; SDS, sodium dodecyl sulfate; shRNA, short hairpin RNA; Tat, transcriptional activator; TFEB, transcription factor EB; TPCs, two-pore channels; Trans-Ned19, (1R,3S)-1- [3][4]piperazin-1-yl] methyl]-4-methoxyphenyl]-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid; TRPM2, transient receptor potential melastatin 2; TRPML1, transient receptor potential mucolipin 1. AbstractHIV-1 Tat is essential for HIV-1 replication and appears to play an important role in the pathogenesis of HIV-associated neurological complications. Secreted from infected or transfected cells, Tat has the extraordinary ability to cross the plasma membrane. In the brain, Tat can be taken up by CNS cells via receptor-mediated endocytosis. Following endocytosis and its internalization into endolysosomes, Tat must be released in order for it to activate the HIV-1 LTR promoter and facilitate HIV-1 viral replication in the nucleus. However, the underlying mechanisms whereby Tat escapes endolysosomes remain unclear. Because Tat disrupts intracellular calcium homeostasis, we investigated the involvement of calcium in Tat endolysosome escape and subsequent LTR transactivation. We demonstrated that chelating endolysosome calcium with high-affinity rhodamine-dextran or chelating cytosolic calcium with BAPTA-AM attenuated Tat endolysosome escape and LTR transactivation.Significantly, we demonstrated that pharmacologically blocking and knocking down the endolysosome-resident two-pore channels (TPCs) attenuated Tat endolysosome escape and LTR transactivation. This calcium-mediated effect appears to be selective for TPCs because knocking down TRPML1 calcium channels was without effect. Our findings suggest that calcium released from TPCs is involved in Tat endolysosome escape and subsequent LTR transactivation. TPCs might represent a novel therapeutic target against HIV-1 infection and HIV-associated neurological complications.

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HIV-1 Tat is essential for HIV-1 replication and plays an important role in latent HIV-1 infection, HIV-1 associated neurological complication, and other HIV-1 comorbidities. Secreted from HIV-1 infected or transfected cells, Tat can be up-taken into cells by receptor-mediated endocytosis and internalized into endolysosomes. To reach nucleus where it can facilitate HIV-1 viral replication, exogenous Tat has to escape the degradation by endolysosomes. Because of findings that endolysosome de-acidification with, for example, the weak-base anti-malarial drug chloroquine prevents exogenous Tat degradation and enhances the amount of Tat available to activate HIV-1 LTR, we hypothesize that acidifying endolysosomes may enhance Tat degradation in endolysosomes and restrict LTR transactivation. Here, we determined the involvement of endolysosome-resident transient receptor potential mucolipin 1 channel (TRPML1) and the big conductance Ca 2+ -activated potassium (BK) channel in regulating endolysosome pH, as well as Tat-mediated HIV-1 LTR transactivation in U87MG cells stably integrated with HIV-1 LTR luciferase reporter. Activating TRPML1 channels with ML-SA1 acidified endolysosomes and restricted Tat-mediated HIV-1 LTR transactivation. These effects of ML-SA1 appeared to be mediated through activation of BK channels, because the effects of ML-SA1 on Tat-mediated HIV-1 LTR transactivation were blocked using pharmacological inhibitors or shRNA knock-down of BK channels. On the other hand, activating TRPML1 and BK channels enhanced cellular degradation of exogenous Tat. These results suggest that acidifying endolysosomes by activating TRPML1 or BK channels may provide therapeutic benefit against latent HIV-1 infection, HIV-1 associated neurocognitive disorders, and other HIV-1 comorbidities.