Abstract
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 Ca2+-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.
Highlights
Two-thirds of cellular Tat can be secreted from HIV-1 infected or transfected cells[17,18,19,20] and extracellular Tat crosses plasma membranes by various mechanisms including endocytosis; a major pathway for Tat entry[21,22] following interactions with specific cell surface proteins and receptors[21,22,23,24,25,26]
Others and we have found that activating endolysosome-resident transient receptor potential mucolipin 1 channel (TRPML1) channels with the agonist ML-SA1 resulted in endolysosome acidification[38,39]
Recent studies have shown that ML-SA1 activation of TRPML1, a cation channel residing on endolysosomes, decreases endolysosome pH38–40
Summary
Two-thirds of cellular Tat can be secreted from HIV-1 infected or transfected cells[17,18,19,20] and extracellular Tat crosses plasma membranes by various mechanisms including endocytosis; a major pathway for Tat entry[21,22] following interactions with specific cell surface proteins and receptors[21,22,23,24,25,26]. Tat has to escape from endolysosomes into the cytosol before it transits to the nucleus and activates the HIV-1 LTR promoter[27,28,29]. Avoiding endolysosome degradation is critical for exogenous Tat to first escape endolysosomes and enter nucleus to activate HIV-1 Tat LTR transactivation. Given that chloroquine does not increase[22], or even decrease[35], HIV-1 LTR transactivation under conditions when Tat is expressed intracellularly, it is generally thought that chloroquine, a weak base, neutralizes the acidic pH of endolysosomes and prevents exogenous HIV-1 Tat degradation, increasing the amount of Tat available to activate HIV-1 LTR in nucleus. Acidifying endolysosomes could enhance HIV-1 Tat degradation in endolysosomes, preventing Tat escape from endolysosomes and blocking subsequent activation of HIV-1 LTR in the nucleus. TRPML1 and BK channels might be targeted therapeutically to prevent re-activation of latent HIV-1 infection, and to decrease the prevalence and severity of HIV-1 associated neurocognitive disorders and other HIV-1 comorbidities
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