We next decided the extent to which TRPML1 knockdown affected endolysosome pH; TRPML1 knockdown by itself did not affect baseline endolysosome pH (data not shown) but did significantly (p? ?0.001) attenuate ML-SA1-induced decreases in endolysosome pH (Fig.?2B). in endolysosomes and restrict LTR transactivation. Here, we decided 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 contamination, HIV-1 associated neurocognitive disorders, and other Rabbit Polyclonal to ABHD12 Acarbose HIV-1 comorbidities. strong class=”kwd-title” Subject terms: Retrovirus, Lysosomes Introduction Infecting 37 million people globally1, HIV-1 enters the CNS within weeks of contamination2,3 and can harbor in CSF, perivascular macrophages, microglia, and astrocytes4. Although combined antiretroviral therapy (ART) effectively suppresses HIV-1 replication, it does not completely eliminate the computer virus. In this ART era, reservoirs of HIV-1 exist centrally and peripherally5,6, low levels of neuroinflammation persist, and the prevalence of HIV associated neurocognitive disorders (HAND) remains high (30C50%)7,8. The presence of viral reservoirs makes complete eradication of HIV-1 extremely challenging9C11. Therefore, additional strategies are needed to block viral reactivation in sanctuary sites and to prevent disease progression including HAND. Because ART does not block Tat secretion from HIV-1 infected cells12, and brain levels of Tat remain elevated even when HIV-1 levels are below detectable levels with ART13, one strategy might be to prevent Tat from activating HIV-1 replication through elongation of the HIV-1 long terminal repeat (LTR)14C16. Two-thirds of cellular Tat can be secreted from HIV-1 infected or transfected cells17C20 and extracellular Tat crosses plasma membranes by various mechanisms including endocytosis; a major pathway for Tat entry21,22 following interactions with specific cell surface proteins and receptors21C26. Once internalized into endolysosomes, Tat has to escape from endolysosomes into the cytosol before it transits to the nucleus and activates the HIV-1 LTR promoter27C29. Typically, strong HIV-1 LTR transactivation requires high concentrations of exogenous Tat and disruption of plasma membranes using, for example, scrape-loading methods22,28,30. When endogenously expressed in cytosol, Tat can be imported directly into nucleus and activate HIV-1 LTR transactivation using importin -dependent nuclear localization signals31. In contrast, secreted Tat or exogenously added Tat has to first enter the endolysosome system via endocytosis. Thus, avoiding endolysosome degradation is critical for exogenous Tat to first escape endolysosomes and then enter nucleus to activate HIV-1 Tat LTR transactivation. Consistent with findings of others22,29,30,32,33, we found that the lysosomotropic agent chloroquine enhanced extracellular Tat-mediated HIV-1 LTR transactivation34. Given that chloroquine does not increase22, or even decrease35, HIV-1 LTR transactivation under conditions when Tat is usually expressed intracellularly, it is generally thought that chloroquine, a weak base, neutralizes the acidic pH of endolysosomes and prevents exogenous HIV-1 Tat degradation, thus increasing the amount of Tat available to activate HIV-1 LTR in nucleus. Thus, 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. The acidic endolysosome luminal pH is usually maintained by the electrogenic pumping of protons by vacuolar-ATPase (v-ATPase) in conjunction with chloride and other ions36,37. Others and we have found that activating endolysosome-resident transient receptor potential mucolipin 1 channel (TRPML1) channels with the agonist ML-SA1.Accordingly, it was important for us to first confirm using U87MG cells the extent to which ML-SA1 affects endolysosome pH. 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 contamination, HIV-1 associated neurocognitive disorders, and other HIV-1 comorbidities. strong class=”kwd-title” Subject terms: Retrovirus, Lysosomes Introduction Infecting 37 million people globally1, HIV-1 enters the CNS within weeks of contamination2,3 and can harbor in CSF, perivascular macrophages, microglia, and astrocytes4. Although combined antiretroviral therapy (ART) effectively suppresses HIV-1 replication, it does not completely eliminate the computer virus. In this ART era, reservoirs of HIV-1 exist centrally and peripherally5,6, low levels of neuroinflammation persist, and the prevalence of HIV associated neurocognitive disorders (HAND) remains high (30C50%)7,8. The presence of viral reservoirs makes complete eradication of HIV-1 extremely challenging9C11. Therefore, additional strategies are needed to block Acarbose viral reactivation in sanctuary sites and to prevent disease progression including HAND. Because ART does not block Tat secretion from HIV-1 infected cells12, and brain levels of Tat remain elevated even when HIV-1 levels are below detectable levels with ART13, one strategy might be to prevent Tat from activating HIV-1 replication through elongation of the HIV-1 long terminal repeat (LTR)14C16. Two-thirds of cellular Tat can be secreted from HIV-1 infected or transfected cells17C20 and extracellular Tat crosses plasma membranes by various mechanisms including endocytosis; a major pathway for Tat entry21,22 following interactions with specific cell surface proteins and receptors21C26. Once internalized into endolysosomes, Tat has to escape from endolysosomes into the cytosol before it transits to the nucleus and activates the HIV-1 LTR promoter27C29. Typically, strong HIV-1 LTR transactivation requires high concentrations of exogenous Tat and disruption of plasma membranes using, for example, scrape-loading methods22,28,30. When endogenously expressed in cytosol, Tat can be imported directly into nucleus and activate HIV-1 LTR transactivation using importin -dependent nuclear localization signals31. In contrast, secreted Tat or exogenously added Tat has to first enter the endolysosome system via endocytosis. Thus, avoiding endolysosome degradation is critical for exogenous Tat to first escape endolysosomes and then enter nucleus to activate HIV-1 Tat LTR transactivation. Consistent with findings of others22,29,30,32,33, we found that the lysosomotropic agent chloroquine enhanced extracellular Tat-mediated HIV-1 LTR transactivation34. Given that chloroquine does not increase22, or even decrease35, HIV-1 LTR transactivation under conditions when Tat is usually expressed intracellularly, it is generally thought that chloroquine, a poor base, neutralizes the acidic pH of endolysosomes and prevents exogenous HIV-1 Tat degradation, thus increasing the amount of Tat available to activate HIV-1 LTR in nucleus. Thus, 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. The acidic endolysosome luminal pH is usually maintained by the electrogenic pumping of protons by vacuolar-ATPase (v-ATPase) in conjunction with chloride and other ions36,37. Others and we have found that activating endolysosome-resident transient receptor potential mucolipin 1 channel (TRPML1) channels with Acarbose the agonist ML-SA1 resulted in endolysosome acidification38,39. In the present.