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Two-pore channels regulate Tat endolysosome escape and Tat-mediated HIV-1 LTR transactivation.

ABSTRACT: HIV-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.

SUBMITTER: Khan N

PROVIDER: S-EPMC7079041 | biostudies-literature |

REPOSITORIES: biostudies-literature

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BK channels regulate extracellular Tat-mediated HIV-1 LTR transactivation.

Project description: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.

| S-EPMC6706582 | biostudies-literature

Endolysosome iron restricts Tat-mediated HIV-1 LTR transactivation by increasing HIV-1 Tat oligomerization and β-catenin expression.

Project description:HIV-1 transactivator of transcription (Tat) protein is required for HIV-1 replication, and it has been implicated in the pathogenesis of HIV-1-associated neurocognitive disorder (HAND). HIV-1 Tat can enter cells via receptor-mediated endocytosis where it can reside in endolysosomes; upon its escape from these acidic organelles, HIV-1 Tat can enter the cytosol and nucleus where it activates the HIV-1 LTR promoter. Although it is known that HIV-1 replication is affected by the iron status of people living with HIV-1 (PLWH), very little is known about how iron affects HIV-1 Tat activation of the HIV-1 LTR promoter. Because HIV-1 proteins de-acidify endolysosomes and endolysosome de-acidification affects subcellular levels and actions of iron, we tested the hypothesis that the endolysosome pool of iron is sufficient to affect Tat-induced HIV-1 LTR transactivation. Ferric (Fe3+) and ferrous (Fe2+) iron both restricted Tat-mediated HIV-1 LTR transactivation. Chelation of endolysosome iron with deferoxamine (DFO) and 2-2 bipyridyl, but not chelation of cytosolic iron with deferiprone and deferasirox, significantly enhanced Tat-mediated HIV-1 LTR transactivation. In the presence of iron, HIV-1 Tat increasingly oligomerized and DFO prevented the oligomerization. DFO also reduced protein expression levels of the HIV-1 restriction agent beta-catenin in the cytosol and nucleus. These findings suggest that DFO increases HIV-1 LTR transactivation by increasing levels of the more active dimeric form of Tat relative to the less active oligomerized form of Tat, increasing the escape of dimeric Tat from endolysosomes, and/or reducing beta-catenin protein expression levels. Thus, intracellular iron might play a significant role in regulating HIV-1 replication, and these findings raise cautionary notes for chelation therapies in PLWH.

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Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation.

Project description:BACKGROUND:Apolipoprotein E (ApoE) is the major carrier protein that mediates the transport and delivery of cholesterol and other lipids in the brain. Three isoforms of ApoE (ApoE2, ApoE3, ApoE4) exist in humans, and their relative expression levels impact HIV-1 infection, HIV-1/AIDS disease progression, and cognitive decline associated with HIV-1-associated neurocognitive disorder. Because HIV-1 Tat, a viral protein essential for HIV-1 replication, can bind to low-density lipoprotein receptor-related protein 1 (LRP1) that controls ApoE uptake in the brain, we determined the extent to which different isoforms of ApoE affected Tat-mediated HIV-1 LTR transactivation. METHODS:Using U87MG glioblastoma cells expressing LTR-driven luciferase, we determined the extent to which LRP1 as well as ApoE2, ApoE3, and ApoE4 affected Tat-mediated HIV-1 LTR transactivation. RESULTS:A specific LRP1 antagonist and siRNA knockdown of LRP1 both restricted significantly Tat-mediated LTR transactivation. Of the three ApoEs, ApoE4 was the least potent and effective at preventing HIV-1 Tat internalization and at decreasing Tat-mediated HIV-1 LTR transactivation. Further, Tat-mediated LTR transactivation was attenuated by an ApoE mimetic peptide, and ApoE4-induced restriction of Tat-mediated LTR transactivation was potentiated by an ApoE4 structure modulator that changes ApoE4 into an ApoE3-like phenotype. CONCLUSIONS:These findings help explain observed differential effects of ApoEs on HIV-1 infectivity and the prevalence of HAND in people living with HIV-1 infection and suggest that ApoE mimetic peptides and ApoE4 structure modulator might be used as a therapeutic strategy against HIV-1 infection and associated neurocognitive disorders.

| S-EPMC5861635 | biostudies-literature

Higher transactivation activity associated with LTR and Tat elements from HIV-1 BF intersubtype recombinant variants.

Project description:HIV-1 is characterized by its rapid genetic evolution and high diversity as a consequence of its error-prone reverse transcriptase and genetic recombination. This latter mechanism is responsible for the creation of circulating recombinant forms (CRFs) found in nature. Previous studies from our lab group have shown that the epidemic in Argentina is characterized by one highly prevalent circulating recombinant form, CRF12_BF, and many related BF recombinant forms. Since transcriptional transactivation of the HIV-1 long terminal repeat (LTR) promoter element requires the essential viral Tat protein, since these genetic structures underwent recombination in variants widely spread in South America, the aim of this work was to study transcriptional activity associated with the recombinant LTR and Tat elements.Differential transcriptional activity was measured for the BF recombinant LTR/Tat complex that is present in widely spread viral variants was demonstrated. This analysis demonstrated a higher activity for the BF complex when compared to its B subtype counterpart.This study indicates structural and functional consequences of recombination events within the LTR promoter and Tat transactivator protein of a naturally occurring HIV-1 recombinant form.

| S-EPMC1402313 | biostudies-literature

Y44A Mutation in the Acidic Domain of HIV-2 Tat Impairs Viral Reverse Transcription and LTR-Transactivation.

Project description:HIV transactivator protein (Tat) plays a pivotal role in viral replication through modulation of cellular transcription factors and transactivation of viral genomic transcription. The effect of HIV-1 Tat on reverse transcription has long been described in the literature, however, that of HIV-2 is understudied. Sequence homology between Tat proteins of HIV-1 and 2 is estimated to be less than 30%, and the main difference lies within their N-terminal region. Here, we describe Y44A-inactivating mutation of HIV-2 Tat, studying its effect on capsid production, reverse transcription, and the efficiency of proviral transcription. Investigation of the mutation was performed using sequence- and structure-based in silico analysis and in vitro experiments. Our results indicate that the Y44A mutant HIV-2 Tat inhibited the activity and expression of RT (reverse transcriptase), in addition to diminishing Tat-dependent LTR (long terminal repeat) transactivation. These findings highlight the functional importance of the acidic domain of HIV-2 Tat in the regulation of reverse transcription and transactivation of the integrated provirions.

| S-EPMC7460587 | biostudies-literature

Two-pore channels regulate endomembrane tension to enable remodeling and resolution of phagolysosomes.

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Project description:Hepatitis C virus (HCV) co-infection occurs in ?30-40% of the HIV-infected population in the US. While a significant body of research suggests an adverse effect of HIV on HCV replication and disease progression, the impact of HCV on HIV infection has not been well studied. Increasing data suggest that hepatocytes and other liver cell populations can serve as reservoirs for HIV replication. Therefore, to gain insight into the impact of HCV on HIV, the effects of the HCV Core protein and infectious hepatitis C virions were evaluated on basal and Tat-induced activation of the HIV long terminal repeat (LTR) in hepatocytes. The HIV LTR was highly induced by the HIV transactivator protein Tat in hepatocytes. Activation varied according to the number of NF-kB binding sites present in the LTRs from different HIV subtypes. Involvement of the NF-kB binding pathway in LTR activation was demonstrated using an NF-kB inhibitor and deletion of the NF-kB binding sites. TNF?, a pro-inflammatory cytokine that plays an important role in HIV pathogenesis, also induced LTR activity in hepatocytes. However, HIV LTR activity was suppressed in hepatocytes in the presence of HCV Core protein, and the suppressive effect persisted in the presence of TNF?. In contrast, infectious hepatitis C virions upregulated HIV LTR activation and gene transcription. Core-mediated suppression remained unaltered in the presence of HCV NS3/4A protein, suggesting the involvement of other viral/cellular factors. These findings have significant clinical implications as they imply that HCV could accelerate HIV disease progression in HIV/HCV co-infected patients. Such analyses are important to elucidate the mechanisms by which these viruses interact and could facilitate the development of more effective therapies to treat HIV/HCV co-infection.

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MCEF is localized to the nucleus by protein sequences encoded within three distinct exons, where it represses HIV-1 Tat-transactivation of LTR-directed transcription.

Project description:Translocations between the human Mixed Lineage Leukemia (MLL) and AF4 Family (AFF) member genes, are implicated in leukemia. Mutations to AFFs can disrupt lymphopoesis, CNS development and spermatogenesis. However, despite the growing list of pathologies linked to AFF members, their evolutionary relationship and the structure/function of individual members, remain to be elucidated. Here, we first report that database mining and phylogenetic analysis with AFF proteins from multiple species, revealed two monophyletic sister clades, suggesting a common Bilateria ancestor. We then examined the structure/function of the most recently discovered AFF member, MCEF (also known as AF5q31 or AFF4). In silico, the human MCEF gene was found to have 21 exons, and code for a protein with seven nuclear localization sequences (NLS). In HeLa cells, an MCEF-EGFP fusion protein, localized exclusively to the nucleus. Consequently, we made twenty constructs, expressing MCEF deletion mutants fused to EGFP and/or DsRed fluorescent proteins. Three distinct protein sequences, encoded by three separate MCEF exons, were found to mediate nuclear localization, only two of which were predicted in silico. Importantly, we also found that ectopic expression of MCEF, repressed HIV-1 LTR-directed RNA Polymerase II transcription, at the level of Tat-transactivation. We suggest that portions of MCEF could be exploited for chimeric transcription factor repression (CTFR) of HIV-1.

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Two-pore channels and disease.

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| S-EPMC6162333 | biostudies-literature

Two-Pore Channels Regulate Inter-Organellar Ca<sup>2+</sup> Homeostasis in Immune Cells.

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| S-EPMC9103377 | biostudies-literature

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