8 results on '"Clementz MA"'
Search Results
2. Resistance to the Tat Inhibitor Didehydro-Cortistatin A Is Mediated by Heightened Basal HIV-1 Transcription.
- Author
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Mousseau G, Aneja R, Clementz MA, Mediouni S, Lima NS, Haregot A, Kessing CF, Jablonski JA, Thenin-Houssier S, Nagarsheth N, Trautmann L, and Valente ST
- Subjects
- Cell Line, HIV-1 genetics, Humans, RNA, Messenger biosynthesis, RNA, Viral biosynthesis, Up-Regulation, tat Gene Products, Human Immunodeficiency Virus genetics, Anti-HIV Agents pharmacology, Drug Resistance, Viral, Gene Expression Regulation, Viral, HIV-1 growth & development, Heterocyclic Compounds, 4 or More Rings pharmacology, Isoquinolines pharmacology, Transcription, Genetic, tat Gene Products, Human Immunodeficiency Virus antagonists & inhibitors
- Abstract
Human immunodeficiency virus type 1 (HIV-1) Tat binds the viral RNA structure transactivation-responsive element (TAR) and recruits transcriptional cofactors, amplifying viral mRNA expression. The Tat inhibitor didehydro-cortistatin A (dCA) promotes a state of persistent latency, refractory to viral reactivation. Here we investigated mechanisms of HIV-1 resistance to dCA in vitro Mutations in Tat and TAR were not identified, consistent with the high level of conservation of these elements. Instead, viruses resistant to dCA developed higher Tat-independent basal transcription. We identified a combination of mutations in the HIV-1 promoter that increased basal transcriptional activity and modifications in viral Nef and Vpr proteins that increased NF-κB activity. Importantly, these variants are unlikely to enter latency due to accrued transcriptional fitness and loss of sensitivity to Tat feedback loop regulation. Furthermore, cells infected with these variants become more susceptible to cytopathic effects and immune-mediated clearance. This is the first report of viral escape to a Tat inhibitor resulting in heightened Tat-independent activity, all while maintaining wild-type Tat and TAR. IMPORTANCE HIV-1 Tat enhances viral RNA transcription by binding to TAR and recruiting activating factors. Tat enhances its own transcription via a positive-feedback loop. Didehydro-cortistatin A (dCA) is a potent Tat inhibitor, reducing HIV-1 transcription and preventing viral rebound. dCA activity demonstrates the potential of the "block-and-lock" functional cure approaches. We investigated the viral genetic barrier to dCA resistance in vitro While mutations in Tat and TAR were not identified, mutations in the promoter and in the Nef and Vpr proteins promoted high Tat-independent activity. Promoter mutations increased the basal transcription, while Nef and Vpr mutations increased NF-κB nuclear translocation. This heightened transcriptional activity renders CD4
+ T cells infected with these viruses more susceptible to cytotoxic T cell-mediated killing and to cell death by cytopathic effects. Results provide insights on drug resistance to a novel class of antiretrovirals and reveal novel aspects of viral transcriptional regulation., (Copyright © 2019 Mousseau et al.)- Published
- 2019
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3. Didehydro-Cortistatin A Inhibits HIV-1 by Specifically Binding to the Unstructured Basic Region of Tat.
- Author
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Mediouni S, Chinthalapudi K, Ekka MK, Usui I, Jablonski JA, Clementz MA, Mousseau G, Nowak J, Macherla VR, Beverage JN, Esquenazi E, Baran P, de Vera IMS, Kojetin D, Loret EP, Nettles K, Maiti S, Izard T, and Valente ST
- Subjects
- Anti-HIV Agents chemical synthesis, Cyclin-Dependent Kinase 8 metabolism, HeLa Cells, Heterocyclic Compounds, 4 or More Rings chemical synthesis, Humans, Isoquinolines chemical synthesis, Molecular Docking Simulation, Protein Binding, Anti-HIV Agents metabolism, HIV-1 drug effects, Heterocyclic Compounds, 4 or More Rings metabolism, Isoquinolines metabolism, tat Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
The intrinsically disordered HIV-1 Tat protein binds the viral RNA transactivation response structure (TAR), which recruits transcriptional cofactors, amplifying viral mRNA expression. Limited Tat transactivation correlates with HIV-1 latency. Unfortunately, Tat inhibitors are not clinically available. The small molecule didehydro-cortistatin A (dCA) inhibits Tat, locking HIV-1 in persistent latency, blocking viral rebound. We generated chemical derivatives of dCA that rationalized molecular docking of dCA to an active and specific Tat conformer. These revealed the importance of the cycloheptene ring and the isoquinoline nitrogen's positioning in the interaction with specific residues of Tat's basic domain. These features are distinct from the ones required for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known ligand of dCA. Besides, we demonstrated that dCA activity on HIV-1 transcription is independent of CDK8. The binding of dCA to Tat with nanomolar affinity alters the local protein environment, rendering Tat more resistant to proteolytic digestion. dCA thus locks a transient conformer of Tat, specifically blocking functions dependent of its basic domain, namely the Tat-TAR interaction; while proteins with similar basic patches are unaffected by dCA. Our results improve our knowledge of the mode of action of dCA and support structure-based design strategies targeting Tat, to help advance development of dCA, as well as novel Tat inhibitors. IMPORTANCE Tat activates virus production, and limited Tat transactivation correlates with HIV-1 latency. The Tat inhibitor dCA locks HIV in persistent latency. This drug class enables block-and-lock functional cure approaches, aimed at reducing residual viremia during therapy and limiting viral rebound. dCA may also have additional therapeutic benefits since Tat is also neurotoxic. Unfortunately, Tat inhibitors are not clinically available. We generated chemical derivatives and rationalized binding to an active and specific Tat conformer. dCA features required for Tat inhibition are distinct from features needed for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known target of dCA. Furthermore, knockdown of CDK8 did not impact dCA's activity on HIV-1 transcription. Binding of dCA to Tat's basic domain altered the local protein environment and rendered Tat more resistant to proteolytic digestion. dCA locks a transient conformer of Tat, blocking functions dependent on its basic domain, namely its ability to amplify viral transcription. Our results define dCA's mode of action, support structure-based-design strategies targeting Tat, and provide valuable information for drug development around the dCA pharmacophore., (Copyright © 2019 Mediouni et al.)
- Published
- 2019
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4. Didehydro-cortistatin A inhibits HIV-1 Tat mediated neuroinflammation and prevents potentiation of cocaine reward in Tat transgenic mice.
- Author
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Mediouni S, Jablonski J, Paris JJ, Clementz MA, Thenin-Houssier S, McLaughlin JP, and Valente ST
- Subjects
- Animals, Anti-HIV Agents pharmacokinetics, Chemokines metabolism, Cocaine adverse effects, Cytokines metabolism, Disease Models, Animal, HIV Infections complications, HIV Infections drug therapy, HIV-1 drug effects, Heterocyclic Compounds, 4 or More Rings pharmacokinetics, Inflammation metabolism, Isoquinolines pharmacokinetics, Mice, Mice, Inbred C57BL, Mice, Transgenic, Neurocognitive Disorders etiology, Neurocognitive Disorders prevention & control, Anti-HIV Agents pharmacology, Cocaine pharmacology, Dopamine Uptake Inhibitors pharmacology, Heterocyclic Compounds, 4 or More Rings pharmacology, Isoquinolines pharmacology, Reward, tat Gene Products, Human Immunodeficiency Virus physiology
- Abstract
HIV-1 Tat protein has been shown to have a crucial role in HIV-1-associated neurocognitive disorders (HAND), which includes a group of syndromes ranging from undetectable neurocognitive impairment to dementia. The abuse of psychostimulants, such as cocaine, by HIV infected individuals, may accelerate and intensify neurological damage. On the other hand, exposure to Tat potentiates cocaine-mediated reward mechanisms, which further promotes HAND. Here, we show that didehydro-Cortistatin A (dCA), an analog of a natural steroidal alkaloid, crosses the blood-brain barrier, cross-neutralizes Tat activity from several HIV-1 clades and decreases Tat uptake by glial cell lines. In addition, dCA potently inhibits Tat mediated dysregulation of IL-1β, TNF-α and MCP-1, key neuroinflammatory signaling proteins. Importantly, using a mouse model where doxycycline induces Tat expression, we demonstrate that dCA reverses the potentiation of cocaine-mediated reward. Our results suggest that adding a Tat inhibitor, such as dCA, to current antiretroviral therapy may reduce HIV-1-related neuropathogenesis.
- Published
- 2015
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5. An analog of the natural steroidal alkaloid cortistatin A potently suppresses Tat-dependent HIV transcription.
- Author
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Mousseau G, Clementz MA, Bakeman WN, Nagarsheth N, Cameron M, Shi J, Baran P, Fromentin R, Chomont N, and Valente ST
- Subjects
- Alkaloids chemical synthesis, Alkaloids chemistry, Alkaloids pharmacokinetics, Animals, Antiretroviral Therapy, Highly Active, Binding Sites, CD4-Positive T-Lymphocytes virology, Cells, Cultured drug effects, Cells, Cultured virology, Female, Gene Expression Regulation, Viral drug effects, HIV Core Protein p24 metabolism, HIV Infections drug therapy, HIV Infections virology, HIV-1 drug effects, HIV-1 physiology, Humans, Male, Mice, Mice, Inbred C57BL, Microsomes, Liver drug effects, Promoter Regions, Genetic, Proviruses drug effects, Proviruses genetics, Transcription, Genetic drug effects, Virus Replication drug effects, tat Gene Products, Human Immunodeficiency Virus antagonists & inhibitors, tat Gene Products, Human Immunodeficiency Virus genetics, Alkaloids pharmacology, Anti-HIV Agents pharmacology, HIV-1 genetics, Heterocyclic Compounds, 4 or More Rings pharmacology, Isoquinolines pharmacology, Polycyclic Compounds chemistry, tat Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
The human immunodeficiency virus type 1 (HIV) Tat protein, a potent activator of HIV gene expression, is essential for integrated viral genome expression and represents a potential antiviral target. Tat binds the 5'-terminal region of HIV mRNA's stem-bulge-loop structure, the transactivation-responsive (TAR) element, to activate transcription. We find that didehydro-Cortistatin A (dCA), an analog of a natural steroidal alkaloid from a marine sponge, inhibits Tat-mediated transactivation of the integrated provirus by binding specifically to the TAR-binding domain of Tat. Working at subnanomolar concentrations, dCA reduces Tat-mediated transcriptional initiation/elongation from the viral promoter to inhibit HIV-1 and HIV-2 replication in acutely and chronically infected cells. Importantly, dCA abrogates spontaneous viral particle release from CD4(+)T cells from virally suppressed subjects on highly active antiretroviral therapy (HAART). Thus, dCA defines a unique class of anti-HIV drugs that may inhibit viral production from stable reservoirs and reduce residual viremia during HAART., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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6. Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of STING-mediated signaling.
- Author
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Sun L, Xing Y, Chen X, Zheng Y, Yang Y, Nichols DB, Clementz MA, Banach BS, Li K, Baker SC, and Chen Z
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- Adaptor Proteins, Signal Transducing metabolism, Animals, Biocatalysis, Cell Membrane enzymology, Chlorocebus aethiops, Coronavirus 3C Proteases, Coronavirus NL63, Human physiology, Cysteine Endopeptidases chemistry, HEK293 Cells, Humans, I-kappa B Kinase metabolism, Interferon Regulatory Factor-3 metabolism, Interferons metabolism, Membrane Proteins chemistry, Protein Multimerization, Protein Structure, Quaternary, Protein Structure, Tertiary, Severe acute respiratory syndrome-related coronavirus physiology, Ubiquitination immunology, Vero Cells, Viral Proteins chemistry, Coronavirus NL63, Human enzymology, Cysteine Endopeptidases metabolism, Immunity, Innate, Membrane Proteins metabolism, Severe acute respiratory syndrome-related coronavirus enzymology, Signal Transduction immunology, Viral Proteins metabolism
- Abstract
Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction.
- Published
- 2012
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7. Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases.
- Author
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Clementz MA, Chen Z, Banach BS, Wang Y, Sun L, Ratia K, Baez-Santos YM, Wang J, Takayama J, Ghosh AK, Li K, Mesecar AD, and Baker SC
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- Cell Line, Coronavirus 3C Proteases, Cytokines metabolism, Humans, Ubiquitins metabolism, Cysteine Endopeptidases metabolism, Interferon Type I antagonists & inhibitors, Severe acute respiratory syndrome-related coronavirus pathogenicity, Ubiquitin metabolism, Viral Proteins metabolism
- Abstract
Coronaviruses encode multifunctional proteins that are critical for viral replication and for blocking the innate immune response to viral infection. One such multifunctional domain is the coronavirus papain-like protease (PLP), which processes the viral replicase polyprotein, has deubiquitinating (DUB) activity, and antagonizes the induction of type I interferon (IFN). Here we characterized the DUB and IFN antagonism activities of the PLP domains of human coronavirus NL63 and severe acute respiratory syndrome (SARS) coronavirus to determine if DUB activity mediates interferon antagonism. We found that NL63 PLP2 deconjugated ubiquitin (Ub) and the Ub-line molecule ISG15 from cellular substrates and processed both lysine-48- and lysine-63- linked polyubiquitin chains. This PLP2 DUB activity was dependent on an intact catalytic cysteine residue. We demonstrated that in contrast to PLP2 DUB activity, PLP2-mediated interferon antagonism did not require enzymatic activity. Furthermore, addition of an inhibitor that blocks coronavirus protease/DUB activity did not abrogate interferon antagonism. These results indicated that a component of coronavirus PLP-mediated interferon antagonism was independent of protease and DUB activity. Overall, these results demonstrate the multifunctional nature of the coronavirus PLP domain as a viral protease, DUB, and IFN antagonist and suggest that these independent activities may provide multiple targets for antiviral therapies.
- Published
- 2010
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8. Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles.
- Author
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Clementz MA, Kanjanahaluethai A, O'Brien TE, and Baker SC
- Subjects
- Amino Acid Substitution genetics, Animals, Cell Line, Cell Membrane ultrastructure, Cell Membrane virology, Cricetinae, Humans, Microscopy, Electron, Transmission, Mitochondria chemistry, Murine hepatitis virus physiology, Murine hepatitis virus ultrastructure, Mutagenesis, Site-Directed, Murine hepatitis virus genetics, Viral Nonstructural Proteins genetics, Virus Assembly
- Abstract
Coronaviruses are positive-strand RNA viruses that replicate in the cytoplasm of infected cells by generating a membrane-associated replicase complex. The replicase complex assembles on double membrane vesicles (DMVs). Here, we studied the role of a putative replicase anchor, nonstructural protein 4 (nsp4), in the assembly of murine coronavirus DMVs. We used reverse genetics to generate infectious clone viruses (icv) with an alanine substitution at nsp4 glycosylation site N176 or N237, or an asparagine to threonine substitution (nsp4-N258T), which is proposed to confer a temperature sensitive phenotype. We found that nsp4-N237A is lethal and nsp4-N258T generated a virus (designated Alb ts6 icv) that is temperature sensitive for viral replication. Analysis of Alb ts6 icv-infected cells revealed that there was a dramatic reduction in DMVs and that both nsp4 and nsp3 partially localized to mitochondria when cells were incubated at the non-permissive temperature. These results reveal a critical role of nsp4 in directing coronavirus DMV assembly.
- Published
- 2008
- Full Text
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