Lisa J Henderson1, Tory P Johnson2, Bryan R Smith1, Lauren Bowen Reoma1, Ulisses A Santamaria1, Muzna Bachani3, Catherine Demarino4, Robert A Barclay4, Joseph Snow5, Ned Sacktor2, Justin Mcarthur2, Scott Letendre6, Joseph Steiner3, Fatah Kashanchi4, Avindra Nath1. 1. Section of Infections of the Nervous System, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda. 2. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore. 3. Translational Neuroscience Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland. 4. Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, Virginia. 5. National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland. 6. University of California San Diego School of Medicine, Division of Infectious Diseases and Global Public Health, San Diego, California, USA.
Abstract
OBJECTIVE: The aim of this study was to measure the protein concentration and biological activity of HIV-1 Tat in cerebrospinal fluid (CSF) of individuals on suppressive antiretroviral therapy (ART). DESIGN: CSF was collected from 68 HIV-positive individuals on ART with plasma viral load less than 40 copies/ml, and from 25 HIV-negative healthy controls. Duration of HIV infection ranged from 4 to more than 30 years. METHODS: Tat levels in CSF were evaluated by an ELISA. Tat protein and viral RNA were quantified from exosomes isolated from CSF, followed by western blot or quantitative reverse transcription PCR, respectively. Functional activity of Tat was assessed using an LTR transactivation assay. RESULTS: Tat protein was detected in 36.8% of CSF samples from HIV-positive patients. CSF Tat concentration increased in four out of five individuals after initiation of therapy, indicating that Tat was not inhibited by ART. Similarly, exosomes from 34.4% of CSF samples were strongly positive for Tat protein and/or TAR RNA. Exosomal Tat retained transactivation activity in a CEM-LTR reporter assay in 66.7% of samples assayed, which indicates that over half of the Tat present in CSF is functional. Presence of Tat in CSF was highly associated with previous abuse of psychostimulants (cocaine or amphetamines; P = 0.01) and worse performance in the psychomotor speed (P = 0.04) and information processing (P = 0.02) cognitive domains. CONCLUSION: Tat and TAR are produced in the central nervous system despite adequate ART and are packaged into CSF exosomes. Tat remains biologically active within this compartment. These studies suggest that Tat may be a quantifiable marker of the viral reservoir and highlight a need for new therapies that directly inhibit Tat.
OBJECTIVE: The aim of this study was to measure the protein concentration and biological activity of HIV-1Tat in cerebrospinal fluid (CSF) of individuals on suppressive antiretroviral therapy (ART). DESIGN: CSF was collected from 68 HIV-positive individuals on ART with plasma viral load less than 40 copies/ml, and from 25 HIV-negative healthy controls. Duration of HIV infection ranged from 4 to more than 30 years. METHODS:Tat levels in CSF were evaluated by an ELISA. Tat protein and viral RNA were quantified from exosomes isolated from CSF, followed by western blot or quantitative reverse transcription PCR, respectively. Functional activity of Tat was assessed using an LTR transactivation assay. RESULTS:Tat protein was detected in 36.8% of CSF samples from HIV-positive patients. CSF Tat concentration increased in four out of five individuals after initiation of therapy, indicating that Tat was not inhibited by ART. Similarly, exosomes from 34.4% of CSF samples were strongly positive for Tat protein and/or TAR RNA. Exosomal Tat retained transactivation activity in a CEM-LTR reporter assay in 66.7% of samples assayed, which indicates that over half of the Tat present in CSF is functional. Presence of Tat in CSF was highly associated with previous abuse of psychostimulants (cocaine or amphetamines; P = 0.01) and worse performance in the psychomotor speed (P = 0.04) and information processing (P = 0.02) cognitive domains. CONCLUSION:Tat and TAR are produced in the central nervous system despite adequate ART and are packaged into CSF exosomes. Tat remains biologically active within this compartment. These studies suggest that Tat may be a quantifiable marker of the viral reservoir and highlight a need for new therapies that directly inhibit Tat.
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