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HIV-Tat Protein: Oxidants and Redox Environment

The Virtual Free Radical School. HIV-Tat Protein: Oxidants and Redox Environment. Sonia C. Flores, Ph.D. Adela Cota-Gomez, Ph.D. University of Colorado Health Sciences Center Webb-Waring Institute for Cancer, Aging and Antioxidant Research phone: (303) 315-0055, fax: (303) 315-8541

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HIV-Tat Protein: Oxidants and Redox Environment

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  1. The Virtual Free Radical School HIV-Tat Protein: Oxidants and Redox Environment Sonia C. Flores, Ph.D. Adela Cota-Gomez, Ph.D. University of Colorado Health Sciences Center Webb-Waring Institute for Cancer, Aging and Antioxidant Research phone: (303) 315-0055, fax: (303) 315-8541 e-mail: Sonia.Flores@uchsc.edu or Adela.Cota-Gomez@uchsc.edu HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 1

  2. What is a virus • Among the smallest replicating microorganisms, 14-25 times smaller than mitonchondria, ranging in size between 40 and 70 nm • The virion size is limited by the structural components; in general larger viruses have more structural proteins and/or larger genomes • Have obligate intracellular growth due to their structural simplicity • Viral morphology is very diverse and is best studied using electron microscopy • Primarily classified based on whether they have RNA or DNA as genetic material • Are major causes of human disease, the most common of human ailments (Fundamental Virology, 2nd edition) HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 2

  3. HIV Structure • Human Immunodeficiency Virus (HIV) belongs to the retrovirus family and the class lentiviridae • Virions have a dense cylindrical core that encases the genomic +strand RNA and viral enzymes (ie. reverse transcriptase, integrase and protease) • A membranous lipid envelope surrounds the core and contains structural proteins • gp120 is the major extracellular envelope glycoprotein (“knob” structure) that binds to the major cellular receptor, CD4 antigen • gp41 is the transmembrane envelope glycoprotein that tethers gp120 to the lipid membrane (Fundamental Virology, 2nd edition) HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 3

  4. HIV Life Cycle 1 2 3 10 8 4 6,7 5 9 (Fundamental Virology, 2nd edition) HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 4

  5. HIV Life Cycle (cont.) • The virion attaches to a specific cell-surface receptor • The virion core penetrates into the cell • Reverse transcription within the virion core structure copies the genomic • viral RNA into DNA • Viral DNA, still associated with incoming virion proteins, translocates into • the nucleus • 5. The viral DNA, more or less randomly, integrates into the cell DNA to • form the provirus • 6. Viral RNA is synthesized by cellular RNA polymerase II using the • integrated provirus as template • Transcripts are processed to mRNA and complete RNA genomes • New virion proteins are synthesized • Virions are assembled at the cell membrane and bud-off • Capsid proteins are processed and the internal structures of the particle • mature after budding (Fundamental Virology, 2nd edition) HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 5

  6. Virus particles budding from a cell Enhanced electron micrograph of budding viral particles. Most retroviral particles assemble at the cell membrane, but additional processing and maturation of the internal core structure occurs after budding. HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 6

  7. HIV Genome • gag, pol, pro and env regions encode structural genes common to all retroviruses • In addition to these, HIV and other lentiviruses contain other genes with diverse roles in the virus life cycle • tat, rev and nef have transcriptional roles in replication • vif and vpu are important for infectivity and virus release • tat-deficient viruses are completely non-infectious • Tat binds to a transactivation- responsive region (TAR) in the HIV LTR and promotes transcriptional elongation (Fundamental Virology, 2nd edition; HIV Sequence Compendium, 2000) HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 7

  8. AIDS patients are oxidatively stressed • AIDS patients have: • Decreased plasma glutathione levels[Eck at al 1989, Staal et al. 1992] • Elevated plasma malondialdehyde levels[Sonnerborg et al. 1988, Revillard et al 1992] • Decreased plasma levels of vitamins A, E, C and beta-carotein [Wang et al 1994, Lacey et al. 1996] • Cultured HIV-Infected Cells: • Have decreased glutathione and vitamin E levels [Staal et al. 1992] • Antioxidants suppress replication of HIV in chronically infected cells [Roederer et al. 1991, Staal et al. 1995] • Treatments that increase oxidative stress (cytokine or UV irradiation) activate HIV replication [Vicenzi and Poli 1994] HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 8

  9. A complication in AIDS • AIDS patients have: • Aseptic cardiovascular and pulmonary complications(cardiac inflammatory infiltrates, myocarditis, interstitial alveolitis, emphysema and pulmonary hypertension). [deLeon et al. 1995] • Elevated levels of sICAM, an important indicator of inflammation. [Rieckmann et al. 1993] • Elevated levels of sICAM correlate with low T-cell counts. [Diez-Ruiz et al. 1993] • Elevated levels of cytokines (TNF, IL-6, IFNg) and chemokines (MCP-1). [Mengozzi et al. 1999, Roulston et al. 1995, Yoo et al. 1996, Israel-Biet et al. 1991] These cardiovascular complications are not associated with viral or opportunistic infections. Therefore, some soluble factor, of viral or cellular origin, must be responsible for this inflammatory response. HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 9

  10. The HIV Tat protein • Tat is absolutely required by the virus for productive infection • Tat protein is made by the virus only in infected cells • Tat is secreted by infected cells and is available to interact with and/or be taken up by un-infected by-stander cells • Internalization of Tat by un-infected cells has been proposed to occur via interactions with cell surface integrins • In un-infected cells Tat affects expression of important cellular genes: 1. Decreases production of superoxide dismutase, glutathione peroxidase and g-glutamyl synthetase 2. Increases expression of cytokines and cell-adhesion molecules in human endothelial cells HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A.10

  11. Functional domains of the HIV-Tat protein Sufficient for MnSOD repression 2nd exon acidic cys-rich core basic 72 86 101 Essential for NF-kB induction HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 11

  12. Tat upsets the oxidant/antioxidant balance • By inhibiting several of the antioxidant enzymes: • MnSOD [Flores et al. 1993] • Glutathione peroxidase [Richard et al. 2001] • g-glutamyl synthetase [Choi et al. 2000] Or by activating NADPH oxidase [Gu et al. 2001] HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 12

  13. Tat increases the “stickiness” of blood vessels by changing the oxidant/antioxidant balance Hypothesis Tat Tat Tat Tat Adhesion Molecules Tat Tat Tat SOD _ O _ SOD O 2 2 HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 13

  14. Tat increases the number of white blood cells that now stick to the blood vessels, SOD SOD HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 14

  15. Many of the regulatory effects of Tat on cellular genes occur through NF-kB activation HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 15

  16. Tat Tat Tat N N C 65 50 P N N Tat activates NF-kB 1 • Tat activates the cell at the surface • Oxidative stress induced leads to • Degradation of cytoplasmic • NF-kB inhibitor, IkB. • Resulting in nuclear localization • of NF-kB and • Up-regulation of cell-adhesion • molecules such as E-selectin N P 2 3 C C 4 Legend NF-kB IkB 5 E-selectin HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 16

  17. Antioxidants inhibit the effects of Tat • N-acetylcysteine (NAC) inhibited the Tat-mediated E- selectin expression in HUVEC • MnSOD mimetics had the same inhibitory effect on Tat HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 17

  18. Tat-Transgenic Mouse Model SV40 smallT poly A * The SPC promoter restricts expression to the lung type II epithelial cells. *SPCP Tat86 Tat • Tat-transgenic mice were engineered to study the in vivo effects of Tat. • Initial characterization of these mice shows decreased levels of lung • MnSOD protein, suggesting possible oxidative stress. • In addition, increased tyrosine nitration and carbonyl protein formation suggest that there is protein damage as a consequence of the oxidative stress. • Hematoxylin and eosin (H&E) staining of transgenic mouse lungs shows increased cellular infiltration indicative of an inflammatory state. HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 18

  19. Tat has similar effects in the central nervous system While our studies have focused on the effects of the Tat in the cardiac and pulmonary vasculature, others have reported similar effects of Tat in other systems such as the central nervous system. • Tat increases oxidative stress in microglia by activating NO synthase [Polazzi et al. 1999] • Tat has pro-inflammatory effects in microglial cells [Bruce-Keller et al. 2001] HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 19

  20. Summary • HIV is a retrovirus of the class lentiviridae and the causative agent of acquired immunodeficiency syndrome (AIDS). • HIV infects CD4+ T-cells via interactions with the gp120 envelope glycoprotein. • The HIV protein Tat is absolutely required by the virus for productive infection, is made only by infected cells but secreted and is taken up by un-infected by-stander cells via cell surface integrins. • In un-infected cells Tat affects expression of important cellular genes including pro-inflammatory cytokines and antioxidant enzymes. • The ability of Tat to decrease levels of antioxidants enzymes and activate oxidant producing enzymes such as NADPH oxidase may directly contribute to the aseptic inflammation and generalized oxidative stress manifested in AIDS patients. HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 20

  21. Summary, cont. • In the vasculature, Tat increases cell-adhesion molecule expression in an NF-kB dependent manner, which may result in increased adhesion of white blood cells to blood vessels. • Antioxidants inhibit the Tat-mediated up-regulation of cell-adhesion molecule expression. • Tat also has pro-inflammatory and pro-oxidant properties in the central nervous system. • Tat transgenic mice with specific expression of Tat in the lung epithelium exhibit evidence of oxidative stress and inflammation. HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 20

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