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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: [email protected] or [email protected]

The Virtual Free Radical School

HIV-1 Tat Oxygen Society Education Program Flores, S and Cota-Gomez, A. 1

HIV-Tat Protein: Oxidants and Redox Environment

Sonia C. Flores, Ph.D. Adela Cota-Gomez, Ph.D.


What is a virus

Among the smallest replicating microorganisms, 14-25 times smaller than mitonchondria, ranging in size between 40 and 70 nmThe virion size is limited by the structural components; in general larger viruses have more structural proteins and/or larger genomesHave obligate intracellular growth due to their structural simplicityViral morphology is very diverse and is best studied using electron microscopyPrimarily 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 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 proteinsgp120 is the major extracellular envelope glycoprotein (“knob” structure) that binds to the major cellular receptor, CD4 antigengp41 is the transmembrane envelope glycoprotein that tethers gp120 to the lipid membrane


HIV Life Cycle


1

2

3

4

5

6,7

8

9

10


HIV Life Cycle (cont.)


1. The virion attaches to a specific cell-surface receptor2. The virion core penetrates into the cell3. Reverse transcription within the virion core structure copies the genomic

viral RNA into DNA4. Viral DNA, still associated with incoming virion proteins, translocates into

the nucleus5. The viral DNA, more or less randomly, integrates into the cell DNA to

form the provirus6. Viral RNA is synthesized by cellular RNA polymerase II using the

integrated provirus as template7. Transcripts are processed to mRNA and complete RNA genomes8. New virion proteins are synthesized9. Virions are assembled at the cell membrane and bud-off10. Capsid proteins are processed and the internal structures of the particle

mature after budding(Fundamental Virology, 2nd edition)

Virus particles budding from a cell


Enhanced electron micrographof budding viral particles.Most retroviral particlesassemble at the cell membrane,but additional processing andmaturation of the internal corestructure occurs after budding.

HIV Genomegag, pol, pro and env regions encode structural genes common to all retrovirusesIn 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 replicationvif and vpu are important for infectivity and virus releasetat-deficient viruses are completely non-infectiousTat binds to a transactivation- responsive region (TAR) in the HIV LTR and promotes transcriptional elongation

(Fundamental Virology, 2nd edition; HIV Sequence Compendium, 2000)


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]


A complication in AIDSAIDS 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, IFN) 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.


The HIV Tat proteinTat 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 -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

acidic cys-rich core basic

72 86 101

2nd exon

Essential forNF-B induction

Sufficient for MnSOD repression

Functional domains of the HIV-Tat protein


Tat upsets the oxidant/antioxidant balance

By inhibiting several of the antioxidant enzymes: MnSOD [Flores et al. 1993]

Glutathione peroxidase [Richard et al. 2001]

-glutamyl synthetase [Choi et al. 2000]

Or by activating NADPH oxidase [Gu et al. 2001]


Tat increases the “stickiness” of blood vessels by changing the oxidant/antioxidant balance

Tat

O2_

AdhesionMolecules

Tat

Tat

Tat

TatTat

Tat

SOD O2_

SOD

Hypothesis


SOD SOD

Tat increases the number of white blood cells that now stick to the blood vessels,


Many of the regulatory effects of Tat on cellular

genes occur through NF-B activation


TatTat

Tat

N

N

C

P

C

N

65

N

C

50

IBP

NF-BN

E-selectin

Tat activates NF-B

Legend

1

2

3

4

5

1. Tat activates the cell at the surface

2. Oxidative stress induced leads to

3. Degradation of cytoplasmicNF-B inhibitor, IB.

4. Resulting in nuclear localizationof NF-B and

5. Up-regulation of cell-adhesionmolecules such as E-selectin


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


Tat-Transgenic Mouse Model


Tat

*SPCP

SV40 smallTpoly ATat86

* The SPC promoter restricts expression to the lung type II epithelial cells.

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.

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]

Tat has similar effects in the central nervous system


SummaryHIV 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.


Summary, cont.In the vasculature, Tat increases cell-adhesion molecule expression in an NF-B 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.


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