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Measuring the Uptake and Transactivation Function of HIV-1 Tat Protein in a Trans-cellular Cocultivation Setup

  • Protocol
HIV Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1354))

Abstract

HIV-1 Tat protein is secreted from infected cells and is endocytosed by uninfected bystander cells. Subsequently, Tat is translocated to the nucleus and binds to promoters of host cell genes, increasing the production of inflammatory host cytokines and chemokines. This inflammatory activation of uninfected cells by HIV-1 Tat protein contributes to the overall inflammatory burden in the central nervous system (CNS) that leads to the development of HIV-associated neurocognitive disorders (HAND). Here we describe methods to evaluate the uptake and transcriptional impact of HIV-1 Tat on uninfected cells by using a trans-cellular transactivation system. Cell lines transiently transfected with Tat expression constructs secrete Tat into the culture medium. Trans-cellular uptake and transactivation caused by secreted Tat can be measured by co-culturing LTR-responsive reporter cells with Tat-transfected cells. Such Tat-producer cells can also be co-cultured with immune cell lines, such as monocytic THP-1 cells or lymphocytic Jurkat T-cells, to evaluate transcriptional changes elicited by Tat taken up by the uninfected cells.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, 10461, USA

    Arthur P. Ruiz & Vinayaka R. Prasad

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  1. Arthur P. Ruiz
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  2. Vinayaka R. Prasad
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Correspondence to Vinayaka R. Prasad .

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Editors and Affiliations

  1. Department of Microbiology & Immunology, Albert Einstein College of Medicine, BRONX, New York, USA

    Vinayaka R. Prasad

  2. Department of Genetics, Albert Einstein College of Medicine, BRONX, New York, USA

    Ganjam V. Kalpana

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Ruiz, A.P., Prasad, V.R. (2016). Measuring the Uptake and Transactivation Function of HIV-1 Tat Protein in a Trans-cellular Cocultivation Setup. In: Prasad, V., Kalpana, G. (eds) HIV Protocols. Methods in Molecular Biology, vol 1354. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3046-3_24

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  • DOI: https://doi.org/10.1007/978-1-4939-3046-3_24

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3045-6

  • Online ISBN: 978-1-4939-3046-3

  • eBook Packages: Springer Protocols

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