Measuring the uptake and transactivation function of HIV-1 tat protein in a trans-cellular cocultivation setup

Arthur P. Ruiz, Vinayaka R. Prasad

Research output: Chapter in Book/Report/Conference proceedingChapter

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 lympho-cytic Jurkat T-cells, to evaluate transcriptional changes elicited by Tat taken up by the uninfected cells.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages353-366
Number of pages14
Volume1354
DOIs
Publication statusPublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1354
ISSN (Print)10643745

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Keywords

  • Chemokine
  • Cytokine
  • Endocytosis
  • HIV-1
  • Tat
  • Trans-cellular transactivation
  • Transcription

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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 Methods in Molecular Biology (Vol. 1354, pp. 353-366). (Methods in Molecular Biology; Vol. 1354). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-3046-3_24