title: Measuring the Uptake and Transactivation Function of HIV-1 Tat Protein in a Trans-cellular Cocultivation Setup.
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.
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, 10461, USA.
authors.forename: Arthur P
authors.initials: AP
authors.lastname: Ruiz
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, 10461, USA. vinayaka.prasad@einstein.yu.edu.
authors.forename: Vinayaka R
authors.initials: VR
authors.lastname: Prasad
mesh_terms: D002460:Cell Line; D018920:Coculture Techniques / Q000379:methods*; D016207:Cytokines / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004705:Endocytosis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015658:HIV Infections / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D015533:Transcriptional Activation; D014162:Transfection / Q000379:methods; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000378:metabolism*
chemical_list: D016207:Cytokines; D054322:tat Gene Products, Human Immunodeficiency Virus
keywords: Chemokine; Cytokine; Endocytosis; HIV-1; Tat; Trans-cellular transactivation; Transcription