Cytoplasmic Distribution of HIV-1 Tat Sensitizes Jurkat T Cells to Sulphamethoxazole-hydroxylamine Induced Toxicity

Abstract

Background: One medication commonly used by HIV-1-infected individuals is the antimicrobial sulphamethoxazole (SMX), which is used in the treatment and prophylaxis of pneumocystis pneumonia. However, SMX is responsible for a very high incidence of hypersensitivity adverse drug reactions (ADRs) in the HIV-1 population. While the pathophysiology of ADRs in general is unknown, sulphamethoxazole-mediated ADRs have been linked to its reactive metabolite sulphamethoxazole-hydroxylamine (SMX-HA). Our previous work has shown that increased expression of the HIV-1 Tat protein in T cells correlated with increased apoptosis after incubation with SMX-HA. In this study we sought to determine the region of the Tat protein responsible for this effect and the mechanism by which Tat contributed to SMX-HA mediated apoptosis. Methods: We established Jurkat T and Cos 7 cell lines that stably expressed full-length Tat (Tat101) and deletion mutants (Tat86, Tat72, Tat48 and Tat∆). These cell lines were then incubated with SMX-HA and assayed for cell viability and production of reactive oxygen species (ROS). We further used confocal microscopy to assess the intracellular distribution of the Tat proteins and to determine if changes in the expression and/or localization of key cytoskeleton proteins contributed to Tat-mediated apoptosis after SMX-HA treatment. Results: Deletion of regions of Tat that lead to increased cytoplasmic accumulation significantly contributed to increased cell death in the presence of SMX-HA. The increased cell death did not require induction of ROS. Quantitative analysis also showed that the Tat-expressing cell lines had significantly lower levels of β-actin and α-tubulin present both before and after treatment with SMX-HA. Increased cytoplasmic localization of Tat correlated with greater disturbances in the distribution of actin filaments. Conclusion: The presence of cytoplasmic Tat in T and epithelial cell lines increases their sensitivity to SMX-HA induced cell death, an effect mediated by the first 48 amino acids of TAT.

HIV-1 tat expression and sulphamethoxazole hydroxylamine mediated oxidative stress alter the disulfide proteome in Jurkat T cells

“…The plasmid construction, their transfection and the subsequent selection of Jurkat E6.1 T cells stably expressing wildtype Tat with and without GFP as well as the Tat deletion mutants fused to GFP was previously described in detail [ 21 , 40 ]. The impact of wildtype Tat and each of the Tat deletion mutants on cell viability in the presence and absence of SMX-HA was also described previously [ 21 ]. Briefly, the Tat gene was PCR-amplified from the plasmid pSVTat that encodes the full-length Tat gene and was a kind gift from Dr. K.T.…”

“…Tat also contains a protein transduction domain (PTD) which enables the protein to be secreted from HIV-1-infected cells and enter uninfected cells to regulate host gene expression [ 16 , 20 ]. The PTD also serves as a nuclear localization sequence (NLS) that restricts Tat to the nucleus preventing Tat from interacting with cellular protein that can result in cytopathic effects as we have recently demonstrated [ 21 ].…”

“…We have previously developed Tat green fluorescent protein (GFP) fusion protein mutants to map the Tat region that contributes to the development of ADRs [ 21 , 40 ]. The mutants include the full-length protein (Tat101GFP) and the product of the first exon (Tat72GFP), both of which have the NLS that restricts them to the nucleus.…”

“…The mutants include the full-length protein (Tat101GFP) and the product of the first exon (Tat72GFP), both of which have the NLS that restricts them to the nucleus. Additionally, a TatΔGFP mutant was created expressing the full-length protein with a deleted NLS that resulted in significant cytoplasmic distribution of the protein [ 21 ]. These constructs are expressed under the control of an inducible promoter so that the expression of these Tat mutant proteins approximated the level of expression achieved in HIV infected T cells.…”

“…These mutant forms of Tat enhance, to varying degrees, SMX-HA-mediated toxicity as we have observed in wildtype HIV infected T cells. These constructs, their induction characteristics, impact on cell viability and the degree to which they induce ROS have been reported in detail elsewhere [ 21 , 40 ].…”