In 2009 approximately 2.6 million people became infected with the Human Immunodeficiency Virus (HIV). In addition to the estimated 33.3 million people currently living with the virus, this makes HIV/ AIDS an epidemic of unprecedented scale in modern times. Treatment of HIV infection requires antiretroviral agents as well as a number of other drugs such as antimicrobials. Hypersensitivity adverse drug reactions (ADRs) to a variety of drugs are common in HIV-infected individuals, but the antimicrobial Sulphamethoxazole remains a major culprit. Hypersensitivity ADRs cause significant morbidity, with the skin and liver most commonly affected and are among the top causes of death in the developed world. While the pathophysiology of drug hypersensitivity in general remains incompletely understood, ADRs to Sulphamethoxazole have been linked to one of its reactive metabolites SMX-HA. Previous work from our lab has also shown that the HIV-1 Tat protein plays a role in SMX-induced hypersensitivity ADRs. We sought to determine if altering the amount of Tat would have an effect on cellular toxicity. We also wanted to find out how Tat affects toxicity and what region of the protein mediated those effects.
We created fusion proteins of Tat and its deletion mutants with green fluorescent protein and placed them in an inducible vector which was subsequently used to create stably transfected Jurkat T cell lines. These cell lines were differentially induced for Tat expression and then used in assays for cellular toxicity and oxidative stress in the absence and presence of SMX-HA. We found that cellular toxicity was dependent on the variant of Tat used. In the preliminary report, the first exon of the Tat protein was able to augment T cell death caused by the addition of SMX-HA, and that the cell death occurred via apoptosis. This cell death took place without alteration to the cellular redox state. In later experiments using a different Tat variant, only the full-length protein affected cell death after SMX-HA treatment. Also, expression of the full-length protein was able to cause an increase in ROS generated after incubation with SMX-HA. None of the deletion mutants had this effect.
To try to further elucidate the effects of HIV-1 Tat on the cellular redox state, a set of experiments were carried out to detect the consequences on thiol proteins of Tat expression in the presence and absence of SMX-HA. Following Tat expression and incubation of the Jurkat cells with either vehicle or SMX-HA, the cells were disrupted in the presence of iodoacetamide and the lysates applied to two-dimensional gel electrophoresis. In the absence of SMX-HA, the Tat-expressing cell lines were already under a fair amount of oxidative stress compared to the parent cell line and the HIV-infected cell line. Also in untreated cells, a small number of protein thiols were already oxidized. Exposure of the Tat-expressing cells to 200µM SMX-HA led to a dramatic increase in thiol protein oxidation
