Neurotoxic HIV-Tat Autocleaves and Forms Novel Amyloid-Like Fibrillar Structures

Abstract

HIV-Tat protein is released extracellularly in large amounts from HIV-infected cells and causes activation of lymphocytes, glial cells and neurotoxicity, even under antiretroviral therapy. Our atomic force microscopy (AFM) and mass spectrometry data showed that HIV-Tat can produce spontaneously shorter peptides, some of which are consistent with the protein's predicted inteins. We found that the HIV-Tat 32-62 piece is neurotoxic by itself. AFM showed, at 32 micrograms/ml, formation of micron-long HIV-Tat 32-62 fibrillar structures. These fibrils present branching and have a persistence length in the 80-90 nm range. With increasing concentration, the fibrils thicken and their branching angles decrease. With further increasing concentration, large irregularly shaped aggregates form in solution and, long fibers and sheet-like aggregates both extending over tens of microns form at air-liquid interface. Birefringence microscopy, FTIR and polarized Raman microspectroscopies of the aggregates suggest that the peptide has highly oriented side chains and backbone and contains 45% beta-sheet, consistent with the amyloid fiber structure. This is different than the full-length HIV-Tat, which is alpha-helical in bound state and in aggregates. Unlike other amyloids, the peptide aggregates have a distinct mechanism of irreversibility. Raman measurements showed that the free cysteines in aggregates form in time S-S bonds, which make the aggregates insoluble. The replacement of Cys34, Phe38 and Ile45 with Ala in the peptide chain led to reduced aggregation, no fibril formation and lower neurotoxicity. We identified microns-long Tat 32-62 fibers in cells expressing the peptide or the full-length Tat. Our model of the thinnest fibril involves a beta sheet backbone of the fibril to which small aggregates of various sizes are bound. These results suggest that HIV-Tat autocleavage makes possible the aggregation of the 32-62 fragment which leads to neurotoxicity through an amyloid type of mechanism.