HIV-TAT 32-62 Protein Fragment Forms Fibrillar Structures

Abstract

HIV-Tat protein, the first protein released extracellularly in large amount after HIV virus enters the cell, causes activation of lymphocytes, glial cells and neurotoxicity. Atomic force microscopy showed that HIV-Tat protein can rupture to smaller pieces, some of them being of similar dimensions with the theoretically predicted inteins of this protein, which can spontaneously excise themselves out of the protein chain. The HIV-Tat 32-62 piece, which we found to be neurotoxic by itself, was synthetized and used in our experiments. Theoretical prediction by PONDR suggested that this fragment is likely unstructured and according to AGGRESCAN, prone to aggregation. Atomic force microscopy showed, aside of round aggregates of various sizes, formation of long fibrillar structures for this peptide. The fibrils are irregular along length and width, with dimensions in the range 0.4-1.3 nm for height and 3.6 - 8.7 nm for width, as measured at half height. The fibrils present branching and their bending is characterized by an average persistence length of 80 nm. Conformational assessment of HIV-Tat 32-62 by circular dichroism indicated a secondary structure dominated by random coil and beta sheet, with minimal alpha helix content, contrary to full length HIV-Tat. The replacement of C34, F38 and I45 with A in the polypeptide chain of HIV-Tat 32-62 led to a similar secondary structure, but to significant reduction in aggregation capacity, no stable fibril formation and preliminary tests indicated less neurotoxicity than for the original peptide. Our results suggest that the aggregation of HIV-Tat 32-62 fragment leads to neurotoxicity, likely through a different mechanism then the full length protein.