HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region

Abstract

Objective: To analyze the mechanisms of release and the extracellular fate of the HIV-1 Tat protein and to determine the Tat domain binding to the extracellular matrix.

Design and methods: Release of Tat was studied by pulse-chase experiments with Tat-transfected COS-1 cells in the presence or absence of different serum concentrations, temperatures and drugs inhibiting the classical secretion pathway or endo-exocytosis, such as brefeldin A and methylamine. The binding of extracellular Tat to heparan sulfate proteoglycans (HSPG) was determined by using trypsin, heparin or heparinase in pulse-chase experiments, by gel shift and competition assays with radiolabeled heparin, and by heparin-affinity chromatography. The mapping of the Tat binding site to heparin was defined by functional assays of rescue of Tat-defective HIV-1 proviruses.

Results: Tat is released in the absence of cell death or permeability changes. Tat release is dependent upon the temperature and serum concentration, and it is not blocked by brefeldin A or methylamine. After release, a portion of the protein remains in a soluble form whereas the other binds to extracellular matrix (ECM)-associated HSPG. The HSPG-bound Tat can be retrieved into a soluble form by heparin, heparinase or trypsin. Binding to heparin is competed out by heparin-binding factors such as basic fibroblast growth factor (bFGF), and it is mediated by the Tat basic region which forms a specific complex with heparin which blocks HIV-1 rescue by exogenous Tat and allows purification of a highly biologically active protein.

Conclusions: These results demonstrate that Tat exits from intact cells through a leaderless secretion pathway which shares several features with that of acid FGF or bFGF. The released Tat binds to HSPG through its basic region and this determines its storage into the ECM, as occurs for bFGF.