HIV-1 Tat Protein Enters Dysfunctional Endothelial Cells via Integrins and Renders Them Permissive to Virus Replication.

Aurelio Cafaro,Barbara Ensoli,Claudia Andreini,Lucia Banci,Maria Rosaria Pavone Cossut,Antonella Tripiciano,Sonia Moretti,Orietta Picconi,Giovanni Barillari,Clelia Palladino,Cecilia Sgadari,Mario Falchi,Angela Arancio,Massimo Campagna,Paolo Monini

doi:10.3390/ijms22010317

Abstract

Previous work has shown that the Tat protein of Human Immunodeficiency Virus (HIV)-1 is released by acutely infected cells in a biologically active form and enters dendritic cells upon the binding of its arginine-glycine-aspartic acid (RGD) domain to the α5β1, αvβ3, and αvβ5 integrins. The up-regulation/activation of these integrins occurs in endothelial cells exposed to inflammatory cytokines that are increased in HIV-infected individuals, leading to endothelial cell dysfunction. Here, we show that inflammatory cytokine-activated endothelial cells selectively bind and rapidly take up nano-micromolar concentrations of Tat, as determined by flow cytometry. Protein oxidation and low temperatures reduce Tat entry, suggesting a conformation- and energy-dependent process. Consistently, Tat entry is competed out by RGD-Tat peptides or integrin natural ligands, and it is blocked by anti-α5β1, -αvβ3, and -αvβ5 antibodies. Moreover, modelling–docking calculations identify a low-energy Tat-αvβ3 integrin complex in which Tat makes contacts with both the αv and β3 chains. It is noteworthy that internalized Tat induces HIV replication in inflammatory cytokine-treated, but not untreated, endothelial cells. Thus, endothelial cell dysfunction driven by inflammatory cytokines renders the vascular system a target of Tat, which makes endothelial cells permissive to HIV replication, adding a further layer of complexity to functionally cure and/or eradicate HIV infection.

Highlights

By mediating the adhesive interactions occurring between endothelial cells and the vessel basement membrane, integrins exert important roles in either the maintenance of vascular integrity or the formation of new vessels [1,2]
To determine whether the Tat protein of Human Immunodeficiency Virus (HIV)-1 could enter quiescent and/or activated endothelial cells, primary human umbilical vein endothelial cells (HUVEC) were cultured in the presence or absence of human recombinant IL-1β, tumor necrosis factor (TNF)-α, and IFN-γ, which were combined at doses as found in inflammatory microenvironments
HUVEC, both inflammatory cytokine (IC)-activated (IC-HUVEC) and non-activated (HUVEC), were incubated in suspension with biologically active Tat protein at concentrations compatible with those detected in specimens from HIV-infected individuals [51,52,53,54]

Summary

Introduction

By mediating the adhesive interactions occurring between endothelial cells and the vessel basement membrane, integrins exert important roles in either the maintenance of vascular integrity or the formation of new vessels [1,2]. Among the inducers of endothelial cell dysfunction are inflammatory mediators including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ; of importance, these cytokines can alter the expression and/or function of integrins, such as α5β1 or αvβ, which are key to endothelial cells [8,9,10,11,12] In this regard, HIV-infected individuals display a chronic systemic inflammation and high IL-1β, TNF-α or IFN -γ plasma levels, even when they are treated with the combination antiretroviral therapy (cART) which reduces Human Immunodeficiency Virus (HIV)-1 replication or infectivity [13,14,15,16]. Abnormal angiogenesis and altered permeability of the blood–brain barrier associated to central and peripheral nervous system vasculopathies occur at high frequency in HIV infected individuals and contribute to the onset of tumors and dementia [22]

Methods

Results

Conclusion