Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology

Abstract

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal f luid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of f luorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potentialsensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal f luid.

Functional and Structural Characterization of Synthetic HIV-1 Vpr That Transduces Cells, Localizes to the Nucleus, and Induces G2 Cell Cycle Arrest

“…Based on these data, several studies have focused on the biological activity of recombinant Vpr. The Vpr expressed in E. coli has been shown to induce apoptosis, an activity recently ascribed to Vpr in HIV-1-infected cells (15,21,51). However, in these studies, Vpr was excluded from the nucleus and embedded in the plasmalemma, possibly leading to the formation of ion conductive membrane pores (51).…”

“…The sVpr produced synthetically and analyzed in this study did not appear to be membrane-integrated but rather transduced cell membranes, was nucleophilic, and induced G 2 cell cycle arrest. The fact that the structure and purity of the recombinant protein studied previously (15,21,51) is unknown and that this product possessed no other known biological activity of viral Vpr implies that it was defective in its ability to fully traverse membranes and thus results in cytotoxic effects. Nevertheless, it will be interesting to examine whether sVpr is able to mediate apoptosis (21,22), an effect that may lead to T cell depletion and immune cell avoidance in vivo.…”

“…The Vpr expressed in E. coli has been shown to induce apoptosis, an activity recently ascribed to Vpr in HIV-1-infected cells (15,21,51). However, in these studies, Vpr was excluded from the nucleus and embedded in the plasmalemma, possibly leading to the formation of ion conductive membrane pores (51). The sVpr produced synthetically and analyzed in this study did not appear to be membrane-integrated but rather transduced cell membranes, was nucleophilic, and induced G 2 cell cycle arrest.…”

Human immunodeficiency virus type 1 (HIV-1) Vpr-regulated cell death: insights into mechanism

“…8 The purified Vpr has been shown to have transactivational and cytotoxic properties in CD4 þ T cells and neurons, respectively. [8][9][10] Such results suggest that Vpr's cellular effects can manifest through various mechanisms including entry of free Vpr into uninfected, activated CD4 þ T cells for destruction. Accordingly, we review the questions surrounding the biochemical mechanism of Vpr-induced cell death.…”

Productive HIV-1 Infection of Primary CD4+ T Cells Induces Mitochondrial Membrane Permeabilization Leading to a Caspase-independent Cell Death

“…Indeed, it has been recently proposed that Vpr (a protein encoded by HIV) is directly targeted to the mitochondrial permeability transition pore complex and permeabilizes mitochondrial membranes in a cell-free system (27). The Vpr protein has been reported to kill lymphocytes, monocytes, and neurons (26,75,76) and to induce mitochondrial dysfunction in the yeast Saccharomyces cerevisiae (77). However, it may also act as a negative regulator of cell death in human cell lines and thymocytes (78,79), and CEM T cells infected with a triple HIV-1-mediated Cell Deathmutant (nef, vpr, and vpu deleted) derived from HIV-1 also undergo cell death (80), suggesting that Vpr is dispensable for HIV-mediated CD4 T cell depletion.…”