Extracellular addition of a domain of HIV‐1 Vpr containing the amino acid sequence motif H(S/F)RIG causes cell membrane permeabilization and death

Abstract

Vpr is a virion-associated protein of human immunodeficiency virus type 1 (HIV-1) whose function in acquired immune deficiency syndrome (AIDS) has been uncertain. We previously employed yeast as a model to examine the effects of Vpr on basic cellular functions; intracellular Vpr was shown to cause cell-growth arrest and structural defects, and these effects were caused by a region of Vpr containing the sequence HFRIGCRHSRIG. Here we show that peptides containing the H(S/F)RIG amino acid sequence motif cause death when added externally to a variety of yeast including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Candida albicans and Schizosaccharomyces pombe. Such peptides rapidly entered the cell from the time of addition, resulting in cell death. Elevated levels of ions, particularly magnesium and calcium ions, abrogated the cytotoxic effect by preventing the Vpr peptides from entering the cells. Extracellular Vpr found in the serum, or breakdown products of extracellular Vpr, may have similar effects to the Vpr peptides described here and could explain the death of uninfected bystander cells during AIDS.

The Cationic Amphipathic α-Helix of HIV-1 Viral Protein R (Vpr) Binds to Nucleic Acids, Permeabilizes Membranes, and Efficiently Transfects Cells

“…Indeed, peptides from the C-terminal region of Vpr including the conserved HFRIGCRHSRIG motif (Fig. 1) can cause permeabilization of yeast cells (41). Recent results showing that Vpr can gain access to intracellular compartments independently from the infection process support the idea that Vpr is membrane-active (24).…”

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

“…Although partial sequences of Vpr have been synthesized for biological (25,26) and structural studies (36 -38), chemical synthesis of full-length soluble forms of Vpr has proven difficult. For example, a Vpr peptide derived from the HIV-1 BRU sequence has been synthesized, but irreversible aggregation precluded purification of the product (42)(43)(44).…”

“…However, when maintained at high concentrations as required for structural investigation, preparations of recombinant Vpr often undergo spontaneous aggregation. In addition, a cytotoxic effect of the protein in both pro-and eukaryotic cells (25,26) limits production of Vpr by recombinant genetics.…”

Inhibition of adenine nucleotide translocator pore function and protection against apoptosis in vivo by an HIV protease inhibitor

“…We pretreated 10 4 cells/ml of Saccharomyces cerevisiae M 22-2-1 (genotype MATa ade2 leu2 lys2 his4 trpl ura3 Canr, por1:LEU2, por2:TRP1; gift from M. Forte, Vollum Institute, Portland, Oregon, USA) (59, 60), S. cerevisiae W301-1B control strain (MATa ade2, leu2, his3, trpl, ura3, can1), and JL1-3 (genotype like W301-1B, aac1:LEU2 aac2:HIS3, aac3:URA; gift from T. Drgon, NIH, Bethesda, Maryland, USA) (61), with NFV, which was followed by treatment with a Vpr-derived peptide (Genemed Synthesis Inc.) as described (62) or H2O2 (1 hour, 28°C). This was followed by plating on standard YPD agarose medium (200 yeasts/plate) and quantification of the percentage of surviving clones after 48 hours of culture at 28°C.…”