The amino-terminal region of Vpr from human immunodeficiency virus type 1 forms ion channels and kills neurons

Abstract

We have previously reported that the accessory protein Vpr from human immunodeficiency virus type 1 forms cation-selective ion channels in planar lipid bilayers and is able to depolarize intact cultured neurons by causing an inward sodium current, resulting in cell death. In this study, we used site-directed mutagenesis and synthetic peptides to identify the structural regions responsible for the above functions. Mutations in the N-terminal region of Vpr were found to affect channel activity, whereas this activity was not affected by mutations in the hydrophobic region of Vpr (amino acids 53 to 71). Analysis of mutants containing changes in the basic C terminus confirmed previous results that this region, although not necessary for ion channel function, was responsible for the observed rectification of wild-type Vpr currents. A peptide comprising the first 40 N-terminal amino acids of Vpr (N40) was found to be sufficient to form ion channels similar to those caused by wild-type Vpr in planar lipid bilayers. Furthermore, N40 was able to cause depolarization of the plasmalemma and cell death in cultured hippocampal neurons with a time course similar to that seen with wild-type Vpr, supporting the idea that this region is responsible for Vpr ion channel function and cytotoxic effects. Since Vpr is found in the serum and cerebrospinal fluids of AIDS patients, these results may have significance for AIDS pathology.

FIG. 1

(A) Amino acid sequence of Vpr. The hydrophobic region is in boldface italics, the H(F/S)RIG motifs are underlined, and the sites of point mutations are indicated in boldface. The synthetic peptides are indicated underneath the sequence, as are the sites of the deletions in VprΔ1 and VprΔ2. (B) Purity of WT Vpr preparation. Lanes 1 and 2, Coomassie blue-stained gel of the Vpr preparation and molecular mass markers, respectively; lane 3, Western blot with antibody specific to the C terminus of Vpr. (C) Western blots of HPLC-purified fractions of Vpr mutant proteins probed with AbN. The arrows indicate the sizes of full-length Vpr and C-terminally truncated forms of Vpr (VprΔ1). Numbers at left show molecular masses in kilodaltons.

FIG. 1

(A) Amino acid sequence of Vpr. The hydrophobic region is in boldface italics, the H(F/S)RIG motifs are underlined, and the sites of point mutations are indicated in boldface. The synthetic peptides are indicated underneath the sequence, as are the sites of the deletions in VprΔ1 and VprΔ2. (B) Purity of WT Vpr preparation. Lanes 1 and 2, Coomassie blue-stained gel of the Vpr preparation and molecular mass markers, respectively; lane 3, Western blot with antibody specific to the C terminus of Vpr. (C) Western blots of HPLC-purified fractions of Vpr mutant proteins probed with AbN. The arrows indicate the sizes of full-length Vpr and C-terminally truncated forms of Vpr (VprΔ1). Numbers at left show molecular masses in kilodaltons.

FIG. 2

C-terminal mutants: examples of currents generated in planar lipid bilayers by VprΔ1 (A) and VprR95Q (C) at different holding potentials. The dashed lines indicate the zero current levels. The average currents are plotted versus holding potential for VprΔ1 (filled circles) (B), VprR95Q (filled squares) (D), and, for comparison, WT Vpr (open circles).

FIG. 3

Hydrophobic region mutants: examples of currents generated in planar lipid bilayers by VprΔ2 (A) and VprE58Q (C) at different holding potentials. The dashed lines indicate the zero current levels. The average currents are plotted versus holding potential for VprΔ2 (filled circles) (B), VprE58Q (filled inverted triangles) (D), and, for comparison, WT Vpr (open circles).

FIG. 4

N-terminal mutants: examples of currents generated in planar lipid bilayers by VprE21Q (A) and VprE24Q (C) at different holding potentials. The dashed lines indicate the zero current levels. The average currents are plotted versus holding potential for VprE21Q (filled triangles) (B), VprE24Q (inverted filled triangles) (D), and, for comparison, WT Vpr (open circles).

FIG. 5

Inhibition of activity by an antibody recognizing the C terminus of Vpr. Shown are examples of currents generated in planar lipid bilayers by WT Vpr (A), VprE58Q (B), and VprE24Q (C) at 0-mV holding potential, before (“0mV”) and after (“+AbC”) addition of 50 μl of affinity-purified antibodies raised against a peptide (C21) comprising the C-terminal 21 amino acids of Vpr (see reference 27) to the trans chamber. The dashed lines indicate the zero current levels. All-points histograms of the data are plotted to the right of each trace.

FIG. 6

Synthetic peptides: examples of currents generated in planar lipid bilayers by N40 (A) and C21 (C and D) at different holding potentials. The dashed lines indicate the zero current levels. The average currents are plotted versus holding potential (B) for N40 (crosses), N21 (closed circles), and, for comparison, WT Vpr (open circles). Currents generated by C21 are shown under normal experimental conditions (C) or when phospholipids contained the negatively charged lipid PS (D).

FIG. 7

Confocal images of hippocampal neurons in response to the external application of purified Vpr (A), C21 (B), N40 (C), N21 (D), 200 mM KCl (E), and CHAPS buffer (F) with the potential-sensitive dye DiBa-C4(3). Purified full-length Vpr or the synthetic peptides were added to a final concentration of 1 μM or 1 mM, respectively. Images were taken between 10 and 13 min after treatment at 37°C.

FIG. 8

Cytotoxic effects of extracellular addition of WT Vpr (1 μM) and Vpr peptides (1 mM) on hippocampal neurons expressed as percentages of dead neurons. The figure shows the effects of exposure to WT Vpr (open circles), N40 (crosses), C21 (filled diamonds), N21 (filled squares), and CHAPS buffer (open squares).