Human immunodeficiency virus type 2 Vpx-Gag interaction

Abstract

Incorporation of Vpx into human immunodeficiency virus type 2 (HIV-2) virus-like particles is mediated by the Gag polyprotein. We have identified residues 15 to 40 of Gag p6 and residues 73 to 89 of Vpx as being necessary for virion incorporation. In addition, we show enhanced in vitro binding of Vpx to a chimeric HIV-1/HIV-2 Gag construct containing residues 2 to 49 of HIV-2 p6 and demonstrate that the presence of residues 73 to 89 of Vpx allows for in vitro binding to HIV-2 Gag.

FIG. 1

Residues 73 to 89 of HIV-2 Vpx are required for particle incorporation. (A) Schematic drawing of wild-type and mutant Vpx constructs cloned into the pTM3 vector. At the top is a diagram of HIV-2 Vpx, with the region predicted to form an amphipathic helix (shaded box), the conserved cysteines (asterisks), and the proline-rich tail (striped box) denoted. a.a., amino acids. Metabolically labeled proteins from BSC40 cell lysates (B) were immunoprecipitated with antisera to Vpx, and cell supernatants (C) were immunoprecipitated with antisera to Vpx and Gag before SDS-PAGE. The locations of the Gag and the Vpx proteins are indicated. M.W., molecular mass.

FIG. 1

Residues 73 to 89 of HIV-2 Vpx are required for particle incorporation. (A) Schematic drawing of wild-type and mutant Vpx constructs cloned into the pTM3 vector. At the top is a diagram of HIV-2 Vpx, with the region predicted to form an amphipathic helix (shaded box), the conserved cysteines (asterisks), and the proline-rich tail (striped box) denoted. a.a., amino acids. Metabolically labeled proteins from BSC40 cell lysates (B) were immunoprecipitated with antisera to Vpx, and cell supernatants (C) were immunoprecipitated with antisera to Vpx and Gag before SDS-PAGE. The locations of the Gag and the Vpx proteins are indicated. M.W., molecular mass.

FIG. 1

Residues 73 to 89 of HIV-2 Vpx are required for particle incorporation. (A) Schematic drawing of wild-type and mutant Vpx constructs cloned into the pTM3 vector. At the top is a diagram of HIV-2 Vpx, with the region predicted to form an amphipathic helix (shaded box), the conserved cysteines (asterisks), and the proline-rich tail (striped box) denoted. a.a., amino acids. Metabolically labeled proteins from BSC40 cell lysates (B) were immunoprecipitated with antisera to Vpx, and cell supernatants (C) were immunoprecipitated with antisera to Vpx and Gag before SDS-PAGE. The locations of the Gag and the Vpx proteins are indicated. M.W., molecular mass.

FIG. 2

Sucrose gradient analysis of virus-like particles. The particles were concentrated by sedimentation through a 20% sucrose cushion at 26,000 rpm for 90 min in an SW28.1 rotor. Particles were resuspended in phosphate-buffered saline, layered onto linear 20 to 60% sucrose gradients, and centrifuged at 20,000 rpm for 16 h. A 20-μl aliquot of each fraction was loaded directly onto an SDS–15% PAGE gel. Fraction 1 is from the top of each gradient, and fraction 11 is from the bottom of each gradient. Protein molecular mass markers are shown in the first lane of each autoradiogram.

FIG. 3

Residues 15 to 40 of HIV-2 p6 are necessary for Vpx virion incorporation. (A) Schematic drawing of the chimeric Gag constructs. Sequences of HIV-2 p6 (indicated numerically) were cloned in frame into the XhoI site of pTM(p6⁻). At the bottom is an alignment of HIV-2 and HIV-1 p6 sequences. (B) Cell lysates of BSC40 cells cotransfected with pTM-X and the Gag chimeric constructs were immunoprecipitated with αVpx and αGag antisera. (C) Cell supernatants from the same experiment. The locations of the Gag proteins and Vpx are indicated. M.W., molecular mass.

FIG. 3

Residues 15 to 40 of HIV-2 p6 are necessary for Vpx virion incorporation. (A) Schematic drawing of the chimeric Gag constructs. Sequences of HIV-2 p6 (indicated numerically) were cloned in frame into the XhoI site of pTM(p6⁻). At the bottom is an alignment of HIV-2 and HIV-1 p6 sequences. (B) Cell lysates of BSC40 cells cotransfected with pTM-X and the Gag chimeric constructs were immunoprecipitated with αVpx and αGag antisera. (C) Cell supernatants from the same experiment. The locations of the Gag proteins and Vpx are indicated. M.W., molecular mass.

FIG. 3

Residues 15 to 40 of HIV-2 p6 are necessary for Vpx virion incorporation. (A) Schematic drawing of the chimeric Gag constructs. Sequences of HIV-2 p6 (indicated numerically) were cloned in frame into the XhoI site of pTM(p6⁻). At the bottom is an alignment of HIV-2 and HIV-1 p6 sequences. (B) Cell lysates of BSC40 cells cotransfected with pTM-X and the Gag chimeric constructs were immunoprecipitated with αVpx and αGag antisera. (C) Cell supernatants from the same experiment. The locations of the Gag proteins and Vpx are indicated. M.W., molecular mass.

FIG. 4

Residues 2 to 49 of HIV-2 p6 are able to enhance Vpx binding in vitro. GST and the GST-p6⁻ and GST-p6_2–49 fusion proteins were incubated with vaccinia virus-expressed Vpx and glutathione beads. Proteins eluted off the beads were detected by Western blotting with Vpx antisera.

FIG. 5

The presence of Vpx residues 73 to 89 allows for in vitro binding of Vpx to GST-Gag. GST and GST-Gag proteins were incubated with ³⁵S-labeled Vpx73 or Vpx89 proteins. M.W., molecular mass.