A conserved dileucine-containing motif in p6(gag) governs the particle association of Vpx and Vpr of simian immunodeficiency viruses SIV(mac) and SIV(agm)

Abstract

Vpr is a small accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is specifically incorporated into virions. Members of the HIV-2/SIV(sm)/SIV(mac) lineage of primate lentiviruses also incorporate a related protein designated Vpx. We previously identified a highly conserved L-X-X-L-F sequence near the C terminus of the p6 domain of the Gag precursor as the major virion association motif for HIV-1 Vpr. In the present study, we show that a different leucine-containing motif (D-X-A-X-X-L-L) in the N-terminal half of p6(gag) is required for the incorporation of SIV(mac) Vpx. Similarly, the uptake of SIV(mac) Vpr depended primarily on the D-X-A-X-X-L-L motif. SIV(mac) Vpr was unstable when expressed alone, but its intracellular steady-state levels increased significantly in the presence of wild-type Gag or of the proteasome inhibitor lactacystin. Collectively, our results indicate that the interaction with the Gag precursor via the D-X-A-X-X-L-L motif diverts SIV(mac) Vpr away from the proteasome-degradative pathway. While absent from HIV-1 p6(gag), the D-X-A-X-X-L-L motif is conserved in both the HIV-2/SIV(sm)/SIV(mac) and SIV(agm) lineages of primate lentiviruses. We found that the incorporation of SIV(agm) Vpr, like that of SIV(mac) Vpx, is absolutely dependent on the D-X-A-X-X-L-L motif, while the L-X-X-L-F motif used by HIV-1 Vpr is dispensable. The similar requirements for the incorporation of SIV(mac) Vpx and SIV(agm) Vpr provide support for their proposed common ancestry.

FIG. 1

Locations of deletions in the p6 domain of the SIV_mac239 Gag polyprotein. The positions of the P-(T/S)-A-P-P and L-X-X-L-F motifs, which are conserved among primate lentiviruses, are indicated. Also shown is the position of the D-X-A-X-X-L-L motif, which is conserved among members of the HIV-2/SIV_sm/SIV_mac and SIV_agm lineages. Numbers refer to the positions of residues, counting from the N terminus of the p6^gag domain. Mutants unable to incorporate SIV_mac Vpx are indicated by shaded boxes.

FIG. 2

Effects of deletions in p6^gag on the incorporation of SIV_mac Vpx into VLP. HeLa cells were cotransfected with constructs expressing wild-type (WT) or mutant SIV_mac Gag polyproteins and a plasmid that provided SIV_mac Vpx in trans, as indicated above each lane. The transfected cells were metabolically labeled with [³⁵S]methionine, and VLP released during the labeling period were pelleted through 20% sucrose. Aliquots of the pelleted material were either analyzed directly by SDS-PAGE to compare the amounts of Gag protein in the samples (A) or immunoprecipitated (IP) with rabbit anti-Vpx serum prior to SDS-PAGE (B).

FIG. 3

Role of a dileucine-containing p6^gag region in SIV_mac Vpx incorporation. HeLa cells were cotransfected with a plasmid providing SIV_mac Vpx and constructs which express either the wild-type (WT) SIV_mac Gag polyprotein or mutant versions that harbor the indicated changes in p6^gag. (A and B) [³⁵S]methionine-labeled VLP material released into the culture medium was sedimented through 20% sucrose and either analyzed directly by SDS-PAGE (A) or immunoprecipitated (IP) with rabbit anti-Vpx serum (B). (C) In parallel, SIV_mac Vpx was immunoprecipitated from the cell lysates.

FIG. 4

Effects of substitutions in a dileucine-containing region of p6^gag on the levels of VLP- and cell-associated SIV_mac Vpr. HeLa cells were transfected with constructs expressing SIV_mac Gag polyproteins with the indicated single-amino-acid substitutions in p6^gag, together with a plasmid which provided HA-tagged SIV_mac Vpr in trans. (A) [³⁵S]methionine-labeled VLP released into the culture medium were sedimented through 20% sucrose and analyzed directly by SDS-PAGE. (B) The transfected cells were lysed, and tagged SIV_mac Vpr was immunoprecipitated (IP) with anti-HA monoclonal antibody 16B12 (Babco, Richmond, Calif.).

FIG. 5

Gag enhances the stability of cell-associated SIV_mac Vpr. (A) HA-tagged SIV_mac Vpr was expressed in the absence of Gag or coexpressed with wild-type (WT) or mutant SIV_mac Gag polyproteins, as indicated above each lane. The cells were metabolically labeled for 12 h, starting at 48 h posttransfection, and lysed, and tagged SIV_mac Vpr was immunoprecipitated to compare the intracellular steady-state levels. (B) HeLa cells expressing HA-tagged SIV_mac Vpr in the absence of Gag were kept for 1 h without or with lactacystin (10 μM; Calbiochem, La Jolla, Calif.) and then subjected to 6 h of metabolic labeling and immunoprecipitation with anti-HA antibody. (C) Cells expressing HA-tagged SIV_mac Vpr together with wild-type or L23A mutant SIV_mac Gag polyprotein were pulse-labeled for 30 min and chased for the times indicated. Cell lysates were immunoprecipitated with anti-HA antibody. (D) HeLa cells expressing HA-tagged SIV_mac Vpr together with wild-type or L23A mutant SIV_mac Gag were kept for 1 h in the presence of 10 μM lactacystin and then subjected to 6 h of metabolic labeling. VLP released into the culture medium were sedimented through 20% sucrose and analyzed directly by SDS-PAGE. Cell-associated SIV_mac Vpr was immunoprecipitated (IP) with anti-HA antibody.

FIG. 6

SIV_agm Vpr incorporation into heterologous SIV_mac particles and requirement for a dileucine-containing region in p6^gag. HeLa cells were cotransfected with constructs expressing the wild-type (WT) SIV_mac Gag polyprotein and either SIV_mac Vpx or SIV_agm Vpr (A) or with constructs expressing SIV_mac Gag polyproteins with the indicated single-amino-acid substitutions in p6^gag and a plasmid providing SIV_agm Vpr (B). The transfected cells were labeled with [³⁵S]cysteine, VLP were sedimented through sucrose, and their protein content was analyzed by SDS-PAGE.

FIG. 7

A conserved dileucine-containing region in p6^gag governs the association of SIV_agm Vpr with autologous VLP. HeLa cells were cotransfected with plasmids expressing SIV_agm Vpr and either the wild-type (WT) SIV_agm Gag polyprotein or mutant Gag precursors with the indicated changes in p6^gag. (A) The protein content of [³⁵S]cysteine-labeled, sucrose-purified VLP was directly analyzed by SDS-PAGE. (B and C) In parallel, cell lysates (B) and lysed VLP (C) were immunoprecipitated (IP) with rabbit anti-SIV_agm Vpr serum (6). The Δ56–66 mutant lacks the C-terminal 11 amino acids of SIV_agm p6^gag, which harbor the conserved L-X-X-L-F motif.