Interaction with the p6 domain of the gag precursor mediates incorporation into virions of Vpr and Vpx proteins from primate lentiviruses

Abstract

Vpr and Vpx proteins from human and simian immunodeficiency viruses (HIV and SIV) are incorporated into virions in quantities equivalent to those of the viral Gag proteins. We demonstrate here that Vpr and Vpx proteins from distinct lineages of primate lentiviruses were able to bind to their respective Gag precursors. The capacity of HIV type 1 (HIV-1) Vpr mutants to bind to Pr55(Gag) was correlated with their incorporation into virions. Molecular analysis of these interactions revealed that they required the C-terminal p6 domain of the Gag precursors. While the signal for HIV-1 Vpr binding lies in the leucine triplet repeat region of the p6 domain reported to be essential for incorporation, SIVsm Gag lacking the equivalent region still bound to SIVsm Vpr and Vpx, indicating that the determinants for Gag binding are located upstream of this region of the p6 domain. Binding to Gag cleavage products showed that HIV-1 Vpr interacted directly with the nucleocapsid protein (NC), whereas SIVsm Vpr and Vpx did not interact with NC but with the p6 protein. These results (i) reveal differences between HIV-1 and SIVsm for the p6 determinants required for Vpr and Vpx binding to Gag and (ii) suggest that HIV-1 Vpr and SIVsm Vpr and Vpx interact with distinct cleavage products of the precursor following proteolytic processing in the virions.

FIG. 1

Interactions of Vpr and Vpx proteins from primate lentiviruses with their homologous Gag precursor. (A) Binding of HIV-1 Vpr to Pr55^Gag in the two-hybrid system. The L40 yeast reporter strain expressing the pairs of indicated hybrids fused to the LexABD and Gal4AD was analyzed for histidine auxotrophy and β-gal activity. Double transformants were patched on selective medium with histidine (+His) (left panel) and then replica plated on medium without histidine (−His) (middle panel) and on Whatman filters for β-gal assay (right panel). Growth in the absence of histidine and expression of β-gal activity indicate interaction between hybrid proteins. The β-gal filter assay was carried out overnight. (B) Vpr-Pr55^Gag interaction in vitro. Chemically synthesized Vpr (lane 3) was incubated with equal amounts of GST-Pr55^Gag (lane 1) or GST (lane 2) immobilized on GSH-agarose beads. Bound Vpr was then analyzed by Western blotting with a rabbit anti-Vpr antiserum. (C and D) Binding of HIV-2, SIV_sm, and SIV_agm Vpr and Vpx to homologous Gag precursors. L40 expressing the pairs of indicated hybrids fused to the LexABD and Gal4AD was analyzed for β-gal activity. The filter assay was incubated for 3 h. Each patch represents an independent transformant.

FIG. 2

HIV-1 Vpr binding to Pr55^Gag and virion incorporation. (A) Selection of Vpr mutants deficient for binding to Pr55^Gag. L40 strain expressing the pairs of indicated hybrids fused to the LexABD and Gal4AD was analyzed for β-gal activity. Vpr*E25K (lane 2) and Vpr*H33L (lane 3) mutants were selected from a random Vpr mutant library, while Vpr*W54R and Vpr*R90K have been described previously (39). (B) Incorporation of HIV-1 Vpr mutants into virions. 293T cells were cotransfected with the pCMVΔR8.91, pMD.G, and pHR′-CMVLacZ plasmids and either a wt (lane 1) or a mutated (lanes 2, 3, 5, and 6) HA-tagged Vpr expression vector. Lysates from transfected cells (Cell) and virions (Supernatant) released into the culture medium and adjusted to contain similar amounts of CAp24 were separated by SDS-PAGE and analyzed by Western blotting with anti-HA (Cell and Supernatant upper panels) or anti-CAp24 (Cell and Supernatant lower panels) monoclonal antibodies. C (lane 4), cell and virion lysates from cells transfected with the pCMVΔR8.91, pMD.G, and pHR′-CMVLacZ vectors and the pAS1B plasmid without an insert.

FIG. 3

Binding of HIV-1 and SIV_sm Vpr and Vpx to the cleavage products of the Gag precursors. L40 strain expressing either HIV-1 Vpr or SIV_sm Vpr or Vpx fused to the LexABD in combination with each of the Gal4AD hybrids indicated on the left was analyzed for histidine auxotrophy and β-gal activity. The interactions between hybrid proteins were scored as follows: +++, cell growth on medium without histidine and development of a blue color in 3 h by β-gal filter assay; ++, cell growth on medium without histidine and development of a blue color after overnight incubation; +, cell growth on medium without histidine and development of a light blue color after overnight incubation; −, no growth on medium without histidine and development of a white color after overnight incubation of the β-gal assay. Quantitative β-gal activities expressed in β-gal units and determined by liquid culture assay are given in parentheses. The background level is approximately 1 to 2 U and corresponds to L40 expressing either the HIV-1 Vpr- or SIVsm Vpr- or Vpx-LexABD hybrid and the Gal4AD-Raf hybrid. ND, not done.

FIG. 4

Role of the LXX repeats of the HIV-1 and SIV_sm Gag p6 domain in Vpr and Vpx binding. (A) Mutation within the p6 (LXX)₄ region disrupts HIV-1 Vpr binding to Pr55^Gag. A diagram shows the HIV-1 wt Pr55^Gag and GagL44A mutant. The p6 (LXX)₄ repeats are underlined, and the Leu residue replaced by Ala in the GagL44A mutant is indicated by an asterisk. Numbering refers to the HIV-1 Lai p6 domain (30). L40 expressing HIV-1 Vpr fused to the LexABD and either GagΔp6 (lane 1), GagL44A (lane 2), or Pr55^Gag wt (lane 3) fused to the Gal4AD was analyzed for β-gal activity. The filter assay was carried out overnight. (B) Quantitative β-gal assay of SIV_sm Vpr and Vpx binding to Gag and p6 mutants. A diagram shows the SIV_sm Gag wt and GagL54A, GagΔL1, GagΔL2, and GagΔL3 mutants. The p6 (LXX)₃ repeats are underlined, and the Leu residue replaced by Ala in the GagL54A mutant is indicated by an asterisk. Numbering refers to the SIVsmPbj1.9 p6 domain (30). L40 expressing either the SIV_sm Vpr (solid bars) or Vpx (white bars) LexABD hybrid in combination with each of the Gal4AD hybrids indicated was assayed for β-gal activity in a liquid culture assay. The results are expressed as the percentages of the β-gal activity determined for each Gag or p6 mutant relative to the activity obtained with the wt Gag and p6, respectively. The background level is approximately 2 U and corresponds to L40 expressing either the SIV_sm Vpr- or Vpx-LexABD hybrid and the Gal4AD-Raf hybrid.

FIG. 5

Characterization of an HIV-1 Vpr mutant displaying a higher avidity for Pr55^Gag in the two-hybrid system. (A) Binding to Pr55^Gag of Vpr*A and Vpr*W18R mutants. The L40 strain expressing either the Gal4AD-Pr55^Gag (lanes 1 to 3) or the irrelevant Gal4AD-Raf (lanes 4 to 6) hybrid in combination with each of the LexABD hybrids indicated was analyzed for β-gal activity. Double transformants were patched on selective medium and then replica plated twice on Whatman filters for β-gal assay. The filter assays were incubated for 2 h (left panel) or 12 h (right panel). Vpr*A (lanes 2 and 5) and Vpr*W18R (lanes 3 and 6) mutants were selected from a random Vpr mutant library. (B) Binding of Vpr*W18R to Pr55^Gag cleavage products. The L40 strain expressing either the wt Vpr (right panels) or Vpr*W18R (left panels) LexABD hybrid in combination with each of the Gal4AD hybrids indicated was analyzed for β-gal activity. The filter assays were incubated for 2 h (lanes 1 to 9, left panel) or 12 h (right panels, and lanes 10 to 11, left panel). (C) Incorporation of Vpr*A and Vpr*W18R into virions. Virion packaging was analyzed as described in the legend to Fig. 2. Lysates from transfected cells (Cell) and from virions (Supernatant) released into the culture medium and adjusted for similar amounts of CAp24 were separated by SDS-PAGE and analyzed by Western blotting with anti-HA (Cell and Supernatant, upper panels) or anti-CAp24 (Cell and Supernatant, lower panels) monoclonal antibodies. HA-tagged Vpr protein has appeared as a doublet (lane 4, Supernatant, upper panel) in some experiments (35). C (lane 1), cell, and virion lysates from cells transfected with the pCMVΔR8.91, pMD.G, and pHR′-CMVLacZ vectors and the pAS1B plasmid without an insert.