Human immunodeficiency virus type 1 Gag contains a dileucine-like motif that regulates association with multivesicular bodies

Abstract

Multivesicular bodies (MVBs) are cholesterol-enriched organelles formed by the endocytic pathway. The topology of vesicle formation in MVBs is identical to that of retroviral budding from the plasma membrane, and budding of human immunodeficiency virus type 1 (HIV-1) into MVBs in macrophages has recently been visualized. The Gag proteins from HIV-1, as well as many other retroviruses, contain short motifs that mediate interactions with MVBs and other endocytic components, suggesting that Gag proteins directly interface with the endocytic pathway. Here, we show that HIV-1 Gag contains an internalization signal that promotes endocytosis of a chimeric transmembrane fusion protein. Mutation of this motif within Gag strongly inhibits virus-like particle production. Moreover, wild-type Gag, but not the internalization-defective mutation, can be induced to accumulate within CD63-positive MVBs by treatment of cells with U18666A, a drug that redistributes cholesterol from the plasma membrane to MVBs. We propose that HIV-1 Gag contains a signal that promotes interaction with the cellular endocytic machinery and that the site of particle production is regulated by the subcellular distribution of cholesterol.

Figures

FIG. 1.

Internalization of a CD4-Gag fusion protein. (A) A schematic of CD4, tailless CD4 [CD4T(−)], and CD4 fusion [CD4T(−).ORF] proteins. The amino acid sequence of the cytoplasmic tail domain (CT) is shown with the dileucine internalization motif underlined and italicized. EC, extracellular domain; TM, transmembrane; ORF, any sequence appended to CD4T(−). (B) Percentage of surface-bound anti-CD4 internalized over the indicated time course by cells expressing CD4 (filled squares), CD4T(−) (filled diamonds), CD4T(−).UL11 (open circles), or CD4T(−).GFP (open triangles). Data are means ± standard errors of the means for quadruplicate experiments repeated three to five times. For CD4 and CD4T(−), the cells were treated with 50 ng of PMA/ml. (C) Internalization of CD4T(−).Gag (open squares) compared to that of CD4T(−) (filled triangles). Data represent means ± standard errors of the means for five to seven quadruplicate experiments.

FIG. 2.

(A) Comparison of VLP production by Gag and that by CD4T(−).Gag. Lysates from cells expressing Gag or CD4T(−).Gag were collected, and VLPs were purified from the tissue culture media after 24 h of incubation. Aliquots of each lysate were assayed for the presence of Gag protein by Western blotting with anti-p24CA. The wild-type Gag band is labeled Pr55 Gag. CD4T(−).Gag migrates at ∼110 kDa. The asterisk indicates an anti-p24CA reactive band that is occasionally seen in CD4T(−).Gag cell lysates. This is probably a degradation product of the fusion protein. (B) Internalization of anti-CD4 MAb by COS-1 cells expressing CD4T(−).Gag. After the uptake of anti-CD4, cells were stained with a green fluorophore prior to permeabilization to detect surface MAbs as described in Materials and Methods. After permeabilization, internalized MAbs were stained with a red fluorophore.

FIG. 3.

Mapping of endocytosis signals within Gag. (A) Internalization of anti-CD4 by cells expressing selected CD4T(−) fusion proteins. Data are means for two to seven quadruplicate experiments. (B) Schematic representation of the set of CD4T(−) fusion proteins used to map internalization determinants within the Gag sequence. The locations of the matrix (MA), CA, (NC, and p6 domains are indicated by shaded boxes. Gag residue numbers for the beginning and end of each fusion construct are shown. A fast rate of internalization is relative to that for CD4T(−). (C) The amino acid sequence of the minimal internalization sequence of Gag, which includes the C-terminal portion of CA, p2, and NC (residues 279 to 432 of Gag). Potential endocytosis signals are underlined and italicized. Residues that have been mutated in this study are indicated by asterisks. (D) Internalization CD4T(−).Gag point mutations. Filled diamonds, CD4T(−); open squares, CD4T(−).Gag; filled triangles, CD4T(−).Gag(Y301K); open triangles, CD4T(−).Gag(LL321,322AA); gray triangles, CD4T(−).Gag(IL-333,334AA). Data represent means ± standard errors of the means for two to seven quadruplicate experiments.

FIG. 4.

Surface expression and internalization of CD4 fusion proteins. (A) CD4 fusion proteins on the surfaces of cells were detected by indirect immunofluorescence without permeabilization. (B) Internalization of CD4T(−).DsRed fusion proteins. Filled squares, CD4; filled diamonds, CD4T(−); open squares, CD4T(−).Gag; open triangles, CD4T(−).GFP; open circles, CD4T(−).DsRed (solid lines) and CD4T(−).dbl DsRed (dashed lines). Data are means of the results for two to four quadruplicate experiments.

FIG. 5.

VLP production of wild-type Gag and Gag with Y301K, LL321,322AA, and IL-333,334AA mutations. (A) Cell lysates and purified VLPs from cells expressing wild-type Gag or the indicated mutations were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with anti-Gag antibodies. (B) Partial rescue of defective Gag mutations by a wild-type Gag protein. Western blots are of a VLP assay of cells coexpressing Gag point mutations and GagGFP (+) or GFP as a negative control (−). Mutated Gag proteins are 55 kDa (lower arrows). GagGFP is ∼80 kDa (upper arrows). Bands indicated by asterisks are apparently degradation products of GagGFP. (C) Quantification of VLP release. Open bars represent VLP release (ratio of Gag signal from VLP fraction to signal from cell lysate) from cells coexpressing Gag mutations and GFP. Closed bars represent VLP release from cells coexpressing Gag mutants and wild-type GagGFP. Each release value is a percentage of the VLP release of GagGFP. Data are means ± standard errors of the means for two to four duplicate experiments.

FIG. 6.

Distribution of GagGFP and early endosomal markers. (A) GFP fluorescence of cells expressing wild-type GagGFP or the indicated mutations. (B) GagGFP fluorescence is depicted in green (left), endosomal markers are depicted in red (middle), and the overlay of the two images is depicted on the right. Colocalization is visible as yellow. EEA1 was detected by indirect immunofluorescence with an anti-EEA1 MAb. The lower panels represent cells that were incubated with Texas Red-transferrin for 1 h. This treatment labels early endosomes and the endosomal recycling compartment.

FIG.7.

The presence of Gag on CD63-positive late endosomes in cells treated with U18666A. (A) GFP fluorescence of cells expressing GagGFP or Gag(IL-333,334AA)-GFP is depicted in the left panels, anti-CD63 immunofluorescence is depicted in the middle panels, and the overlay of red and green fluorescence is depicted in the right panels with colocalization appearing as yellow. The indicated images are from cells treated with U18666A. (B) Colocalization of GagGFP or Gag(IL-333,334AA)-GFP in control cells (open bars) or U18666A-treated cells (filled bars) as determined by analysis of confocal images of 6 to 18 cells for each condition. Means and standard deviations are depicted. (C) Filipin fluorescence of control and U18666A-treated cells detected by confocal microscopy.

FIG. 8.

VLP production in cells treated with U18666A. (A) Gag was immunoprecipitated from 35S-labeled cells treated or untreated with U18666A and from VLPs collected after 4 h of labeling. (B) Quantification of results shown in panel A. Data are means ± standard errors of the means for two duplicate experiments.