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. 2001 Sep;75(17):7913-24.
doi: 10.1128/jvi.75.17.7913-7924.2001.

Multimerization of human immunodeficiency virus type 1 Gag promotes its localization to barges, raft-like membrane microdomains

O W Lindwasser  1 , M D Resh
Affiliations

Multimerization of human immunodeficiency virus type 1 Gag promotes its localization to barges, raft-like membrane microdomains

O W Lindwasser et al. J Virol. 2001 Sep.

Abstract

The Gag polyprotein of human immunodeficiency virus type 1 (HIV-1) organizes the assembly of nascent virions at the plasma membrane of infected cells. Here we demonstrate that a population of Gag is present in distinct raft-like membrane microdomains that we have termed "barges." Barges have a higher density than standard rafts, most likely due to the presence of oligomeric Gag-Gag assembly complexes. The regions of the Gag protein responsible for barge targeting were mapped by examining the flotation behavior of wild-type and mutant proteins on Optiprep density gradients. N-myristoylation of Gag was necessary for association with barges. Removal of the NC and p6 domains shifted much of the Gag from barges into typical raft fractions. These data are consistent with a model in which multimerization of myristoylated Gag proteins drives association of Gag oligomers into raft-like barges. The functional significance of barge association was revealed by several lines of evidence. First, Gag isolated from virus-like particles was almost entirely localized in barges. Moreover, a comparison of wild-type Gag with Fyn(10)Gag, a chimeric protein containing the N-terminal sequence of Fyn, revealed that Fyn(10)Gag exhibited increased affinity for barges and a two- to fourfold increase in particle production. These results imply that association of Gag with raft-like barge membrane microdomains plays an important role in the HIV-1 assembly process.

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Figures

FIG. 1
FIG. 1
Raft fractionation of Pr55Gag and endogenous cellular proteins in Jurkat cells. Jurkat cells were transfected with the proviral vector pHXB2ΔBalID25S and harvested 48 h posttransfection. Following extraction on ice with 0.5% TX-100, the lysates were subjected to flotation on Optiprep gradients as described in Materials and Methods. Five fractions were collected, trichloroacetic acid precipitated, and analyzed by SDS-PAGE followed by Western blotting. (A) Detection of Pr55Gag with rabbit anti-p24CA serum. A representative gel is shown. The top of the gradient (fraction 1) contains the raft fraction. At right is the quantitation of several independent experiments. Data are means and standard deviations. (B) Endogenous Jurkat proteins from the same fractions were detected using monoclonal antibodies to the indicated proteins.
FIG. 2
FIG. 2
Raft fractionation of Pr55Gag in COS-1 cells. COS-1 cells were transfected with pHXB2ΔBalID25S or pCMV5 Gag and subjected to extraction on ice with 0.5% TX-100 and flotation on five-fraction Optiprep gradients. Representative Western blots are shown on the left. Pr55Gag was detected with rabbit anti-p24CA serum. Caveolin-1 was detected with anti-caveolin-1 polyclonal antibody. Quantitations of several independent experiments are depicted graphically on the right.
FIG. 3
FIG. 3
Gag-Gag interactions interfere with the isolation of Gag proteins from low-buoyant-density raft fractions. (A) Diagram of the Fyn-Gag chimeric proteins. The hatched box represents the coding sequence derived from Fyn (squiggles represent posttranslational modification of this sequence with myristate and palmitate). Open boxes designate the boundaries of the domains of Pr55Gag. Also shown are the p6 domain and p15 region, which are deleted from FynGagΔp6 and FynGagΔp15, respectively. (B) COS-1 cells were transfected with the indicated constructs and subjected to raft flotation. Representative Western blots are shown on the left, and quantitations are on the right. (C) COS-1 cells were transfected with pCMV5 GagΔp15 or cotransfected with both pCMV5 Gag and pCMV5 GagΔp15 (+ wt Gag). Where indicated, cells were treated with 10 mg of mβCD/ml prior to the experiment. Cell extracts were processed according to the five-fraction flotation protocol and analyzed by Western blotting. Representative blots are on the left, and quantitations of the percentage of GagΔp15 in each fraction are on the right. The distribution of wild-type Gag was essentially unchanged from that seen in single-transfection experiments (data not shown). Approximately equal amounts of wild-type Gag and Gag mutant proteins were expressed.
FIG. 3
FIG. 3
Gag-Gag interactions interfere with the isolation of Gag proteins from low-buoyant-density raft fractions. (A) Diagram of the Fyn-Gag chimeric proteins. The hatched box represents the coding sequence derived from Fyn (squiggles represent posttranslational modification of this sequence with myristate and palmitate). Open boxes designate the boundaries of the domains of Pr55Gag. Also shown are the p6 domain and p15 region, which are deleted from FynGagΔp6 and FynGagΔp15, respectively. (B) COS-1 cells were transfected with the indicated constructs and subjected to raft flotation. Representative Western blots are shown on the left, and quantitations are on the right. (C) COS-1 cells were transfected with pCMV5 GagΔp15 or cotransfected with both pCMV5 Gag and pCMV5 GagΔp15 (+ wt Gag). Where indicated, cells were treated with 10 mg of mβCD/ml prior to the experiment. Cell extracts were processed according to the five-fraction flotation protocol and analyzed by Western blotting. Representative blots are on the left, and quantitations of the percentage of GagΔp15 in each fraction are on the right. The distribution of wild-type Gag was essentially unchanged from that seen in single-transfection experiments (data not shown). Approximately equal amounts of wild-type Gag and Gag mutant proteins were expressed.
FIG. 3
FIG. 3
Gag-Gag interactions interfere with the isolation of Gag proteins from low-buoyant-density raft fractions. (A) Diagram of the Fyn-Gag chimeric proteins. The hatched box represents the coding sequence derived from Fyn (squiggles represent posttranslational modification of this sequence with myristate and palmitate). Open boxes designate the boundaries of the domains of Pr55Gag. Also shown are the p6 domain and p15 region, which are deleted from FynGagΔp6 and FynGagΔp15, respectively. (B) COS-1 cells were transfected with the indicated constructs and subjected to raft flotation. Representative Western blots are shown on the left, and quantitations are on the right. (C) COS-1 cells were transfected with pCMV5 GagΔp15 or cotransfected with both pCMV5 Gag and pCMV5 GagΔp15 (+ wt Gag). Where indicated, cells were treated with 10 mg of mβCD/ml prior to the experiment. Cell extracts were processed according to the five-fraction flotation protocol and analyzed by Western blotting. Representative blots are on the left, and quantitations of the percentage of GagΔp15 in each fraction are on the right. The distribution of wild-type Gag was essentially unchanged from that seen in single-transfection experiments (data not shown). Approximately equal amounts of wild-type Gag and Gag mutant proteins were expressed.
FIG. 4
FIG. 4
The myristoylated M domain of Gag is necessary but not sufficient for raft partitioning of Gag proteins. (Top) Cells were transfected with pVALO Gag31eGFP and subjected to raft fractionation. (Bottom) Cells were transfected with pCMV 5 GagΔp15 and treated on the following day for 12 h with 100 μM 2-OH-myr. Treated (open bars) and untreated (filled bars) control cells were then subjected to the five-fraction extraction and flotation protocol to detect raft-associated protein.
FIG. 5
FIG. 5
Separation of barges from rafts on eight-fraction Optiprep gradients. COS-1 cells were transfected with pCMV5 Gag or pCMV5 GagΔp15 or were mock transfected. After extraction on ice in 0.5% TX-100, cell extracts were subjected to the TX-100 eight-fraction Optiprep flotation protocol described in Materials and Methods. The lipid markers cholesterol and GM1 were detected as described in Materials and Methods (these lipids showed no significant difference in fractionation when isolated from mock-transfected [this figure] or Gag-transfected [data not shown] cells). Gag proteins were detected by Western blotting with rabbit anti-p24CA. Endogenous caveolin-1 was detected with anti-caveolin-1 polyclonal antibody, and endogenous Na+-K+ ATPase was detected using anti-Na+-K+ ATPase α3 subunit monoclonal antibody. The data for cholesterol and GM1 are means of four experiments; those for protein markers are means of two to four experiments.
FIG. 6
FIG. 6
Characterization of eight-fraction Optiprep gradients in the absence of detergent. These experiments were conducted as described in the legend to Fig. 5, with the exception that no detergent was present in the extraction buffers or the gradients. Note the altered distributions of cholesterol and Gag. Data for cholesterol and GM1 are means of four experiments, and those for protein markers are means of two experiments.
FIG. 7
FIG. 7
Attachment of the myristoylated, palmitoylated N terminus of Fyn to Gag increases localization to barges and VLP production. (A) VLPs were isolated from COS-1 cells transfected with pCMV5 Gag. After resuspension of the VLP pellet in cold TNET and extraction on ice, the lysate was fractionated using the eight-fraction Optiprep flotation protocol. Note that TX-100-extracted VLPs float to fraction 5, the barge fraction. (B) COS-1 cells were transfected with pCMV5 Gag (open bars) or with pCMV5 Fyn(10)Gag (filled bars). At 48 h posttransfection, the cells were subjected to the eight-fraction detergent gradient described above. (C) COS-1 cells were transfected with pCMV5 Gag or with pCMV5 Fyn(10)Gag. At 2, 4, or 24 h prior to each experiment the medium was replaced with fresh DMEM plus 10% fetal bovine serum. At the indicated times, the medium was removed from the plates and VLPs were isolated. The cells were harvested in ice-cold RIPA buffer. Gag protein in VLP and cell fractions was analyzed by SDS-PAGE followed by Western blotting. VLP production was quantitated by dividing the amount of Gag in VLPs by the amount of Gag in the cells. This number was set to 1 for each time point (open bars). VLP production by Fyn(10)Gag (filled bars) was determined as discussed above and directly compared to that of Gag. Error bars indicate standard deviations. n = 2 (2 and 4 h) and 4 (24 h).
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