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. 2003 Sep;77(17):9474-85.
doi: 10.1128/jvi.77.17.9474-9485.2003.

The Mason-Pfizer monkey virus PPPY and PSAP motifs both contribute to virus release

Eva Gottwein  1 , Jochen Bodem, Barbara Müller, Ariane Schmechel, Hanswalter Zentgraf, Hans-Georg Kräusslich
Affiliations

The Mason-Pfizer monkey virus PPPY and PSAP motifs both contribute to virus release

Eva Gottwein et al. J Virol. 2003 Sep.

Abstract

Late (L) domains are required for the efficient release of several groups of enveloped viruses. Three amino acid motifs have been shown to provide L-domain function, namely, PPXY, PT/SAP, or YPDL. The retrovirus Mason-Pfizer monkey virus (MPMV) carries closely spaced PPPY and PSAP motifs. Mutation of the PPPY motif results in a complete loss of virus release. Here, we show that the PSAP motif acts as an additional L domain and promotes the efficient release of MPMV but requires an intact PPPY motif to perform its function. Examination of HeLaP4 cells expressing PSAP mutant virus by electron microscopy revealed mostly late budding structures and chains of viruses accumulating at the cell surface with little free virus. In the case of the PPPY mutant virus, budding appeared to be mostly arrested at an earlier stage before induction of membrane curvature. The cellular protein TSG101, which interacts with the human immunodeficiency virus type 1 (HIV-1) PTAP L domain, was packaged into MPMV in a PSAP-dependent manner. Since TSG101 is crucial for HIV-1 release, this result suggests that the Gag-TSG101 interaction is responsible for the virus release function of the MPMV PSAP motif. Nedd4, which has been shown to interact with viral PPPY motifs, was also detected in MPMV particles, albeit at much lower levels. Consistent with a role of VPS4A in the budding of both PPPY and PTAP motif-containing viruses, the overexpression of ATPase-defective GFP-VPS4A fusion proteins blocked both wild-type and PSAP mutant virus release.

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Figures

FIG. 1.
FIG. 1.
Comparison of MPMV expression from pMPMV and pSHRM15. HeLaP4 cells were transfected with equal amounts of pMPMV (lanes 1 and 3) or pSHRM15 (lanes 2 and 4), and levels of intracellular (lanes 1 and 2) and virus-associated CA-reactive proteins (lanes 3 and 4) were analyzed by Western blotting with anti-CA antiserum. Molecular mass standards in kilodaltons are shown on the right; MPMV Gag and CA are identified on the left.
FIG. 2.
FIG. 2.
(a) Schematic representation of MPMV Gag, showing the L-domain sequence within pp24 and the mutations introduced. (b) wt and L-domain mutant MPMV release. Cells were transfected with the indicated pMPMV constructs and metabolically labeled with [35S]methionine for 12 h. Cell-associated (upper panel) and virus-associated (lower panel) CA protein was immunoprecipitated with anti-CA antiserum, resolved by SDS-PAGE, and subjected to phosphorimage analysis. Note that no precursor or intermediate processing product was observed in the virus samples. (c) Quantification of wt and mutant release efficiencies. CA-containing bands were quantified. The release efficiency of wt and mutant virus as well as that of mixed virus populations was calculated by dividing the signal from virus-associated CA protein by the sum of the signals from cell- and virus-associated CA-containing proteins. The obtained numbers were normalized for wt release efficiency. n = 4 for columns 1, 2, 3, and 8; n = 2 for all other columns. (d) Steady-state levels of wt and mutant virus release. Cells were cotransfected with the indicated pSHRM15 plasmids, and cell- and virus-associated viral proteins (upper and lower panel, respectively) were analyzed by Western blotting with an anti-CA antiserum. Again, no precursor or intermediate processing product was observed in the virus samples.
FIG. 3.
FIG. 3.
Effect of PPPY mutant Gag protein on wt virus release. HeLaP4 cells were cotransfected with the indicated pSHRM15 (lanes 1 to 6) or pMPMV (lanes 7 to 9) wt and PY(−) plasmids. In each case, the amount of total DNA was adjusted to 15 μg. Steady-state levels of cell- and virus-associated CA protein (upper and lower panel, respectively) were analyzed by Western blotting with anti-CA antiserum.
FIG. 4.
FIG. 4.
Kinetic analysis of wt and mutant Gag processing. (a) Kinetics of wt Gag processing and virus release. HeLaP4 cells were transfected with pMPMV and pulse-labeled with [35S]methionine for 30 min. Cells were chased in DMEM for the indicated time. Cell- and virus-associated CA-containing proteins (lanes 1 to 6 and 7 to 12, respectively) were immunoprecipitated with anti-CA antiserum, resolved by SDS-PAGE, and visualized by phosphorimage analysis. (b) wt and L-domain mutant processing kinetics. HeLaP4 cells were transfected with the indicated pMPMV constructs, labeled as described above, and chased for 0, 2, or 8 h. At the 2-h time point, a Gag-related band (arrow) that migrates slower than Gag was detected in cells expressing PPPY mutant and double mutant Gag.
FIG. 5.
FIG. 5.
Thin-section EM analysis of HeLaP4 cells transfected with pMPMV (wt). Bars, 500 nm (a) and 200 nm (b).
FIG. 6.
FIG. 6.
Thin-section EM analysis of HeLaP4 cells transfected with PPPY mutant pMPMV. Bars, 1 μm (a) and 100 nm (b).
FIG. 7.
FIG. 7.
Thin-section EM analysis of HeLaP4 cells transfected with PSAP mutant pMPMV. Bars, 500 nm (a) and 200 nm (b to d).
FIG. 8.
FIG. 8.
Incorporation of cellular proteins into MPMV. (a) wt and PSAP mutant virus was purified by centrifugation through sucrose cushions followed by sedimentation in an Optiprep velocity gradient. wt (lanes 1 and 2) and PSAP mutant (lane 3) virus protein content was analyzed by silver staining. (b) Comparable amounts of lysates from wt and mutant virus-expressing cells (lanes 1 and 2) as well as highly purified virus (the same amounts as in panel a, lanes 1 and 3) were probed for the cellular proteins TSG101 (upper panel), 14-3-3γ (middle panel), and Nedd-4 (lower panel) by Western blotting. In the lower panel, the major Nedd-4-related bands migrating at 120 and 110 kDa are indicated. The arrow marks a band reacting with Nedd-4 antiserum which does not comigrate with one of the major viral proteins detected in the Ponceau S stain (not shown). (c) To analyze whether TSG101 and Nedd-4 are released from 293T cells in the absence of MPMV expression, we probed sucrose pellets from culture media of cells transfected with pMPMV or EGFP expression vectors (lanes 3 and 4) for TSG101 (upper panel) and Nedd-4 (lower panel). The arrow marks the same product as in panel b.
FIG. 9.
FIG. 9.
Effect of coexpression of wt or ATPase-defective GFP-VPS4A fusion proteins on MPMV release. 293T cells were cotransfected with the indicated pMPMV plasmids. In each case, the amount of total DNA was adjusted to 4 μg. Steady-state levels of cell- and virus-associated CA protein (upper and lower panel, respectively) were analyzed by Western blotting with anti-CA antiserum, and the expression of the VPS4A constructs was confirmed by Western blotting with an anti-GFP antiserum (middle panel).
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