Related Experiment Videos
The Mason-Pfizer monkey virus PPPY and PSAP motifs both contribute to virus release
Eva Gottwein1, Jochen Bodem, Barbara Müller
1Abteilung Virologie, Universitätsklinikum Heidelberg, D-69120 Heidelberg, Germany.
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.
Insights
The Mason-Pfizer monkey virus (MPMV) PSAP motif aids virus release, but only if the PPPY motif is intact. This suggests a coordinated L-domain function essential for enveloped virus release.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Enveloped viruses utilize late (L) domains for efficient release.
- PPXY, PT/SAP, and YPDL are known L-domain motifs.
- Mason-Pfizer monkey virus (MPMV) possesses adjacent PPPY and PSAP motifs.
Purpose of the Study:
- To investigate the role of the PSAP motif in MPMV release.
- To determine the functional relationship between the PPPY and PSAP motifs in MPMV.
- To elucidate the cellular factors involved in MPMV L-domain function.
Main Methods:
- Site-directed mutagenesis of MPMV L-domain motifs (PSAP and PPPY).
- Electron microscopy to visualize virus budding and release.
- Analysis of cellular protein (TSG101, Nedd4, VPS4A) incorporation into viral particles.
- Overexpression of dominant-negative VPS4A to assess budding defects.
Main Results:
- The PSAP motif promotes efficient MPMV release but requires an intact PPPY motif.
- PSAP mutant viruses showed accumulation of budding structures and cell-surface viruses.
- PPPY mutant viruses exhibited budding arrest at an early stage.
- TSG101 incorporation into MPMV particles was PSAP-dependent.
- Nedd4 was detected at lower levels in MPMV particles.
- VPS4A ATPase-defective mutants blocked release of wild-type and PSAP mutant viruses.
Conclusions:
- The PSAP motif functions as an L domain in MPMV, dependent on the PPPY motif.
- The Gag-TSG101 interaction likely mediates the PSAP motif's role in virus release.
- VPS4A plays a critical role in the budding of viruses with PPPY and PTAP motifs.