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Published on: February 21, 2019
Tsg101 can replace Nedd4 function in ASV Gag release but not membrane targeting.
Gisselle Medina1, Andrew Pincetic, Lorna S Ehrlich
1Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222, USA.
Avian sarcoma virus Gag protein release utilizes distinct pathways involving Nedd4 and Tsg101. Tsg101 can substitute for Nedd4 in viral budding, but through a different membrane-associated mechanism.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- The Late (L) domain of avian sarcoma virus (ASV) Gag protein is crucial for efficient virus release.
- Nedd4 ubiquitin ligase E3 family members interact with the ASV Gag L domain.
- Nedd4 and Tsg101 interaction suggests Nedd4 links ASV Gag to the ESCRT machinery.
Purpose of the Study:
- To investigate the distinct roles of Tsg101 and Nedd4 in ASV Gag protein-mediated virus release.
- To determine if Tsg101 can functionally replace Nedd4 in ASV budding.
- To elucidate the membrane trafficking pathways utilized by ASV Gag during release.
Main Methods:
- Covalent linkage of Tsg101 to ASV Gag lacking the Nedd4 binding site (Deltap2b-Tsg101).
- Assessing virus-like particle (VLP) release in DF-1 avian cells using dominant-negative Tsg101 mutants and siRNA-mediated depletion.
- Analyzing VLP association with endosome-like membrane domains enriched in CD63 or fluorescent phosphatidylethanolamine analogues.
Main Results:
- Covalently linking Tsg101 to ASV Gag (Deltap2b-Tsg101) bypassed the need for Nedd4 for efficient budding.
- VLP release from DF-1 cells was resistant to Tsg101 interference and depletion, suggesting an alternative pathway.
- Wild-type ASV Gag VLPs localized to specific membrane domains, while Deltap2b and Deltap2b-Tsg101 VLPs did not, despite efficient release of the latter.
Conclusions:
- Tsg101 and Nedd4 facilitate ASV Gag release through functionally interchangeable but independent pathways.
- Tsg101 can substitute for Nedd4 in promoting viral budding but directs the process via different membrane domains.
- ASV Gag release mechanisms are more complex than previously understood, involving distinct ESCRT-dependent and independent routes.
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