Tsg101 can replace Nedd4 function in ASV Gag release but not membrane targeting.
The Late (L) domain of the avian sarcoma virus (ASV) Gag protein binds Nedd4 ubiquitin ligase E3 family members and is the determinant of efficient virus release in avian and mammalian cells. We previously demonstrated that Nedd4 and Tsg101 constitutively interact raising the possibility that Nedd4 links ASV Gag to the ESCRT machinery. We now demonstrate that covalently linking Tsg101 to ASV Gag lacking the Nedd4 binding site (Deltap2b-Tsg101) ablates the requirement for Nedd4, but the rescue of budding occurs by use of a different budding mechanism than that used by wild type ASV Gag. The evidence that Tsg101 and Nedd4 direct release by different pathways is: (i) Release of the virus-like particles (VLPs) assembled from Gag in DF-1, an avian cell line, was resistant to dominant-negative interference by a Tsg101 mutant previously shown to inhibit release of both HIV and Mo-MLV. (ii) Release of VLPs from DF-1 cells was resistant to siRNA-mediated depletion of the endogenous pool of Tsg101 in these cells. (iii) VLPs assembled from wild-type ASV Gag exhibited highly efficient release from endosome-like membrane domains enriched in the tetraspanin protein CD63 or a fluorescent analogue of the phospholipid phosphatidylethanolamine. However, the VLPs assembled from the L domain mutant Deltap2b or a chimeric Deltap2b-Tsg101 Gag lacked these domain markers even though the chimeric Gag was released efficiently compared to the Deltap2b mutant. These results suggest that Tsg101 and Nedd4 facilitate Gag release through functionally exchangeable but independent routes and that Tsg101 can replace Nedd4 function in facilitating budding but not directing through the same membranes.
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222, USA.
Gisselle
G
Medina
Andrew
A
Pincetic
Lorna S
LS
Ehrlich
Yongjun
Y
Zhang
Yi
Y
Tang
Jonathan
J
Leis
Carol A
CA
Carter
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics* / Q000502:physiology* / Q000648:ultrastructure; D001717:Birds; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure / Q000821:virology; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D015683:Gene Products, gag / Q000235:genetics* / Q000378:metabolism*; D015750:Genes, gag; D046529:Microscopy, Electron, Transmission; D000075702:Nedd4 Ubiquitin Protein Ligases; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D000099177:Tsg101 Protein
D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D015683:Gene Products, gag; D000075702:Nedd4 Ubiquitin Protein Ligases; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases; D000099177:Tsg101 Protein