The functionally exchangeable L domains in RSV and HIV-1 Gag direct particle release through pathways linked by Tsg101.
The functionally exchangeable L domains of HIV-1 and Rous sarcoma virus (RSV) Gag bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 function in endocytic trafficking, and studies show that expression of Tsg101 or Nedd4 fragments interfere with release of HIV-1 or RSV Gag, respectively, as virus-like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, we tested the effect on RSV Gag release of co-expression with mutated forms of Vps4, Nedd4 and Tsg101. A dominant-negative mutant of Vps4A, an AAA ATPase required for utilization of endosomal sorting proteins that was shown previously to interfere with HIV-1 budding, also inhibited RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacted in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacted with Tsg101. Deletion of the N-terminal region of Tsg101 or the HECT domain of Nedd4 did not prevent interaction; however, three-dimensional spatial imaging suggested that the interaction of RSV Gag with full-length Tsg101 and N-terminally truncated Tsg101 was not the same. Co-expression of RSV Gag with the Tsg101 C-terminal fragment interfered with VLP release minimally; however, a significant fraction of the released VLPs was tethered to each other. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. We conclude that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through Tsg101-Nedd4 interaction.
Medina
Gisselle
G
Departments of Molecular Genetics & Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794-5222, USA.
Zhang
Yongjun
Y
Tang
Yi
Y
Gottwein
Eva
E
Vana
Marcy L
ML
Bouamr
Fadila
F
Leis
Jonathan
J
Carter
Carol A
CA
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006388:Hemagglutinins / Q000378:metabolism; D006801:Humans; D000075702:Nedd4 Ubiquitin Protein Ligases; D010446:Peptide Fragments / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases; D033921:Vesicular Transport Proteins; D000099177:Tsg101 Protein
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D015683:Gene Products, gag; D006388:Hemagglutinins; D000075702:Nedd4 Ubiquitin Protein Ligases; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases; D025262:Vacuolar Proton-Translocating ATPases; D033921:Vesicular Transport Proteins; D000099177:Tsg101 Protein; C000618483:Nedd4 protein, human; C490305:VPS4A protein, human