NEDD4L overexpression rescues the release and infectivity of human immunodeficiency virus type 1 constructs lacking PTAP and YPXL late domains.
The cellular ESCRT pathway functions in membrane remodeling events that accompany endosomal protein sorting, cytokinesis, and enveloped RNA virus budding. In the last case, short sequence motifs (termed late domains) within human immunodeficiency virus type 1 (HIV-1) p6(Gag) bind and recruit two ESCRT pathway proteins, TSG101 and ALIX, to facilitate virus budding. We now report that overexpression of the HECT ubiquitin E3 ligase, NEDD4L/NEDD4-2, stimulated the release of HIV-1 constructs that lacked TSG101- and ALIX-binding late domains, increasing infectious titers >20-fold. Furthermore, depletion of endogenous NEDD4L inhibited the release of these crippled viruses and led to cytokinesis defects. Stimulation of virus budding was dependent upon the ubiquitin ligase activity of NEDD4L and required only the minimal HIV-1 Gag assembly regions, demonstrating that Gag has ubiquitin-dependent, cis-acting late domain activities located outside of the p6 region. NEDD4L stimulation also required TSG101 and resulted in ubiquitylation of several ESCRT-I subunits, including TSG101. Finally, we found that TSG101/ESCRT-I was required for efficient release of Mason-Pfizer monkey virus, which buds primarily by using a PPXY late domain to recruit NEDD4-like proteins. These observations suggest that NEDD4L and possibly other NEDD4-like proteins can ubiquitylate and activate ESCRT-I to function in virus budding.
Chung
Hyo-Young
HY
University of Utah, Department of Biochemistry, 15 N. Medical Drive, East, Salt Lake City, UT 84112-5650, USA.
Morita
Eiji
E
von Schwedler
Uta
U
Müller
Barbara
B
Kräusslich
Hans-Georg
HG
Sundquist
Wesley I
WI
D020816:Amino Acid Motifs; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D015497:HIV-1 / Q000235:genetics / Q000254:growth & development*; D006801:Humans; D016093:Mason-Pfizer monkey virus / Q000254:growth & development; D000075702:Nedd4 Ubiquitin Protein Ligases; D014157:Transcription Factors / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*; D000099177:Tsg101 Protein
D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D000075702:Nedd4 Ubiquitin Protein Ligases; D014157:Transcription Factors; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D054301:gag Gene Products, Human Immunodeficiency Virus; D000099177:Tsg101 Protein; C083002:p6 gag protein, Human immunodeficiency virus 1; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human