The ESCRT-associated protein Alix recruits the ubiquitin ligase Nedd4-1 to facilitate HIV-1 release through the LYPXnL L domain motif.
The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPX(n)L, that bind Tsg101 and Alix, respectively. Interactions with these two cellular proteins recruit members of the host's fission machinery (ESCRT) to facilitate HIV-1 release. Other retroviruses gain access to the host ESCRT components by utilizing a PPXY-type L domain that interacts with cellular Nedd4-like ubiquitin ligases. Despite the absence of a PPXY motif in HIV-1 Gag, interaction with the ubiquitin ligase Nedd4-2 was recently shown to stimulate HIV-1 release. We show here that another Nedd4-like ubiquitin ligase, Nedd4-1, corrected release defects resulting from the disruption of PTAP (PTAP(-)), suggesting that HIV-1 Gag also recruits Nedd4-1 to facilitate virus release. Notably, Nedd4-1 remediation of HIV-1 PTAP(-) budding defects is independent of cellular Tsg101, implying that Nedd4-1's function in HIV-1 release does not involve ESCRT-I components and is therefore distinct from that of Nedd4-2. Consistent with this finding, deletion of the p6 region decreased Nedd4-1-Gag interaction, and disruption of the LYPX(n)L motif eliminated Nedd4-1-mediated restoration of HIV-1 PTAP(-). This result indicated that both Nedd4-1 interaction with Gag and function in virus release occur through the Alix-binding LYPX(n)L motif. Mutations of basic residues located in the NC domain of Gag that are critical for Alix's facilitation of HIV-1 release, also disrupted release mediated by Nedd4-1, further confirming a Nedd4-1-Alix functional interdependence. In fact we found that Nedd4-1 binds Alix in both immunoprecipitation and yeast-two-hybrid assays. In addition, Nedd4-1 requires its catalytic activity to promote virus release. Remarkably, RNAi knockdown of cellular Nedd4-1 eliminated Alix ubiquitination in the cell and impeded its ability to function in HIV-1 release. Together our data support a model in which Alix recruits Nedd4-1 to facilitate HIV-1 release mediated through the LYPX(n)L/Alix budding pathway via a mechanism that involves Alix ubiquitination.
Sette
Paola
P
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Jadwin
Joshua A
JA
Dussupt
Vincent
V
Bello
Nana F
NF
Bouamr
Fadila
F
D002135:Calcium-Binding Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D015497:HIV-1 / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation; D000075702:Nedd4 Ubiquitin Protein Ligases; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057074:Virus Release; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C117020:PDCD6IP protein, human; D054301:gag Gene Products, Human Immunodeficiency Virus; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases