Tsg101 and Alix interact with murine leukemia virus Gag and cooperate with Nedd4 ubiquitin ligases during budding.
Retroviruses use endosomal machinery to bud out of infected cells, and various Gag proteins recruit this machinery by interacting with either of three cellular factors as follows: ubiquitin ligases of the Nedd4 family, Tsg101, or Alix/Aip1. Here we show that the murine leukemia virus Gag has the unique ability to interact with all three factors. Small interfering RNAs against Tsg101 or Alix and dominant-negative forms of Nedd4 can all reduce production of virus-like particles. However, inactivating the Nedd4-binding site abolishes budding, whereas disrupting Tsg101 or Alix binding has milder effects. Nedd4 ubiquitin ligases are therefore essential, and Tsg101 and Alix play auxiliary roles. Most interestingly, overexpression of Alix can stimulate the release of Gag, and this occurs independently of most Alix partners Tsg101, Cin85, Alg-2, and endophilins. In addition, Gag mutants that do not bind Tsg101 or Alix concentrate on late endosomes and become very sensitive to dominant-negative forms of Nedd4 that do not conjugate ubiquitin. This suggests that the direct interaction of Gag with Tsg101 and Alix favors budding from the plasma membrane and relieves a requirement for ubiquitination by Nedd4.1. Other Nedd4-dependent Gag proteins also contain binding sites for Tsg101 or Alix, suggesting that this could be a common feature of retroviruses.
Segura-Morales
Carolina
C
IGMM-CNRS, 1919 Route de Mende 34293, Montpellier Cedex 5, France.
Pescia
Christina
C
Chatellard-Causse
Christine
C
Sadoul
Remy
R
Bertrand
Edouard
E
Basyuk
Eugenia
E
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D009052:Leukemia Virus, Murine / Q000737:chemistry / Q000502:physiology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D000075702:Nedd4 Ubiquitin Protein Ligases; D051381:Rats; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014779:Virus Replication; D000099177:Tsg101 Protein
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D015683:Gene Products, gag; D000075702:Nedd4 Ubiquitin Protein Ligases; D014157:Transcription Factors; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000099177:Tsg101 Protein; C492144:Pdcd6ip protein, mouse; C000618479:NEDD4L protein, rat; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618461:Nedd4l protein, mouse