title: Ubiquitin conjugation to Gag is essential for ESCRT-mediated HIV-1 budding.
abstract: BACKGROUND
  HIV-1 relies on the host ESCRTs for release from cells. HIV-1 Gag engages ESCRTs by directly binding TSG101 or Alix. ESCRTs also sort ubiquitinated membrane proteins through endosomes to facilitate their lysosomal degradation. The ability of ESCRTs to recognize and process ubiquitinated proteins suggests that ESCRT-dependent viral release may also be controlled by ubiquitination. Although both Gag and ESCRTs undergo some level of ubiquitination, definitive demonstration that ubiquitin is required for viral release is lacking. Here we suppress ubiquitination at viral budding sites by fusing the catalytic domain of the Herpes Simplex UL36 deubiquitinating enzyme (DUb) onto TSG101, Alix, or Gag.
  
  
  RESULTS
  Expressing DUb-TSG101 suppressed Alix-independent HIV-1 release and viral particles remained tethered to the cell surface. DUb-TSG101 had no effect on budding of MoMLV or EIAV, two retroviruses that rely on the ESCRT machinery for exit. Alix-dependent virus release such as EIAV's, and HIV-1 lacking access to TSG101, was instead dramatically blocked by co-expressing DUb-Alix. Finally, Gag-DUb was unable to support virus release and dominantly interfered with release of wild type HIV-1. Fusion of UL36 did not effect interactions with Alix, TSG101, or Gag and all of the inhibitory effects of UL36 fusion were abolished when its catalytic activity was ablated. Accordingly, Alix, TSG101 and Gag fused to inactive UL36 functionally replaced their unfused counterparts. Interestingly, coexpression of the Nedd4-2s ubiquitin ligase suppressed the ability of DUb-TSG101 to inhibit HIV-1 release while also restoring detectable Gag ubiquitination at the membrane. Similarly, incorporation of Gag-Ub fusion proteins into virions lifted DUb-ESCRT inhibitory effect. In contrast, Nedd4-2s did not suppress the inhibition mediated by Gag-DUb despite restoring robust ubiquitination of TSG101/ESCRT-I at virus budding sites.
  
  
  CONCLUSIONS
  These studies demonstrate a necessary and natural role for ubiquitin in ESCRT-dependent viral release and indicate a critical role for ubiquitination of Gag rather than ubiquitination of ESCRTs themselves.
authors.affiliation: Viral Budding Unit, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4 Center Dr, Bethesda, MD 20894, USA.
authors.forename: Paola
authors.initials: P
authors.lastname: Sette
authors.forename: Kunio
authors.initials: K
authors.lastname: Nagashima
authors.forename: Robert C
authors.initials: RC
authors.lastname: Piper
authors.forename: Fadila
authors.initials: F
authors.lastname: Bouamr
mesh_terms: D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D057074:Virus Release; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*; D000099177:Tsg101 Protein
chemical_list: D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025801:Ubiquitin; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D000099177:Tsg101 Protein; C117020:PDCD6IP protein, human; C078035:UL36 protein, Human herpesvirus 1
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