title: Rescue of HIV-1 release by targeting widely divergent NEDD4-type ubiquitin ligases and isolated catalytic HECT domains to Gag.
abstract: Retroviruses engage the ESCRT pathway through late assembly (L) domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA). The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release.
authors.affiliation: Program in Gene Function and Expression, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Weiss
authors.forename: Elena
authors.initials: E
authors.lastname: Popova
authors.forename: Hikaru
authors.initials: H
authors.lastname: Yamanaka
authors.forename: Hyung Cheol
authors.initials: HC
authors.lastname: Kim
authors.forename: Jon M
authors.initials: JM
authors.lastname: Huibregtse
authors.forename: Heinrich
authors.initials: H
authors.lastname: Göttlinger
mesh_terms: D056827:Endosomal Sorting Complexes Required for Transport / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D000075702:Nedd4 Ubiquitin Protein Ligases; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D057074:Virus Release / Q000502:physiology*; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D054301:gag Gene Products, Human Immunodeficiency Virus; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases