title: ESCRT-independent budding of HIV-1 gag virus-like particles from Saccharomyces cerevisiae spheroplasts.
abstract: Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs) that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs) in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated) in the generation of VLPs. Our data reveal: 1) characterized Gag-ESCRT interaction motifs (late domains) are not required for VLP budding, 2) loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3) ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Norgan
authors.forename: Jacqueline R E
authors.initials: JR
authors.lastname: Lee
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Oestreich
authors.forename: Johanna A
authors.initials: JA
authors.lastname: Payne
authors.forename: Eugene W
authors.initials: EW
authors.lastname: Krueger
authors.forename: David J
authors.initials: DJ
authors.lastname: Katzmann
mesh_terms: D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013104:Spheroplasts / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism; D057074:Virus Release; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae