title: The host protein Staufen1 participates in human immunodeficiency virus type 1 assembly in live cells by influencing pr55Gag multimerization.
abstract: Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. The activities of several host cell proteins and machineries are also critical to the completion of virus assembly and the release of infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55Gag. In this report, we tested whether Stau1 modulates pr55Gag assembly using a new and specific pr55Gag oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both the overexpression and knockdown of Stau1 increase the pr55Gag-pr55Gag BRET levels, suggesting a role for Stau1 in regulating pr55Gag oligomerization during assembly. This effect of Stau1 on pr55Gag oligomerization was observed only in membranes, a cellular compartment in which pr55Gag assembly primarily occurs. Consistently, expression of Stau1 harboring a vSrc myristylation signal led to a 6.5-fold enrichment of Stau1 in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55Gag-pr55Gag BRET, demonstrating that Stau1 acts on assembly when targeted to membranes. A role for Stau1 in the formation of particles is further supported by the detection of membrane-associated detergent-resistant pr55Gag complexes and the increase of virus-like particle release when Stau1 expression levels are modulated. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55Gag-pr55Gag interactions, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.
authors.affiliation: Département de biochimie, Université de Montréal, and HIV-1 RNA Trafficking Laboratory, Lady Davis Institute for Medical Research-Sir Mortimer B. Davis Jewish General Hospital, Montréal, QC, Canada H3C 3J7.
authors.forename: Laurent
authors.initials: L
authors.lastname: Chatel-Chaix
authors.forename: Levon
authors.initials: L
authors.lastname: Abrahamyan
authors.forename: Céline
authors.initials: C
authors.lastname: Fréchina
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Mouland
authors.forename: Luc
authors.initials: L
authors.lastname: DesGroseillers
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000502:physiology*; D015967:Gene Expression Regulation, Viral; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D015683:Gene Products, gag; D008164:Luminescent Proteins; D011498:Protein Precursors; D016601:RNA-Binding Proteins; C492185:STAU1 protein, human; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; C054562:yellow fluorescent protein, Bacteria