Interaction and co-encapsidation of human immunodeficiency virus type 1 Gag and Vif recombinant proteins.
Human immunodeficiency virus type 1 (HIV-1) wild-type (WT) virion infectivity factor (Vif) protein (Vifwt) and full-length Gag precursor (Pr55Gag) were found to be co-encapsidated into extracellular, membrane-enveloped virus-like particles released by budding from Sf9 cells co-expressing the two recombinant proteins in trans, with an average copy number of 3.5+/-0.6 Vifwt per 100 Pr55Gag molecules. No preferential localization at the plasma membrane was observed for recombinant Vif in the absence of Gag expression, and a significant proportion of Vif accumulated within the nucleus. Two conserved motifs, W89RKRRY94 and P156KKIKP161, seemed to act as nuclear addressing signals. The Pr55Gag and Vifwt interacting domains were analysed by biopanning of a phage-displayed hexapeptide library. The Vif-binding domain, which spanned residues H421-T470 in Pr55Gag, corresponded to the C-terminal region of nucleocapsid (NC), including the second zinc finger, the intermediate spacer peptide sp2 and the N-terminal half of the p6 domain. Deletions in these Gag domains significantly decreased the Vif encapsidation efficiency, and complete deletion of NC abolished Vif encapsidation. In Vif, four discrete Gag-binding sites were identified, within residues T68-L81 (site I) and W89-P100 (site II) in the central domain, and within residues P162-R173 (III) and P177-M189 (IV) at the C terminus. Substitutions in site I and deletion of site IV were detrimental to Vif encapsidation, whereas substitution of basic residues for alanine in sites III and IV had a positive effect. The data suggest a direct intracellular Gag-Vif interaction and the occurrence of a Pr55Gag-mediated membrane-targeting pathway for Vif in Sf9 cells.
Huvent
I
I
Laboratoire de Virologie Moléculaire et Pathogénèse Virale (CNRS UMR-5812), Institut de Biologie, Faculté de Médecine, Montpellier, France.
Hong
S S
SS
Fournier
C
C
Gay
B
B
Tournier
J
J
Carrière
C
C
Courcoul
M
M
Vigne
R
R
Spire
B
B
Boulanger
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages; D002460:Cell Line; D015870:Gene Expression; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D016346:Gene Products, vif / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014771:Virion; D019065:Virus Assembly / Q000502:physiology*; D054320:vif Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D016346:Gene Products, vif; D019151:Peptide Library; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D054320:vif Gene Products, Human Immunodeficiency Virus