Assembly of HIV GAG-B-galactosidase fusion proteins into virus particles.
We have studied the assembly of human immunodeficiency virus (HIV-1) Gag-B-galactosidase (Gag-B-gal; GBG) fusion proteins into HIV particles in the presence of HIV Gag proteins. Release of fusion proteins from cells was measured by assay of media versus cellular B-gal activities and was dependent on co-expression of unfused Gag proteins. Gag-B-gal incorporation into virus particles was demonstrated by detergent treatment and density gradient fractionation studies and was dependent on protein-protein interactions requiring the C-terminal two-thirds of the HIV CA domain. The central MA domain appeared unimportant for fusion protein incorporation; a nonmyristylated GBG protein was incorporated but at a relatively reduced level, while the NC and p6 domains slightly affected the assembly of fusion proteins into particles. Subcellular fractionation studies showed that all fusion proteins including the nonmyristylated one were enriched in the cytoplasmic pellet fraction. However, assembly into particles did not correlate with subcellular fractionation patterns. Similarly, virion incorporation levels of Gag-B-gal proteins did not correlate with their immunofluorescence localization patterns. However, we observed that while most fusion proteins displayed a perinuclear ring with heterogeneous staining throughout cells, short fusion proteins appeared enriched on the intracellular membranes, and fusion proteins with intact MA but deleted NC domains showed an enhanced surface staining without a clear perinuclear ring. Altogether, our data suggest that the CA domain is the primary determinant for assembly of HIV fusion proteins into virus particles.
Wang
C T
CT
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
Stegeman-Olsen
J
J
Zhang
Y
Y
Barklis
E
E
D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000254:growth & development*; D008969:Molecular Sequence Data; D009024:Morphogenesis / Q000235:genetics; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014771:Virion / Q000254:growth & development*; D001616:beta-Galactosidase / Q000032:analysis / Q000378:metabolism
D015683:Gene Products, gag; D009227:Myristic Acids; D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D001616:beta-Galactosidase