ASSEMBLY OF HIV GAG-B-GALACTOSIDASE FUSION PROTEINS INTO VIRUS-PARTICLES

We have studied the assembly of human immunodeficiency virus (HIV-I) G ag-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 treat ment and density gradient fractionation studies and was dependent on p rotein-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 slightl y affected the assembly of fusion proteins into particles. Subcellular fractionation studies showed that all fusion proteins including the n onmyristylated one were enriched in the cytoplasmic pellet fraction. H owever, assembly into particles did not correlate with subcellular fra ctionation patterns. Similarly, virion incorporation levels of Gag-B-g al proteins did not correlate with their immunofluorescence localizati on patterns. However, we observed that while most fusion proteins disp layed a perinuclear ring with heterogeneous staining throughout cells, short fusion proteins appeared enriched on the intracellular membrane s, and fusion proteins with intact MA but deleted NC domains showed an enhanced surface staining without a clear perinuclear ring. Altogethe r, our data suggest that the CA domain is the primary determinant for assembly of HIV fusion proteins into virus particles. (C) 1994 Academi c Press, Inc.