No abstract

Here we use this assay to demonstrate homomeric interactions between Gag polyproteins encoded by six different retroviruses. Of the Gag polyproteins tested, only those encoded by closely related retroviruses formed heteromultimers. To determine the primary sequence requirements for human immunodeficiency virus type 1 Gag polyprotein multimerization, we studied the effects on multimerization of deletion and linker insertion mutations. Sequences necessary for this process were located between the C-terminal one-third of the capsid domain and the C terminus of the nucleocapsid domain.

We have established a genetic assay for the multimerization of retroviral gag polyproteins. This assay is based on the GAL4 two-hybrid system for studying protein-protein interactions (S. Fields and 0. Song, Nature (London) 340:245-246, 1989). In our initial experiments, we generated Saccharomyces cerevisiae plasmids that separately express the GAL4 DNA-binding and GAL4 activation domains fused to the human immunodeficiency virus type 1 (HIV-1) gag polyprotein, Pr55w*. The coexpression of these two hybrid proteins in S. cerevisiae results in the association of the GAL4 domains and the potent activation of an integrated GAL4-responsive lacZ indicator gene. Similar results were obtained with plasmids encoding GAL4-Moloney murine leukemia virus (M-MuLV) gag polyprotein hybrid proteins. In contrast, the heterologous GAL4-HlV-1 gag and GAL4-M-MuLV gag fusion proteins were unable to interact with each other to induce lacZ expression. The results suggest that this yeast system provides a rapid and specific assay for the interactions of retroviral gag proteins that occur during virion assembly.