TARGETING OF MOLONEY MURINE LEUKEMIA-VIRUS GAG PRECURSOR TO THE SITE OF VIRUS BUDDING

Retrovirus Moloney murine leukemia virus (M-MuLV) matures by budding a t the cell surface, Central to the budding process is the myristoylate d viral core protein precursor Gag which, even in the absence of all o ther viral components, is capable of associating with the cytoplasmic leaflet of the plasma membrane and assembling into extracellular virus -like particles, In this paper we have used heterologous, Semliki Fore st virus-driven, expression of M-MuLV Gag to study the mechanism by wh ich this protein is targeted to the cell surface, In pulse-chase exper iments, BFA, monensin, and 20 degrees C block did not affect incorpora tion of Gag into extracellular particles thereby indicating that the s ecretory pathway is not involved in targeting of Gag to the cell surfa ce. Subcellular fractionation studies demonstrated that newly synthesi zed Gag became rapidly and efficiently associated with membranes which had a density similar to that of plasma membrane-derived vesicles, Pr otease-protection studies confirmed that the Gag-containing membranes were of plasma membrane origin, since in crude cell homogenates, the b ulk of newly synthesized Gag was protease-resistant as expected of a p rotein that binds to the cytoplasmic leaflet of the plasma membrane, T aken together these data indicate that targeting of M-MuLV Gag to the cell surface proceeds via direct insertion of the protein to the cytop lasmic side of the plasma membrane, Furthermore, since the membrane in sertion reaction is highly efficient and specific, this suggests that the reaction is dependent on as-yet-unidentified cellular factors.