Proteolytic activity of purified avian sarcoma and leukemia virus NC-PR protein expressed in Escherichia coli.

Processing of the internal structural and enzymatic proteins of retroviruses occurs during or shortly after budding and is accomplished by the viral protease (PR), which belongs to the large family of aspartic proteases. It is not known how the activity of PR is regulated so that proteolysis occurs at this time. Cellular aspartic proteases are synthesized as zymogens with short N-terminal extensions that are proteolytically removed to generate the free active enzyme. In the avian sarcoma and leukosis viruses (ASLV), PR is expressed as the carboxy-terminal domain of the Gag polyprotein, which thus has a structure analogous to such a zymogen. We have investigated the enzymatic properties of ASLV PR when it is part of a longer protein, NC-PR, serving as a model for Gag. This protein represents about one-third of Gag and consists of the nucleocapsid (NC) domain fused to the N-terminus of PR. NC-PR and derivatives of NC-PR were expressed in bacterial cells and purified. In short-term assays, these fusion proteins lacked measurable protease activity toward an exogenous substrate prepared by in vitro translation. In contrast to PR, which is a homodimer, NC-PR migrated as a monomer both by glycerol gradient sedimentation and by gel filtration chromatography. Thus the NC domain appears to inhibit enzymatic activity by altering the dimerization potential of the PR domains. However, upon long incubations NC-PR was found to cleave itself to generate free and fully active PR, implying that dimerization was not prevented entirely. On the basis of these results, we hypothesize that the Gag protein in vivo is also incompletely active as a protease, because upstream portions of Gag interfere with proper interaction of the PR domains. The eventual dimerization, perhaps triggered by other events, then could lead to a cascade whereby PR is proteolytically freed from Gag and thereby gains enzymatic activity.