Alkaline Protease Associated with Virus Particles of a Nuclear Polyhedrosis Virus: Assay, Purification, and Properties

Abstract

Proteolytic activity was detected within polyhedra of the nuclear polyhedrosis virus of Spodoptera littoralis . The enzyme activity was detected by its ability to degrade the major structural polypeptide of polyhedra (polyhedrin). A quantitative assessment of activity was made by a radioassay technique using 3 H-labeled polyhedrin as the substrate. Of the structural components of polyhedra, virus particles showed the greatest specific proteolytic activity. Preparations of purified nucleocapsids were inactive. The virus particle enzyme displayed a temperature optimum for proteolysis of 30 to 40°C and a pH optimum of 9.6. Its activity was inhibited by H 2+ and Cu 2+ , but not by 2-mercaptoethanol. The enzyme was purified from detergent-treated virus particles by affinity column chromatography, using polyhedrin linked to cyanogen bromide-activated Sepharose. Three major envelope polypeptides (L107, L85, and L71) bound to the column at 4°C, but after incubation at 31°C, polypeptide L71 alone was eluted. The fractions containing this protein exhibited a specific enzyme activity more than 80-fold greater than that present in polyhedra. The possible significance of the alkaline protease, and other proteins with affinity for polyhedrin, is discussed.