Investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus-infected Sf-9 insect cell culture by inhibition assay

Proteases produced during the culture of Spodoptera frugiperda Sf-9 cells i nfected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis reveale d that: (1) carboxyl and cysteine proteases were predominantly produced by the insect cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus. and (2) the protease activity was almost completely bl ocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protea se inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay di scriminated between the two protease activities and elucidated the sequenti al behavior of the carboxyl and cysteine proteases produced in the virus-in fected Sf-9 cell culture. The carboxyl protease(s) existed in the virus-inf ected cells all the time and their level in the medium continuously increas ed. Uninfected cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected cells. At a certain tim e after virus infection. the cysteine protease activity was largely increas ed in the virus-infected cells and a significant amount of the protease(s) was released into the medium, due to the cell membranes losing their integr ity. The behavior of intracellular and extracellular cysteine protease acti vities coincided with that of a recombinant protein whose expression was un der the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected insect cells showed that the inhibition-b ased protease assay was useful for analyzing the carboxyl protease and cyst eine protease activities emerging in the insect cell (Sf-9)/baculovirus exp ression system.