Enzymic inactivation of vaccinia virus with a bacterial protease.

pepsin (Dawson and McFarlane, 1948 ; Peter and Stoeckenius, 1954). For the purpose of finding out any enzyme to affect the surface structure of vaccinia virus under less radical conditions than with pepsin (pH 3.0), attempts were made to test various enzymes for the effect on the infectivity of the virus. Virus suspension was prepared from the homogenate of infected chorioallantoic membranes (CAM) by one cycle of differential centrifugation at 7.500•~g for 40min. and at 500•~g for 10min. To 1.0cc of virus suspension in 1/150M phosphate-buffered saline (pH 7.4), 0.5cc of enzyme solution was added and the final volume of the mixture was made to 2.0cc with phosphate-buffered saline. The mixture was incubated at 37•Ž for specified period being followed by a high speed centrifugation to separate virus from enzyme. The following enzymes were investigated ; snake venom •glecithinase•h1), bacterial protease •gNagarse•h2), pepsin3), and streptokinase-streptodornase •gVaridase•h4). Titration of infectivity was carried out by pock count on the CAM of 12-day old fertile hen's eggs. Nagarse, a crystalline protease prepared from Bacillus subtilis, was the sole enzyme which inactivated significantly vaccinia virus. Table 1 shows the effect of Nagarse at different concentrations on the infectivity of vaccinia virus. After 2 hours' incubation with 250ƒÊg per cc of Nagarse the infectivity dropped from 8.8 log10 to 6.3 log10. The inactivation power of this solution is 2.5 in log difference (‡TM log), which is considered to be statistically significant for the titration method employed. In the next experiment an approach was made to elucidate the mode of action of Nagarse on vaccinia virus, employing DFP (Diisopropylfluorophosphate), which is known as a specific inhibitor of the protease and cholinesterase. The results are summarized in Table 2, indicating that the inactivation effect of Nagarse was inhibited by the co existence of DFP in the reaction mixture. The data led the authors to consider that the digestion of the protein-moiety which might be surface-constituent of virus particle accounted for the loss of infectivity of vaccinia virus. The other interesting finding was that no further inactivation was demonstrated by repeated digestions, as shown in Table 3. One possibility may be that two types of viruses are mixed in the preparation, one of which, Nagarse resistant virus, occupies about 1% of the total virus population. Another possibility that infectious nucleic acid might be released by enzymic digestion, to which the remaining infectivity may be due, should be considered as well. Based on the following observations, however, the latter