416 S. GARD AND O. MAAL0E 



and Kleczkowski (1944) presented conclusive evidence of an in vitro combina- 

 tion of the two. Neither the enzyme nor the virus was inactivated and the 

 combination was readily reversible on dilution. Thus, as in the case of phage 

 ribonuclease, two factors — a direct reduction of avidity and damage to the 

 host cell — might cooperate in reducing infectivity. Bawden and Pirie (1936) 

 had shown that potato virus X was readily hydrolyzed and inactivated by 

 trypsin. Kleczkowki (1944), confirming this observation, found that this 

 virus, in spite of its sensitivity, did not bind more enzyme than the resistant 

 TMV. Most animal viruses seem to be trypsin-resistant; for that reason, 

 trypsin digestion has been applied as a means of purification. Hovrever, 

 Merrill (1936) presented conclusive evidence to show that pseudorabies 

 virus was readily inactivated by trypsin; the infectivity of vaccinia virus 

 was slowly reduced by trypsin alone, more rapidly by trypsin + chymotryp- 

 sin; EEE and swine influenza virus were resistant. The effect of chymotrypsin 

 upon the same four viruses followed a different pattern: EEE and pseudo- 

 rabies virus were inactivated, vaccinia and swine influenza virus were resistant. 



The effect of pepsin is less easily studied, as comparatively few viruses are 

 stable at the pH optimal for the activity of this enzyme. Stanley (1934b) 

 observed a slow inactivation of TMV, presumably the result of enzyme action 

 on virus protein gradually denatured by low pH. Bawden and Pirie (1937) 

 and Kleczkowski (1944), on the other hand, found this virus to be resistant. 

 Kleczkowski showed that pepsin, which unlike trypsin combines specifically 

 only with proteins serving as substrates, did not combine with native but did 

 so readily with denatured virus, which also was readily digested. In contrast, 

 potato virus X combined with the enzyme and was inactivated. Of animal 

 viruses, poHovirus (Barski et ah, 1954) and presumably the other members of 

 the group of intestinal viruses are pepsm-resistant. 



Bawden and Pirie (1937) observed that commercial papain formed pre- 

 cipitates with plant virus suspensions as with various nonviral nucleoproteins 

 and nucleic acids. The precipitate included virus as weU as enz3nne. With 

 certain proportions of the reactants, precipitation was complete. TMV was 

 thus removed from the solution by papain but not inactivated. The infecti- 

 vity of potato virus X, on the other hand, was destroyed by papain in the 

 presence of KCN but not in its absence, an indication that the proteolytic 

 action of the enzyme was the factor responsible for inactivation. According 

 to Lepine (1948), rabies virus is inactivated by papain. Polioviruses are 

 resistant to papain and ficin (Gard and Ostlund, 1951). 



Other enzjnaaes have not attracted much interest. Thus, nothing is known 

 about the effect of pure Hpolytic enzymes on lipid-containing viruses. 



It is not yet possible to tell to what extent specific enzyme resistance- 

 sensitivity patterns of virus proteins exist. Merrill's observations might 

 be an indication that such is the case. As to the kinetics and the mechanism 



