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SUMMARY Argininosuccinate synthetase-lyase activity in HeLa or mouse L cells increased markedly following infection by vaccinia virus. A repression mechanism determines specific activities in uninfected cells and greater activity was obtained in the presence of 0-2 mM-citrulline than of 0-6 mM-arginine. However, maximum activity in infected cells was obtained with 0.6 mM-arginine. The development of increased enzyme activity in virus-infected cells was inhibited by puromycin or actinomycin D. Treatment of infected cells with FUdR resulted in higher levels of activity than in similar cells in the absence of the inhibitor. The Michaelis constants of enzymes in HeLa and mouse L cells were o.II × I0-aM and o-o7 x m-3M, respectively; the same value of 1.o × T0-3M was determined for both species of infected ceils. Amino acid analysis of purified vaccinia virus prepared from HeLa cell cultures infected in the presence of (14C)-carbamoyl-citruUine showed that radioactivity was associated exclusively with arginine. It is concluded that the anabolism of arginine from citrulline in vaccinia virus-infected cells is determined by virus-coded enzymes. INTRODUCTION The accompanying paper (Cooke & Williamson, ~973) has described enhanced utilization of citrulline in certain continuous cell lines following poxvirus infection. In particular, the replication of rabbitpox virus in mouse sarcoma 18o cells and of vaccinia virus in human citrullinaemia cells in the presence of citrulline has been shown to occur under conditions not permissive for cell growth. Inhibition by canavanine of rabbitpox virus replication under such conditions indicated the necessity for arginine biosynthesis. Anabolism of arginine from citrulline is determined by the activity of two enzymes, argininosuccinate synthetase (E.C. 6.3.4.5-) and argininosuccinate lyase (E.C. 4.3- 2. I.). This paper describes changes in the activity of these enzymes during vaccinia virus replication and presents evidence showing that these changes depend specifically upon virus infection. METHODS Virus. The Lister strain of vaccinia virus was used throughout this work. Infectivity titrations were made by plaque formation in cultures of RK~a cells. Cell cultures. Methods used for the growth and maintenance of HeLa cells and their equilibration to medium containing either arginine or citruUine have been described (Cooke & Williamson, I973). The same methods were used with mouse L cells.

Argininosuccinate Synthetase-lyase Activity in Vaccinia Virus-infected HeLa and Mouse L Cells