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A novel protein kinase that requires protamine as an activator to catalyze the phosphorylation of viral acceptor proteins was extracted from vaccinia virus cores with deoxycholate and purified 250-fold by DNA-cellulose and DEAE-cellulose column chromatography. The enzyme has a molecular weight of 62,000 as determined by sucrose gradient sedimentation. Two heat-stable phosphate acceptor proteins were extracted from virus particles with a nonionic detergent and purified by heat treatment, precipitation with organic solvents, and CM-cellulose chromatography. The molecular weights of the phosphate acceptor proteins, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, are 38,500 and 11,700.

A protein synthesis inhibitor, solubilized from vaccinia virus (Ben-Hamida, F., Person, A., and Beaud, G. (1983) J. Virol. 45, 452-455), has been purified to homogeneity, yielding a basic protein with molecular mass of 11 kDa. This purified protein migrates as a single spot in two-dimensional gel analysis (isoelectric point above 8.6). It is phosphorylated by the vaccinia-associated protein kinase, and it aggregates in the absence of reducing agents. This 11-kDa protein inhibits protein synthesis when added to a reticulocyte lysate at a stoichiometric ratio of approximately one protein molecule/ribosome, and it associates with the ribosome fraction after incubation in reticulocyte lysates or in Ehrlich ascites tumor cell lysates. As previously described for the inhibitor associated with vaccinia cores, the purified inhibitor inhibits the formation of the 40 S ribosomal subunit X Met-tRNAi ribosomal initiation complex. It has no detectable effect on the formation of the ternary complex (Met-tRNAi X GTP X eucaryotic initiation factor 2). This inhibitor associated with vaccinia virus particles may be involved in the shutoff of host protein synthesis and may also be responsible for the absence of virus replication in some cell-virus systems.