SUMMARYProtein kinase has been extracted in soluble form from virions of pseudorabies virus using 10~ NP40, 0.6 M-NaC1. Chromatographic analysis of the extract on DEAEcellulose and on phosphocellulose showed it to contain more than one kinase. The activity responsible for the phosphorylation of the major phosphoproteins (mol. wts. 120000, 115000 and 72000) of virions was found to be similar in its properties to the host enzyme casein kinase I1. Purified casein kinase II from ascites cells or from pig liver was able to phosphorylate heat-inactivated virions. In addition to the major phosphoproteins, active virion preparations were able to phosphorylate a minor low molecular weight phosphoprotein, incorporation into which could be stimulated by the addition of cyclic AMP to the assay. Purified host cyclic AMP-dependent protein kinase also phosphorylated this protein in heat-inactivated virions. Analysis of herpes simplex virus type 1 showed that the major phosphoproteins (VP12 and VP23) could be phosphorylated in heat-inactivated virions by added casein kinase II. One of these (VP12) together with a further minor phosphoprotein (VP 14) could be phosphorylated by cyclic AMP-dependent protein kinase.

Cytoplasmic fractions from normal baby hamster kidney fibroblasts and from fibroblasts infected with pseudorabies virus were fractionated by DEAE-cellulose chromatography and fractions assayed for protein kinase activity. In preparations from uninfected and infected cells protein kinase activities identified as casein kinase I and 11, the two isoforms of the cyclic-AMP-dependent protein kinase, protein kinase C, and a presumed protcolytic fragment of protein kinase C were present in comparable amounts. However in infected cells a new protein kinase activity was detected, appearing about 4 h after infection and increasing during the following 6 h at least. This new protein kiiiase was purified 100-fold by high-performance gel-permeation and ion-exchange chromatography, and characterized. It has an apparent relative molecular mass of 68000 on the basis of gel-permeation chromatography, and a sedimentation coefficient of 4.3 S. It catalysed the phosphorylation of serine residues of basic proteins in vitro, with protamine a better substrate than mixed histones; and used ATP (apparent K, = 60 pM), but not GTP, as phosphoryl donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, C a 2 + + calmodulin, C a 2 + + phospholipid, double-stranded RNA, and heparin) did not significantly alter the activity of this enzyme. A distinguishing characteristic of the protein kinase was a high KCI concentration optimum with the persistence of activity up to 800 mM KCI, at least.The phosphorylations catalysed by phosphoprotein kinases have been shown to be of great importance in the regula-. tion of normal cellular metabolism. The possibility arises that protein kinases may also play a role in the altered metabolism of cells infected with certain animal viruses. In the case of thc complex herpes viruses, infected cells show a profile of phosphorylated proteins which differs markedly from that in uninfected cells [l]. The phosphorylation encompasses both proteins absent from uninfected cells (generally virally coded proteins) [l -41, and pre-existing host-cell proteins [5, 61. If such phosphorylations were part of the infective strategy of the virus one could conceive of their catalysis -at least in some cases -by virally coded enzymes, and the possible existence of such viral protein kinases has, in fact, been suggested by the finding of protein kinase activity associated with the purified virions of several herpes viruses [7 -1 I].In the studies cited above, the protein kinases associated with the virion were not purified. Hence it is not possible to conclude whether they differ from the host enzymes which have been shown to be associated with virions [12]. The reasons for this lack of purification are the difficulties of extraction and quantity of starting material presented by virions. It therefore seemed prudent to turn to the cytoplasm of infected cells (where some, at least, of the new phosphorylations occur) to look for new or altered proteinCorrespoizdmce to D. P. Leader, Department of ...

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Ord & Stocken (1966) have shown that [32P]_ phosphate injected into rats is incorporated into the histones of the thymus gland. It has now been found that incorporation also takes place in vitro. Rat-thymus nuclei from six to eight rats of 120-140g. body wt. were prepared as described by Ord, Raaf, Smit & Stocken (1965). These were suspended in 10ml. of the isolation medium

1. The capacity of the histone-DNA complex to act as a template for RNA synthesis is increased by phosphorylation of histone F1. 2. In regenerating liver, DNA synthesis is preceded by phosphorylation of histone F1. 3. Exposure to ionizing radiation in vivo decreases and delays the phosphorylation of histone F1 in regenerating liver, and decreases it in the hyperplastic kidney. 4. Histone F1 is phosphorylated to a greater extent in dividing than in resting tissues.