William S. Stevely

PROTEIN-KINASE ACTIVITIES ASSOCIATED WITH THE VIRIONS OF PSEUDORABIES AND HERPES-SIMPLEX VIRUS

Protein kinase has been extracted in soluble form from virions of pseudorabies virus using 10% NP40, 0.6 M-NaCl. Chromatographic analysis of the extract on DEAE-cellulose and on phosphocellulose showed it to contain more than one kinase. The activity responsible for the phosphorylation of the major phosphoproteins (mol. wts. 120 000, 115 000 and 72 000) of virions was found to be similar in its properties to the host enzyme casein kinase II. 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 (VP14) could be phosphorylated by cyclic AMP-dependent protein kinase.

Characteristics of the Induction of a New Protein Kinase in Cells Infected with Herpesviruses

The appearance of a recently described protein kinase activity (virus-induced protein kinase, ViPK) has been studied during infection of hamster fibroblasts with pseudorabies virus or with herpes simplex virus type 1 (HSV-1). An enzyme activity with comparable catalytic properties was induced in both cases, and had broadly similar kinetics of appearance to that of the viral DNA polymerase. The amount of active ViPK detected depended on the multiplicity of infection, and no ViPK was induced after the viruses had been subjected to irradiation with u.v. light. When cells were infected with the tsK mutant of HSV-1, ViPK was induced at the permissive but not at the restrictive temperature. The ViPK preparations obtained from cells infected with each virus differed in chromatographic properties on anion-exchange and gel-permeation resins. These results indicate that expression of the viral genome is required for induction of ViPK. They suggest that the enzyme may be encoded by the viral genome, but do not provide proof of this.

The phosphorylation of ribosomal protein S6 in hamster fibroblasts infected with pseudorabies virus inactivated by ultraviolet radiation.

Infection of baby hamster fibroblasts with pseudorabies virus at high multiplicities resulted in a substantial increase in the phosphorylation of ribosomal protein S6. However, the phosphorylation was still observed with virus that had been completely inactivated by u.v. irradiation. We therefore conclude that expression of the viral genome is not required for the virus to elicit this effect.

The characteristics of the cell-free translation of mRNA from cells infected with the herpes virus pseudorabies virus.

The translation in vitro of mRNA from pseudorabies virus infected cells was studied using systems derived from wheat germ and from rabbit reticulocyte. The mRNA was shown by molecular hybridization to contain sequences complementary to virus DNA. Products of in vitro translation co-migrating with virus proteins on polyacrylamide gel electropherograms were detected and the major immune precipitation. Optimum conditions for the stimulation of amino acid incorporation in vitro were determined and found to be similar for mRNA from both infected and mock-infected cells.

The effect of hypertonic conditions on protein synthesis in cells infected with herpes virus

When HeLa cells are placed in a growth medium containing an elevated concentration of sodium chloride there is a rapid cessation of protein synthesis accompanied by a complete breakdown of polyribosomes [I]. However protein synthesis in HeLa cells infected with poliovirus, vesicular stomatitis virus or vaccinia virus is relatively resistant to hypertonic medium [2,3]. In such infected cells it is well established that there is, in normal medium, an inhibition of host protein synthesiswith preferential synthesis of virus-specified proteins. Thus a parallel was drawn between the selective effect on initiation of increased sodium chloride and the suppression of host protein synthesis by these viruses. A similar examination of simian virus 40-infected cells [4] indicated that in this case virus-infected cell protein synthesis was again more resistant to hypertonic medium than protein synthesis in uninfected cells. In addition to these experiments in vivo cell-free protein synthesising systems have also been used to examine the mechanism of inhibition of host protein synthesis by virus. These indicated that for some virus messenger RNA species addition of sodium to the cellfree system inhibited the initiation of translation of host protein synthesis whereas it was stimulatory for the translation of the virus messenger RNA [ 5,6]. On the basis of these and other studies a general mechanism was proposed [6] for the inhibition of cell functions following virus infection. It was suggested that on infection the cell membrane is damaged and that this leads to an alteration in ion transport

HSV-1 infection inhibits procollagen and protein secretion from normal and ataxia telangiectasia cultured skin fibroblasts.

Human skin fibroblasts derived from a healthy individual and from a child with the genetic disorder ataxia telangiectasia were infected with herpes simplex virus type 1 (HSV-1). The virus infection did not affect the synthesis of procollagen but inhibited its release from the cells.