The phosphorylation of ribosomal protein S6 by protein kinases from cells infected with pseudorabies virus.

We examined the ability of protein kinase activities from BHK (baby-hamster kidney) cells infected with pseudorabies virus to catalyse the phosphorylation of ribosomal protein S6 in vitro. When the cytosol from infected cells was fractionated on DEAE-cellulose, 40S ribosomal protein kinase activity was found associated with the two isoforms of the cyclic AMP-dependent protein kinase, protein kinase C and a protein kinase (ViPK, virus-induced protein kinase) only detected in infected cells. The phosphorylation of ribosomal protein by ViPK was of particular interest because the appearance of the protein kinase and the increase in the phosphorylation of protein S6 in infected cells shared a similar time course. At moderate concentrations of KCl the major ribosomal substrate for ViPK was ribosomal protein S7, a protein not found to be phosphorylated in vivo. However, at 600 mM-KCl, or in the presence of 5-10 mM-spermine at 60-150 mM-KCl, the phosphorylation of ribosomal protein S7 was suppressed and ribosomal protein S6 became the major substrate. The maximum stoichiometry of phosphorylation obtained under the latter conditions was 1-2 mol of phosphate/mol of S6, and only mono- and di-phosphorylated forms of S6 were detected on two-dimensional gel electrophoresis. As the infection of BHK cells by pseudorabies virus results in the appearance of phosphorylated species of S6 containing up to 5 mol of phosphate/mol of S6 protein, it appears unlikely that ViPK alone can be responsible for the multiple phosphorylation seen in vivo. Nevertheless, tryptic phosphopeptide analysis did indicate that in vitro ViPK catalysed the phosphorylation of at least one of the sites on ribosomal protein S6 phosphorylated in vivo, so that a contributory role for the enzyme in the phosphorylation in vivo cannot be excluded.

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.

Partial purification and characterization of a new phosphoprotein kinase from cells infected with pseudorabies virus.

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 II, the two isoforms of the cyclic-AMP-dependent protein kinase, protein kinase C, and a presumed proteolytic 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 kinase was purified 100-fold by high-performance gel-permeation and ion-exchange chromatography, and characterized. It has an apparent relative molecular mass of 68 000 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 Km = 60 microM), but not GTP, as phosphoryl donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, Ca2+ + calmodulin, Ca2+ + 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 KCl concentration optimum with the persistence of activity up to 800 mM KCl, at least.

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.

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.

Synthesis and methylation of ribosomal RNA in HeLa cells infected with the herpes virus pseudorabies virus.

The effects of infection with the herpes virus pseudorabies virus on the metabolism of HeLa cell ribosomal RNA were examined. There is a decline both in the synthesis of nucleolar 45S ribosomal precursor RNA and in its processing to mature cytoplasmic RNA. The methylated oligonucleotides in the ribosomal RNA species were studied. The methylation of cytoplasmic ribosomal RNA was essentially unchanged. However there was some undermethylation of the nucleolar precursor. If undermethylated RNA does not mature then this may partly explain the reduced processing in the infected cells.

The effect of herpesvirus infection on ribosomal RNA synthesis and on nucleolar size and number in HeLa cells.

The effects of infection by the herpesvirus pseudorabies virus on the nucleolus and on ribosomal RNA synthesis in HeLa cells were examined. The size of individual nucleoli and the total nucleolar content per cell are reduced to approximately 35 per cent of normal by 6 hours of infection. The proportion of cells containing one nucleolus rises from 10 to 50 per cent in the same time. The synthesis of ribosomal precursor RNA declines to approximately 40 per cent in this period.

Phosphorylation of ribosomal proteins in hamster fibroblasts infected with pseudorabies virus or herpes simplex virus.

In BHK cells infected with pseudorabies virus, there was a substantial increase in the phosphorylation of ribosomal protein S6. This increase occurred between 2 and 4 h after infection and persisted at least until 9 h. We estimated that in mock-infected cells S6 contained, on an average, one phosphate group per protein chain, whereas in infected cells this rose to between four and five phosphate groups per protein chain. A second ribosomal protein, either S16 or S18, was also phosphorylated after infection. No increase in cyclic AMP was found at the time of phosphorylation. We also found an increased phosphorylation of S6 in herpes simplex virus-infected BHK cells.

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.

Inverted repetition in the chromosome of pseudorabies virus.

An electron microscope examination of pseudorabies virus DNA single strands after self-annealing shows a loop of single-stranded DNA at one end of the molecule contiguous to a double-strand region. The molecule then terminates in a further single-stranded region that does not form a loop. It is suggested that the DNA contains a sequence of 13.3 x 106 daltons at one end, which is repeated internally with opposite polarity. The segment of the genome separating the repeats has a double-strand molecular weight of 5.4 x 106. The whole native DNA has a molecular weight of 90 x 106 to 95 x 106.

Virus-induced proteins in pseudorabies-infected cells. II. Proteins of the virion and nucleocapsid.

Analysis of purified naked and enveloped nucleocapsids of pseudorabies virus with high-resolution techniques has allowed a reassessment of their protein composition. Enveloped particles are shown to contain at least 20 proteins whose molecular weights are in the range 20,000 to 230,000. Naked nucleocapsids contain one major and seven minor proteins in the molecular weight range 20,000 to 155,000. Phosphorylation of at least one virion protein is shown to take place in vivo. These results demonstrate that pseudorabies virus is similar in its protein complement to other herpesviruses which have recently been examined.

Virus-induced proteins in pseudorabies-infected cells. I. Acid-extractable proteins of the nucleus.

Three basic proteins have been identified in chromatin preparations from pseudorabies virus-infected cells. They appear to be virus specified and are similar in size and charge to host histones; one difference however is that they contain tryptophan. All are produced by 3 h postinfection, and two (IP II and III) seem to be arginine rich. Three similar proteins are also found in herpes simplex MP 17-infected cells, and two of these co-electrophorese with two of the pseudorabies proteins. Partially purified preparations of pseudorabies virus contain low amounts of all three proteins.

Variations in the phosphate content of histone F1 in normal and irradiated tissues.

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.