Vaccinia virus morphogenesis is interrupted when expression of the gene encoding an 11-kilodalton phosphorylated protein is prevented by the Escherichia coli lac repressor.

A conditional lethal vaccinia virus mutant, which constitutively expresses the Escherichia coli lac repressor and has the lac operator controlling the F18R gene (the 18th open reading frame of the HindIII F fragment of the vaccinia virus strain WR genome) encoding an 11-kDa protein, was previously shown to be dependent on the inducer isopropyl-beta-D-thiogalactoside (IPTG) for replication (Y. Zhang and B. Moss, Proc. Natl. Acad. Sci. USA 88:1511-1515, 1991). Further studies indicated that the yield of infectious virus could be regulated by titration with IPTG and that virus production was arrested by IPTG removal at appropriate times. Under nonpermissive conditions, an 11-kDa protein reactive with antiserum raised to a previously described DNA-binding phosphoprotein (S. Y. Kao and W. R. Bauer, Virology 159:399-407, 1987) was not synthesized, indicating that the latter is the product of the F18R gene. In the absence of IPTG, replication of viral DNA and the subsequent resolution of concatemeric DNA molecules appeared normal. Omission of IPTG did not alter the kinetics of early and late viral protein synthesis, although the absence of the 11-kDa polypeptide was noted by labeling infected cells with [35S]methionine or [32P]phosphate. Pulse-chase experiments revealed that proteolytic processing of the major viral structural proteins, P4a and P4b, was inhibited under nonpermissive conditions, suggesting a block in virus maturation. Without addition of IPTG, the failure of virus particle formation was indicated by sucrose gradient centrifugation of infected cell lysates and by the absence of vaccinia virus-mediated pH-dependent cell fusion. Electron microscopic examination of infected cells revealed that immature virus particles, with aberrant internal structures, accumulated when synthesis of the 11-kDa DNA-binding protein was prevented.     

Identification of Phosphorylation Sites within the Herpes Simplex Virus Tegument Protein VP22

The herpes simplex virus protein VP22 is a major phosphoprotein of infected cells. In this study, we identify two serine phosphorylation sites within VP22 and show that the N-terminal site is a substrate for casein kinase II, while the extreme C-terminal site is a substrate for another, as yet unidentified, cellular kinase. Furthermore, we show that a mutant of VP22 which has both sites altered is unable to incorporate phosphate in vivo, confirming that there are no other phosphorylation sites within VP22.     

Phosphorylation of ribosomal proteins in rat cerebral cortex in vitro.

Incubation of cerebral cortical tissue from immature rats in the presence of [32P]orthophosphate resulted in similar rates of incorporation of radioactivity into the proteins of free and membrane-bound ribosomes. Incorporation of label into ribosomal proteins of both species continued actively for at least 3 hours. Since recovery of membrane-bound ribosomes from rat cerebral cortex is quite low, further analyses of the radioactive phosphoproteins were restricted to the free ribosome population. A significant fraction of the radioactivity which was precipitated with trichloroacetic acid was not removed by heating in trichloroacetic acid at 90 degrees or extracted with organic solvents and therefore was presumed to be covalently bound to protein. The radioactive phosphoryl groups present in the ribosomal proteins were mainly in ester linkages since they were readily removed by exposure to 1 N NaOH, relatively unaltered by 1N HCl, and unaffected by hydroxylamine. This conclusion was supported by the isolation of labeled o-phosphoserine and o-phosphothreonine residues from hydrolysates of ribosomal proteins. A significant fraction of the labeled phosphoproteins in the purified ribosomes appeared to be bound tightly to the ribosome structure since only 40% of the radioactivity could be removed by extraction of these ribosomes with 1 M KCl. Phosphorylation of proteins of cerebral monoribosomes was more rapid than the same process in polyribosomes from the same source. Eight radioactive phosphoprotein bands could be detected by electrophoresis of proteins obtained from unfractionated cerebral ribosomes on unidimensional polyacrylamide gels containing sodium dodecyl sulfate. The protein nature of these materials was confirmed by pronase digestion. Proteins of subribosomal particles isolated from the total free ribosomal population were labeled differentially. When dissociation was carried out in the presence of EDTA, the small subunit contained four radioactive phosphoprotein bands, whereas the large subunit contained five. Three of the radioactive phosphoprotein components of the small subunit were removed when dissociation of cerebral ribosomes which were previously washed with high salt media was carried out in the presence of puromycin and high salt. However, only the largest labeled phosphoprotein band of the large subunit was removed by this procedure. This component exhibited the same electrophoretic mobility as one of the radioactive phosphoprotein bands which was removed from the small subunit by high salt treatment..     

Characterization of a protein kinase and two phosphate acceptor proteins from vaccinia virions.

The phosphorylation of two purified vaccinia virus proteins (Acceptors I and II) by a protein kinase isolated from vaccinia virus cores has been studied. Phosphorylation of viral acceptor proteins by the purified enzyme was dependent on the presence of ATP, Mg2+, and protamine or other basic proteins, and was maximal at alkaline pH values. Cyclic mononucleotides did not stimulate the vaccinia protein kinase under a variety of conditions. Protamine, however, was shown to function as an enzyme activator. In its presence, the purified vaccinia protein kinase phosphorylated mainly serine residues in Acceptor I, and predominantly threonine residues in Acceptor II. Phosphorylation of protamine accounted for less than 1% of the total 23P incorporation. Tryptic peptide maps prepared from 32P-labeled Acceptors I and II demonstrated that they contained different labeled peptide sequences and were, therefore, distinct protein species. From additional studies on both purified and virus-associated protein kinase it was concluded that various proteins affected the protein kinase reaction in one of three ways. One class of proteins served as phosphate acceptors, but only when another activator protein was present. A second class consisted of proteins that were strong activators but poor phosphate acceptors. The third class contained proteins that were fair phosphate acceptors, but which also activated the phosphorylation of other acceptor proteins.     

Serine protein kinase activity associated with rotavirus phosphoprotein NSP5.

The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine. We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11. Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity. After metabolic labeling of NSP5 with 32Pi, only serine residues were phosphorylated. Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products. Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified. Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa. Moreover, there were polypeptides migrating between 28 and 35 kDa. Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28- to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation. Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A. The immunoprecipitated NSP5 possessed protein kinase activity. Incubation with [gamma-32P]ATP resulted in 32P labeling of 28- to 35-kDa NSP5. The distribution of 32P radioactivity between the components of the complex was similar to the phosphorylation in vivo. Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.     

Synthesis, posttranslational modifications, and nuclear transport of polyomavirus major capsid protein VP1.

Polyomavirus major capsid protein VP1 synthesis was studied in infected primary baby mouse kidney cells. A standard curve of VP1 protein was used to quantitate VP1 in the cytoplasm and nucleus of infected cells during the time course of infection. Polyomavirus VP1 continued to be accumulated in the cytoplasm of the cells until 27 h postinfection, at which time the synthesis of VP1 leveled off. VP1 continued to accumulate in the nucleus of the infected cells throughout the course of infection. The presence of the six isospecies, A to F, of polyomavirus VP1 was also studied to determine the relative quantity of each species during the time course of infection. All six species were found in the cytoplasm and nucleus of infected cells at various times postinfection. However, the relative quantity of each species was different at early as compared with later times of infection. In addition, phosphorylated VP1 was found in isolated polyribosomes of infected cells, suggesting that phosphorylation of VP1 is a cotranslational modification. Examination of the effect of macromolecular synthesis on the transport of VP1 into the nucleus of infected baby mouse kidney cells as well as the rate of its nuclear accumulation during and after protein synthesis inhibition revealed that the continual transport and accumulation of VP1 in the nucleus required protein synthesis.     

Temporal regulation of herpes simplex virus type 2 VP22 expression and phosphorylation

The VP22 protein of herpes simplex virus type 2 (HSV-2) is a major component of the virion tegument. Previous work with HSV-1 indicated that VP22 is phosphorylated during infection, and phosphorylation may play a role in modulating VP22 localization in infected cells. It is not clear, however, when phosphorylation occurs in infected cells or how it is regulated. Less is known about the synthesis and phosphorylation of HSV-2 VP22. To study the complete biosynthetic history of HSV-2 VP22, we generated a monoclonal antibody to the carboxy terminus of VP22. Using immunoprecipitation and Western blot analyses, we show that HSV-2 VP22 can be found in three distinct isoforms in infected cells, two of which are phosphorylated. Like HSV-1 VP22, HSV-2 VP22 is synthesized ca. 4 h after infection, and the isoform later incorporated into virions is hypophosphorylated. In addition, we demonstrate for the first time (i) that newly synthesized VP22 is phosphorylated rapidly after synthesis, (ii) that this phosphorylation occurs in a virus-dependent manner, (iii) that the HSV-2 kinase UL13 is capable of inducing phosphorylation of VP22 in the absence of other viral proteins, (iv) that phosphorylated VP22 is very stable in infected cells, (v) that phosphorylated isoforms of VP22 are gradually dephosphorylated late in infection to produce the virion tegument form, and (vi) that this dephosphorylation occurs independently of viral DNA replication or virion assembly. These results indicate that HSV-2 VP22 is a stable protein that undergoes highly regulated, virus-dependent phosphorylation events in infected cells.     

Analysis of the protein-kinase activity of Escherichia coli cells.

When growing E. coli cells in a minimal medium containing radioactive orthophosphate, several proteins appear to be significantly labeled after analysis by polyacrylamide gel electrophoresis and autoradiography : at least four in the soluble cellular fraction, five in crude ribosomes and one in salt-washed ribosomes. In all cases, phosphorylation occurs at the level of threonine and serine residues as shown by paper electrophoresis of acid hydrolysates of these proteins. It is concluded that bacteria do contain a protein kinase activity.     

A nucleolus-specific phosphoprotein in mouse ascites tumor cells

Analysis of proteins in nucleoli and chromatin of mouse ascites tumor cells labeled with [³²P]orthophosphate by SDS-polyacrylamide gel electrophoresis showed that a highly radioactive protein was localized in the nucleoli. This protein was purified and the final preparation appeared as a single component on hydroxylapatite column chromatography with or without SDS. This protein was found to be a nucleolus-specific phosphoprotein with a molecular weight of 120 000. The phosphate moiety in this protein turned over very rapidly whereas the protein itself was stable. When the nucleoli were disrupted by EDTA treatment, this unique protein was found as a major protein constituent of the ultracentrifugal supernatant.     

Phosphoprotein Component of Vaccinia Virions

The recent discovery of a protein kinase activity in vaccinia virions led us to search for a viral protein which is phosphorylated in vivo. Vaccinia virus was radioactively labeled by infecting cells in the presence of (32)P(1). A phosphoprotein was isolated from purified delipidated virions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The phosphoprotein appeared to be a specific viral component induced after infection. More than 60% of the phosphoprotein was associated with viral cores. The electrophoretic mobility of the protein suggested that it has a molecular weight of 11,000 to 12,000. Phosphoserine was liberated by acid hydrolysis and identified by electrophoresis with known standards. Tryptic digests of the purified phosphoprotein were analyzed by two-dimensional electrophoresis and chromatography on thin-layer cellulose plates, and a single major phosphopeptide was resolved. The high selectivity of phosphorylation suggested that the process has a specific function.     

The vaccinia virus A33R protein provides a chaperone function for viral membrane localization and tyrosine phosphorylation of the A36R protein

The products of the A33R and A36R genes of vaccinia virus are incorporated into the membranes of intracellular enveloped virions (IEV). When extracts of cells that had been infected with vaccinia virus and labeled with H(3)(32)PO(4) were immunoprecipitated with antibodies against the A33R protein, two prominent bands were resolved. The moderately and more intensely labeled bands were identified as phosphorylated A33R and A36R proteins, respectively. The immunoprecipitated complex contained disulfide-bonded dimers of A33R protein that were noncovalently linked to A36R protein. Biochemical analysis indicated that the two proteins were phosphorylated predominantly on serine residues, with lesser amounts on threonines. The A36R protein was also phosphorylated on tyrosine, as determined by specific binding to an anti-phosphotyrosine antibody. Serine phosphorylation and A33R-A36R protein complex formation occurred even when virus assembly was blocked at an early stage with the drug rifampin. Tyrosine phosphorylation was selectively reduced in cells infected with F13L or A34R gene deletion mutants that were impaired in the membrane-wrapping step of IEV formation. In addition, tyrosine phosphorylation was specifically inhibited in cells infected with an A33R deletion mutant that still formed IEV. Immunofluorescence and immunoelectron microscopy indicated that in the absence of the A33R protein, the A36R protein was localized in Golgi membranes but not in IEV. In the absence of the A36R protein, however, the A33R protein still localized to IEV membranes. These studies together with others suggest that the A33R protein guides the A36R protein to the IEV membrane, where it subsequently becomes tyrosine phosphorylated as a signal for actin tail formation.     

Identification of induced protein kinase activities specific for the ribosomal proteins uniquely phosphorylated during infection of HeLa cells with vaccinia virus

We have examined the ribosomal protein kinase activities in partially purified cytoplasmic extracts from HeLa cells infected with vaccinia virus. We found an activity or activities, absent from mock-infected cells, that was capable of phosphorylating the proteins S2 and S13 in vitro. The ribosomes phosphorylated in vitro exhibited the same multiple phosphorylation of S2 found in vivo, at least 3 phosphoryl residues being seen, and the same mono-phosphorylation of S13. Also as in vivo, ribosomal protein S2 contained phosphothreonine as well as phosphoserine, whereas S13 contained only phosphoserine. This strongly suggests that these new protein kinase activities are responsible for the ribosomal protein phosphorylations that occur during infection with vaccinia virus.     

Stoichiometry of phosphorylation of hepatic ATP-citrate lyase by protein kinase

Hepatic ATP-citrate lyase prepared with a fluoride-free step to allow endogenous phosphatases to dephosphorylate the enzyme was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase and [γ-³²P]ATP. After electrophoresis the radioactive phosphate was located predominantly in the gel slice containing the Coomassie blue stained protein corresponding to ATP-citrate lyase. The Stoichiometry of phosphorylation of hepatic ATP-citrate lyase in vitro by the catalytic subunit was such that 0.53 ± 0.02 molecules of phosphate were incorporated per subunit. The degree of phosphorylation was independent of the amount of ATP-citrate lyase present as substrate in the concentration range 1.2–6.4 μm. In the absence of catalytic subunit there was very little labeled phosphate incorporated into ATP-citrate lyase. Phosphorylation of ATP-citrate lyase by catalytic subunit was abolished by the specific protein inhibitor of cyclic AMP-dependent protein kinase. When ATP-citrate lyase was subjected to electrophoresis under nondenaturing conditions, lyase activity was recovered from the gel slice corresponding to the Coomassie blue staining phosphoprotein of a stained gel run in parallel.     

Phosphorylation in vivo and in vitro of the arginine-ornithine periplasmic transport protein of Escherichia coli

The arginine-ornithine periplasmic binding protein, an essential component of the arginine-ornithine transport system of Escherichia coli, was isolated in a phosphorylated form and in a non-phosphorylated form from the periplasmic fluid, after incubation of intact cells with (32P)orthophosphate under conditions similar to those used for arginine transport studies. The binding protein could also be labeled with 32Pi by incubation in vitro of the periplasmic fluid with [gamma-32P]ATP, or by incubation in vitro of the purified binding protein with radioactive ATP, Mg2+ and a phosphokinase enzyme released by osmotic-shock treatment. The two forms of the protein were separated by DEAE-Sephacel chromatography. By several different criteria, which included binding studies, analyses of the amino acid composition of the two forms of the protein, analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and testing for other components of the periplasmic space with affinities for inorganic phosphate, it was concluded that the 32P-labeled protein corresponds to a phosphorylated form of the arginine-ornithine-binding protein. The phosphorylation reaction required Mg2+ and a phosphokinase from the periplasmic fluid. The dissociation constant of the phosphorylated protein for arginine was 5.0 microM (dissociation constant of the unmodified protein equals 0.1 microM), suggesting that the chemically modified protein is the active form of the molecule which releases the ligand for its translocation through the cytoplasmic membrane. The pH-stability profile of the phosphoprotein has a 'U'-shape characteristic of acyl phosphates. Reaction of the phosphorylated binding protein with hydroxylamine at pH 5.4, also released Pi from the phosphoprotein. These properties suggest that the phosphoryl group of the phosphoprotein is linked covalently to a carboxyl function of the protein. This information indicates that ATP is a direct energy donor for the active transport of arginine and ornithine in E. coli, and a step of phosphorylation of the arginine-ornithine-binding protein appears to be involved in the utilization of the phosphate bond energy by the arginine-ornithine transport system.     

Inhibition of the Peptide Bond Synthesizing Cycle by Chloramphenicol

To study the mechanism by which chloramphenicol inhibits bacterial protein synthesis, we examined the kinetics of the puromycin-induced release of peptides from transfer ribonucleic acid (tRNA) in the presence and in the absence of chloramphenicol. Washed Escherichia coli ribosomes with nascent peptides which had been radioactively labeled in vivo were used for this study. When such ribosomes were incubated in the presence of 10 mug of puromycin per ml, approximately one-fourth of the radioactive peptide material was rapidly released from tRNA. This rapid, puromycin-dependent reaction is assumed to be equivalent to the peptidyl transferase reaction. Chloramphenicol inhibited the extent of the puromycin-induced release of peptides by only 50%, demonstrating that some of the peptide chains which are present on active ribosomes react with puromycin, even in the presence of chloramphenicol. The addition of the supernatant fraction and guanosine triphosphate (GTP) increased the extent of the puromycin-induced release; this additional release was completely inhibited by chloramphenicol. Peptidyl chains on washed ribosomes prepared from chloramphenicol-inhibited cells were not released by puromycin in the presence of chloramphenicol and reacted slowly with puromycin in the absence of chloramphenicol. The release of peptidyl groups from these ribosomes became largely insensitive to chloramphenicol after preincubation of the ribosomes with GTP and the supernatant fraction. We conclude that chloramphenicol does not inhibit the peptidyl transferase reaction as measured by the puromycin-induced release of peptides from tRNA, but rather inhibits some step in the peptide synthesis cycle prior to this reaction.     

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.     

A polysome-membrane binding system from rat liver. I. Basic characterization of the binding system.

A simple reaction system was developed to examine the binding of polysomes to membranes of the endoplasmic reticulum and to investigate the fate of ribosomes and nascent chains during protein synthesis in vitro. The system conssited of Sephadex G-25 treated post-mitochondrial fraction prepared from rat liver (Sephadex-PM) as a source of membranes, and radioactive free polysomes prepared from another rat liver. The following results were obtained. 1. Nascent chains on free polysomes labeled in vivo were transferred to membranes in vitro. The process required protein synthesis. 2. This reaction occurred in two steps: a) Binding of the free polysomes to membranes in the absence of protein synthesis. b) Release of ribosomes, leaving nascent chains on the membranes, requiring protein syntehsis. 3. A portion of the ribosomes found on membranes in vivi (membrane-bound ribosomes) was also released from the membranes during incubation in vitro, leaving their nascent chains on the membranes. The significance of the transfer of nascent chains from free polysomes to membranes in vitro is discussed in the light of known polysome-membrane interaction in vivo.     

Vaccinia virus recombinants expressing the SA11 rotavirus VP7 glycoprotein gene induce serotype-specific neutralizing antibodies.

A cDNA copy of the gene coding for the major outer neutralizing protein (VP7) of simian 11 rotavirus was incorporated into the vaccinia virus genome under the control of the vaccinia promoter (molecular weight, 7,500). A deletion mutant of this gene which codes for a secreted form of VP7 when expressed under the control of the simian virus 40 late promoter (M. S. Poruschynsky, C. Tyndall, G. W. Both, F. Sato, A. R. Bellamy, and P. H. Atkinson, J. Cell Biol. 101:2199-2209, 1985) was also inserted. Each recombinant vaccinia virus directed the synthesis of a rotavirus protein in infected cells, and the product encoded by the mutated gene was secreted. Rabbits immunized with the two types of recombinant vaccinia virus generated antibodies that were able both to recognize simian 11 rotavirus in an enzyme-linked immunosorbent assay and to neutralize the virus in a plaque-reduction test. Antibodies induced by the recombinant vaccinia viruses expressing either form of VP7 were serotype specific.     

Calcium gradient-dependent and calcium gradient-independent phosphorylation of sarcoplasmic reticulum by orthophosphate. The role of magnesium.

Phosphorylation of the calcium-transport ATPase of skeletal muscle sarcoplasmic reticulum by inorganic phosphate was investigated in the presence or absence of a calcium gradient. The maximum phosphoprotein formation in the presence of a calcium gradient at 20 degrees C and pH 7.0 is approximately 4 nmol/mg sarcoplasmic reticulum protein, but only between 2.4 and 2.8 nmol/mg protein in the absence of a calcium gradient, using Ionophore X-537 A or phospholipase-A-treated sarcoplasmic reticulum vesicles. Maximum phosphoprotein formation independent of calcium gradient at 20 degrees C and pH 6.2 is in the range of 3.6--4 nmol/mg protein. Half-maximum phosphoprotein formation dependent on calcium gradient was achieved with 0.1--0.2 mM free orthophosphate at 10 mM free magnesium or at 0.1--0.2 mM free magnesium at 10 mM free orthophosphate. Phosphoprotein formation independent of calcium gradient is in accordance with a model which assumes, firstly, the formation of a ternary complex of the ATPase protein with orthophosphate and magnesium (E . Pi . Mg) in equilibrium with the phosphoprotein (E-Pi . Mg) and, secondly, an interdependence of both ions in the formation of the ternary complex. The apparent equilibrium constant was 0.6 and the apparent dissociation constants KMg, KMg', KPi and KPi' were 8.8, 1.9, 7.2 and 1.5 mM respectively, assuming a total concentration of the phosphorylation site per enzyme of 7 nmol/mg protein.     

Enhanced phosphorylation of ribosomal protein s6 and other cytoplasmic proteins after mengovirus infection of Ehrlich ascites tumor cells.

After mengovirus infection of Ehrlich ascites tumor cells, an increased incorporation of radioactive phosphate into ribosomal protein S6 was detected. As judged from the shape of the radioautography spot and the location of the modified protein on the two-dimensional polyacrylamide gel, it must be concluded that more than one phosphate group was incorporated into ribosomal protein S6. Concomitantly, multiple cytoplasmic proteins, particularly those with high affinity for ribosomes, were phosphorylated. Compared with the controls (mock infection), mengovirus infection induced only a slight additional phosphorylation of the proteins of the ribosome-free supernatant. Employing the radio isotope dilution test with 3', 5'-cyclic AMP-binding protein, no enhancement of the intracellular concentration of 3', 5'-cyclic AMP in response to mengovirus infection could be deomonstrated.