Herpes simplex virus type-1 immediate-early gene expression and shut off of host protein synthesis are inhibited in neomycin-treated human epidermoid carcinoma 2 cells

Infection of human epidermoid carcinoma-2 (HEp-2) cells by Herpes simplex virus type 1 (HSV-1) leads to significant activation of inositol phospholipid turnover after 15 min. The effect of neomycin, an inhibitor of inositol phospholipid turnover, has been investigated for its effect on HSV-1 multiplication in HEp-2 cells. HSV-1 multiplication is inhibited by neomycin. This inhibition is not due to a block of virus adsorption or penetration. Neomycin inhibits the expression of virus immediate-early genes, as well as expression of early genes and viral DNA synthesis. In neomycin-treated cells, the usual virion-associated shut off of host protein synthesis does not occur. These results indicate that the inositol phospholipid pathway is involved in immediate-early gene expression and shut off of host protein synthesis in HEp-2 cells.     

Ribosome and protein synthesis modifications after infection of human epidermoid carcinoma cells with herpes simplex virus type 1

Summary Modifications of ribosomes have been investigated in human epidermoid carcinoma-2 cells at different stages of herpes simplex virus type 1 infection. Very early in infection, there is an increase in ribosomal protein S6 phosphorylation even in the absence of serum. The same result is obtained in the presence of actinomycin D. At early infection time, ribosomal proteins S2, S3a and Sa are newly phosphorylated. At early and early-late times, three phosphorylated non-ribosomal proteins (v1, v2 and v3) are differently associated temporally to ribosomes. Analyses of proteins extracted from 40S subunits, 80S ribosomes and polysomes show that v1 and v2 are distributed differently among the different ribosomal populations. S6 phosphopeptides were found to be identical after serum stimulation and after viral infection. In every case phosphoserine and phosphothreonine were identified in S6. Only phosphoserine was found in other phosphorylated proteins. Our results indicate that herpes simplex virus type 1 is able to modify pre-existing ribosomes: (i) by stimulating a pre-existing kinase for S6 phosphorylation even in the absence of serum and of viral genome expression; (ii) by inducing new specific kinase activity(ies); and (iii) by association of new, phosphorylated proteins to ribosomes. These ribosomal modifications are correlated with changes in protein synthesis, as shown by two-dimensional electrophoretic analyses of newly synthesized ³⁵S-labelled proteins.     

Regulation of ribosomal protein S6 phosphorylation in heat-shocked HeLa cells

Decreases in energy charge, ribosomal protein phosphorylation and rate of protein synthesis are well-documented facets of the cellular response to hyperthermia in non-vertebrates. We have tried to reproduce this response pattern in 32P-labelled HeLa cells in order to investigate the hypothetical causal relationship between these effects. In HeLa cells shifted from 36 degrees C to 42 degrees C, dephosphorylation of S6 and inhibition of protein synthesis, owing to a decreased initiation rate, were observed, but could not have been mediated by changes in the cells' general energy charge since the ATP and GTP levels were not reduced. In addition, we found that the hyperthermic translation block developed faster than the overall dephosphorylation of S6, showing that S6 dephosphorylation cannot be responsible for the translation block unless site-specific effects play a critical role.     

Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts

The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.     

Herpes simplex virus type-1-induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type I IFN secretion

Adaptive cellular immunity is required to clear HSV-1 infection in the periphery. Myeloid dendritic cells (DCs) are the first professional Ag-presenting cell to encounter the virus after primary and secondary infection and thus the consequences of their infection are important in understanding the pathogenesis of the disease and the response to the virus. Following HSV-1 infection, both uninfected and infected human DCs acquire a more mature phenotype. In this study, we demonstrate that type I IFN secreted from myeloid DC mediates bystander activation of the uninfected DCs. Furthermore, we confirm that this IFN primes DCs for elevated IL-12 p40 and p70 secretion. However, secretion of IFN is not responsible for the acquisition of a mature phenotype by HSV-1-infected DC. Rather, virus binding to a receptor on the cell surface induces DC maturation directly, through activation of the NF-kappaB and p38 MAPK pathways. The binding of HSV glycoprotein D is critical to the acquisition of a mature phenotype and type I IFN secretion. The data therefore demonstrate that DCs can respond to HSV exposure directly through recognition of viral envelope structures. In the context of natural HSV infection, the coupling of viral entry to the activation of DC signaling pathways is likely to be counterbalanced by viral disruption of DC maturation. However, the parallel release of type I IFN may result in paracrine activation so that the DCs are nonetheless able to mount an adaptive immune response.     

Herpes simplex virus type 1-induced modifications in the distribution of nucleolar B-36 protein

Nucleolar B-36 protein was localized ultrastructurally by immunocytochemistry with monoclonal antibody P2G3 and colloidal gold label in rabbit fibroblast cells before and during infection with herpes simplex virus (HSV) type 1. In non-infected cells, labeling was sparse and restricted to the fibrillar component of the nucleoli. During the infectious cycle, B-36 protein appeared to be somewhat more abundant within the morphologically altered fibrillar component of the nucleoli. In addition, the protein was also detected in some but not all virus-induced intranuclear dense bodies. These observations suggest the presence of functionally distinct dense bodies. The association of B-36 protein with both structures was not disrupted by a hypotonic shock and detergent treatment, which suggest that these sites do not represent areas of passive intranuclear diffusion. Inhibition of protein synthesis late in infection, viral DNA replication or RNA synthesis did not alter the distribution of B-36 protein. We suggest that this protein may play a role in the increased compaction of the ribonucleoprotein fibrils induced by HSV infection, perhaps in association with some of the virus-encoded proteins which also have been detected in the nucleoli.     

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.     

The stimulation of the phosphorylation of ribosomal protein S6 by cycloheximide and puromycin

The administration of cycloheximide or puromycin to rats in amounts that all but completely inhibited hepatic protein synthesis caused an increase in the amount of radioactive phosphate incorporated into the liver ribosomal protein S6; there was also an increase in the prominence of the derivatives of S6 which contain increasing numbers of phosphorylated serine residues.     

Role of AF6 protein in cell-to-cell spread of Herpes simplex virus 1

AF6 and its rat homologue afadin are multidomain proteins localized at cell junctions and involved in intercellular adhesion. AF6 interacts via its PDZ domain with nectin-1 at epithelial adherens junctions. Nectin-1 serves as a mediator of cell-to-cell spread for Herpes simplex virus 1 (HSV-1). We analyzed the role of AF6 protein in the viral spread and nectin-1 clustering at cell-cell contacts by knockdown of AF6 in epithelial cells. AF6 knockdown reduced efficiency of HSV-1 spreading, however, the clustering of nectin-1 at cell-cell contacts was not affected. Thus, AF6 protein is important for spreading of HSV-1 in epithelial cells, independently of nectin clustering, possibly by stabilization of the E-cadherin-dependent cell adhesion.     

Kinetics of ribosomal protein S6 phosphorylation by HepG-2 cells in response to insulin

The effect of insulin on the phosphorylation of ribosomal protein S6 was studied in a human liver cell line (HepG-2), using [32P] inorganic phosphate. Increased rate of protein S6 phosphorylation was detected 8 min following the addition of insulin to serum starved cells. Maximum enhancement of phosphorylation was observed at 80 nM insulin. Minimum level of insulin required to produce measurable increase of S6 phosphorylation was 20 nM. Radioactivity of protein S6 increased most in the native subunit and polysome fractions. Significant increase in radioactivity of this protein was not observed in the monosome fraction during the first 30 min of insulin stimulation. Increase in the specific radioactivity of native 40S subunit was higher than that of polysomes. These results suggest that phosphorylation takes place in the subunit compartment and moves preferentially into the polysomes.     

Regulation of ribosomal protein S6 phosphorylation by casein kinase 1 and protein phosphatase 1

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5'-m(7)GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.     

Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1)

p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1.     

Ethionine and the phosphorylation of ribosomal protein S6.

Ribosome phosphorylation was studied by monitoring the phosphorylation state of small subunit protein S6 as visualized on two-dimensional electrophoretograms of ribosomal proteins isolated from rat liver. No phosphorylation of S6 was observed under conditions of ethionine-induced inhibition of protein synthesis. Moderate phosphorylation, detected as the appearance of S6 and four or five phosphorylated derivatives, was observed in saline-treated animals. Reversal of ethionine-induced inhibition of protein synthesis by treatment with adenine led to extensive phosphorylation of S6. A model for protein synthesis which includes requisite phosphorylation of ribosomes during initiation is proposed. Cyclic adenosine 3':5'-monophosphate concentration was significantly elevated in liver of both ethionine- and ethionine plus adenine-treated rats, relative to that of saline-treated animals.     

Herpes simplex virus type 1 UL51 protein is involved in maturation and egress of virus particles

The UL51 gene of herpes simplex virus type 1 (HSV-1) encodes a phosphoprotein whose homologs are conserved throughout the herpes virus family. Recently, we reported that UL51 protein colocalizes with Golgi marker proteins in transfected cells and that targeting of UL51 protein to the Golgi apparatus depends on palmitoylation of its N-terminal cysteine at position 9 (N. Nozawa, T. Daikoku, T. Koshizuka, Y. Yamauchi, T. Yoshikawa, and Y. Nishiyama, J. Virol. 77:3204-3216, 2003). However, its role in the HSV replication cycle was unknown. Here, we generated UL51-null mutants (FDL51) in HSV-1 to uncover the function of UL51 protein. We show that the mutant plaques were much smaller in size and that maximal titers were reduced nearly 100-fold compared to wild-type virus. Electron microscopy indicated that the formation of nucleocapsids was not affected by the deletion of UL51 but that viral egress from the perinuclear space was severely compromised. In FDL51-infected cells, a large number of enveloped nucleocapsids were observed in the perinuclear space, but enveloped mature virions in the cytoplasm, as well as extracellular mature virions, were rarely detected. These defects were fully rescued by reinsertion of the UL51 gene. These results indicate that UL51 protein is involved in the maturation and egress of HSV-1 virus particles downstream of the initial envelopment step.     

Herpes simplex virus types 1 and 2 modulate autophagy in SIRC corneal cells

Autophagy and apoptosis function as important early cellular defense mechanisms in infections and other diseases. The outcome of an infection is determined by a complex interplay between the pathogenic microorganism and these intracellular pathways. To better understand the cytopathogenicity of Herpes simplex virus types 1 and 2 (HSV-1 and -2), we studied the effect of these viruses on the autophagic and apoptotic processes in the SIRC corneal cell line. Infection with the KOS strain of HSV-1 and a wild-type strain of HSV-2 enhanced autophagosome formation, triggered cytoplasmic acidification, increased LC3B lipidation and elevated the ratio of apoptotic cells. The autophagy inhibitor bafilomycin A1 triggered a significant increase in the apoptotic responses of HSV-1- and HSV-2-infected cells. Thus, both HSV types affect autophagy and apoptosis in a coordinated fashion, and autophagy plays cytoprotective role in HSV-infected cells via antagonizing apoptosis. Together these data implicate autophagy in the pathogenic mechanism of herpetic keratitis.     

Insulin- and phorbol ester-stimulated phosphorylation of ribosomal protein S6

The relative abilities of insulin and the phorbol ester tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) to lead to the phosphorylation of ribosomal protein S6 in vivo were compared in a Reuber H35 hepatoma cell line shown previously to be highly responsive to these agents. In quiescent (serum-starved) cultures of H35 cells incubated with 32Pi, both insulin (10(-7) M) and TPA (1.6 X 10(-6) M) resulted in the marked phosphorylation of S6 compared to the unstimulated cultures as evidenced by an increase in radioactivity associated with S6 and by a corresponding shift in the mobility of phosphorylated S6 during two-dimensional electrophoresis. Following incubation with insulin or TPA, greater than 95% of the phosphate was in derivatives containing four to five phosphate groups. The site-specific phosphorylation of S6 in response to both optimal and suboptimal concentrations of insulin and/or TPA was examined by two-dimensional peptide mapping of the trypsin-digested ribosomal protein S6. The tryptic phosphopeptides of S6 obtained following treatment of the H35 cells with insulin and/or TPA were identical and were the same phosphopeptides as those observed previously following the phosphorylation in vitro of 40 S ribosomal subunits from reticulocytes with purified protease-activated kinase II (Perisic, O., and Traugh, J. A. (1983) J. Biol. Chem. 258, 13998-14002).     

Increased phosphorylation of ribosomal protein S6 in hamster fibroblasts transformed by polyoma virus and simian virus 40

The extent of phosphorylation of ribosomal protein S6 was compared in normal hamster fibroblasts and in fibroblasts transformed by polyoma virus or simian virus 40. In both strains of transformed cells the protein was more highly phosphorylated than in the normal cells.     

TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.     

Regulation of 40S ribosomal protein S6 phosphorylation in Swiss mouse 3T3 cells

Addition of serum to resting cultures of Swiss mouse 3T3 cells causes an immediate multiple phosphorylation of 40S ribosomal protein S6. After 60 min of stimulation, changing to medium containing no serum led to the net dephosphorylation of S6. During this same period, a second protein, as yet unidentified, became increasingly phosphorylated. Incubation of cells with cycloheximide prior to the addition of serum almost completely blocked the activation of protein synthesis. There was no effect on the serum-induced phosphorylation of S6. If cells were stimulated in the presence of cAMP phosphodiesterase inhibitors theophylline or SQ 20006, both S6 phosphorylation and the activation of protein synthesis were inhibited. Stimulation of cells with serum also led to an immediate drop in total intracellular cAMP levels. This was blocked by prostaglandin E1 (PGE1), which caused a 10 fold increase in total intracellular cyclic AMP. However, PGE1 had no effect on protein synthesis or S6 phosphorylation.