Inhibition of the Phosphorylation of the Regulatory Non-structural Protein of Vesicular Stomatitis Virus by an Antiviral Xanthate Compound

Müller‐Decker, Karin; Amtmann, Eberhard; Sauer, Gerhard

doi:10.1099/0022-1317-68-12-3045

Cited by 26 publications

(9 citation statements)

References 20 publications

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“…In this connection, the antiviral xanthate compound D609 (tricyclo-decane-9-yl-xanthogenate), an inhibitor of VSV growth in cell cultures, is worth mentioning. The drug strongly inhibited phosphorylation of newly synthesized P protein in VSV-infected cells, with concomitant inhibition of secondary transcription and replication (18). However, primary transcription-mediated by the input viral ribonucleoprotein containing P protein that is already phosphorylated-remained unaffected.…”

Section: Discussionmentioning

confidence: 97%

Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P.

Barik

Banerjee

1992

Proc. Natl. Acad. Sci. U.S.A.

127

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The precise mechanism of transactivation by phosphoprotein and the role of its phosphorylation status in this process have been the subject of intensive research in the past few years. We have recently cloned and expressed large quantities of phosphate-free P protein in Escherichia coli (2). The VSV transcription reaction mixture reconstituted in vitro with purified L protein, N-RNA template, and unphosphorylated P protein (P0) was found to be defective. Addition of uninfected cell extract to the transcription reaction mixture or prior phosphorylation of the P protein by the cell extract restored transcription, suggesting an essential role of cellular protein kinase-mediated phosphorylation in transcriptional activity of P protein (3). The phosphorylated product (P,) of the cell kinase reaction was further phosphorylated by an L protein-associated kinase to produce the fully phosphorylated form (P2). Thus, it was proposed that complete activation of P protein is mediated through a cascade phosphorylation pathway involving two protein kinase activities-the cell kinase and the L kinase-acting sequentially (3). Substrate specificity and other biochemical parameters ofthe two kinases revealed that they were distinct and different from each other. In this communication, we report the purification and detailed characterization of the cellular protein kinase and show that it is a single enzyme with properties identical to that of cellular casein kinase II (CKII). Phosphorylation of the phosphate-free P protein (PO) by purified CKII and L kinase in vitro fully restored transcriptional activity of P protein. MATERIALS AND METHODSViral (VSV, New Jersey serotype, Ogden subtype) L protein and N-RNA template and bacterially expressed P protein of the same serotype were purified as described (3). CKII, purified from bovine testis, was a kind gift from Edwin G. Krebs and David Litchfield (Howard Hughes Institute, Seattle). Oligopeptides were synthesized in a Beckman 990B automated solid-phase peptide synthesizer and purified through HPLC. Dephosphorylated casein, heparin, and protamine were purchased from Sigma. Rabbit anti-human CKII antibody (Upstate Biotechnology, Lake Placid, NY) was raised against a 23-mer synthetic peptide corresponding to residues 70-91 of the a (catalytic) subunit of human CKII coupled to keyhole limpet hemocyanin.Purification of Po Kinase (PK). The P protein phosphorylating activity (PK) was purified essentially as described for CKII (4) except that the fractions were monitored by their ability to phosphorylate bacterially expressed P protein (PO) as substrate. In brief, the postribosomal supernatant (S100) of baby hamster kidney (BHK) cell extract (10 g of protein) in buffer A [50 mM Tris-HCl, pH 7.5/0.1 mM EDTA/5% (vol/vol) glycerol/0.02% NaN3/1 mM dithiothreitol] containing 50 mM NaCl was loaded onto a 20-ml DEAE-cellulose column. After washing with 60 ml of the same buffer, the column was developed with a linear gradient of 0.1-0.4 M NaCl in buffer A (total vol, 120 ml; collected in 60 fraction...

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Section: Discussionmentioning

confidence: 97%

Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P.

Barik

Banerjee

1992

Proc. Natl. Acad. Sci. U.S.A.

127

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“…In this regard, it is noteworthy that D609 has been previously shown to inhibit the replication of a variety of unrelated DNA and RNA viruses in vitro. [58][59][60][61] Its broad spectrum of activity suggested that it may affect a fundamental metabolic process required for virus replication. In particular, Mellert et al reported that a D609/undecanoid acid combination inhibited HIV-1 replication in infected lymphoma cells, likely interfering with certain steps of virus assembly.…”

Section: Discussionmentioning

confidence: 99%

Phosphatidylcholine-specific phospholipase C activation is required for CCR5-dependent, NF-kB–driven CCL2 secretion elicited in response to HIV-1 gp120 in human primary macrophages

Fantuzzi

Spadaro

Purificato

et al. 2008

Blood

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CCL2 (MCP-1) has been shown to enhance HIV-1 replication. The expression of this chemokine by macrophages is up-modulated as a consequence of viral infection or gp120 exposure. In this study, we show for the first time that the phosphatidylcholinespecific phospholipase C (PC-PLC) is required for the production of CCL2 triggered by gp120 in human monocyte-derived macrophages (MDMs). Using a combination of pharmacologic inhibition, confocal laserscanner microscopy, and enzymatic activity assay, we demonstrate that R5 gp120 interaction with CCR5 activates PC-PLC, as assessed by a time-dependent modification of its subcellular distribution and a concentration-dependent increase of its enzymatic activity. Furthermore, PC-PLC is required for NF-kB-mediated CCL2 production triggered by R5 gp120. Notably, PC-PLC activation through CCR5 is specifically induced by gp120, since triggering CCR5 through its natural ligand CCL4 (MIP-1␤) does not affect PC-PLC cellular distribution and enzymatic activity, as well as CCL2 secretion, thus suggesting that different signaling pathways can be activated through CCR5 interaction with HIV-1 or chemokine ligands. The identification of PC-PLC as a critical mediator of well-defined gp120-mediated effects in MDMs unravels a novel mechanism involved in bystander activation and may contribute to define potential therapeutic targets to block Env-triggered pathologic responses. (Blood. 2008;111:3355-3363)

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“…The specific phosphorylation of certain virus-encoded proteins that participate in transcriptional activity was inhibited by D609. In the case of vesicular stomatitis virus, phosphorylation of the viral nonstructural protein, which is part of the polymerase complex, and secondary virus transcription were inhibited upon treatment of infected cells with D609 (23).…”

mentioning

confidence: 99%

Inhibition of c-fos transcription and phosphorylation of the serum response factor by an inhibitor of phospholipase C-type reactions.

Schalasta¹,

Doppler²

1990

Mol. Cell. Biol.

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Phospholipase C activity is necessary for transcriptional c-fos activation by providing diacylglycerol as an activator of protein kinase C. We found that transcriptional activation of c-fos and the phosphorylation of its major transcription factor were inhibited by tricyclodecan-9-yl xanthogenate, which blocks phospholipase C-type reactions. Transcription of the c-ras and ,-actin genes in the same cells remained unaffected.Rapid transcriptional activation of the c-fos proto-oncogene can be achieved by a variety of growth factors and other extracellular stimuli such as epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (5,12,16,21). Induction occurs even in the presence of inhibitors of protein synthesis, suggesting a posttranslational alteration of preexisting transcription factors. Inducibility depends on the presence of the serum response element (SRE) that is located in the proximity of the mRNA cap site of c-fos (10,11,35,36). It does so by interacting with at least two phosphoproteins, the 67-kilodalton serum response factor (SRF) (32, 37) and the recently identified 62-kilodalton ternary complex factor (31, 34). Ternary complex formation between SRE-bound SRF and the ternary complex factor (the latter does not bind by itself to SRE [34]) is thought to be necessary for inducible expression of c-fos (33, 34). Complex formation between SRE and SRF has been observed in A431 cells after stimulation with EGF (28), and phosphorylation of SRF has been implicated in its binding activity (27). Phosphorylation of proteins is a common regulatory alteration mediated by kinases (4). To assess whether phosphorylation of SRF renders the protein biologically active, we have used an inhibitor of phospholipase C-type reactions that has been shown to preclude indirectly also the activity of a protein kinase C (PKC) (24). The compound D609 was shown to prevent formation of diacylglycerol (22), which acts as a second messenger and induces

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Inhibition of the Phosphorylation of the Regulatory Non-structural Protein of Vesicular Stomatitis Virus by an Antiviral Xanthate Compound

Cited by 26 publications

References 20 publications

Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P.

Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P.

Phosphatidylcholine-specific phospholipase C activation is required for CCR5-dependent, NF-kB–driven CCL2 secretion elicited in response to HIV-1 gp120 in human primary macrophages

Inhibition of c-fos transcription and phosphorylation of the serum response factor by an inhibitor of phospholipase C-type reactions.

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