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

Abstract

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...

Rabies virus matrix protein regulates the balance of virus transcription and replication

“…Association of VSV L with different host cell or virus factors might affect L polymerase activity (Barik & Banerjee, 1992;Gupta & Banerjee, 1997;Das et al, 1998). Indeed, VSV P phosphorylation mutants able to support either replication or transcription of defective interfering particles (Pattnaik et al, 1997;Hwang et al, 1999) and P antibodies inhibiting replication but not transcription have been described (Richardson & Peluso, 1996).…”

N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation

“…The conclusions derived from these investigations have demonstrated that the phosphorylation of P is important for its functionality in various ways. Several studies employing in vitro and in vivo experiments have suggested that N-terminal phosphorylation of P of the VSV Indiana serotype by casein kinase II (CKII) is essential for its transcription function (11,12,(14)(15)(16)18). In contrast, Spadafora et al (17) showed that the N-terminal phosphorylation of P may not be essential for VSV RNA synthesis but may play a role in the self-association of P and interaction with L. In addition, an infectious VSV in which N-terminal phosphorylation of P was absent at all three sites could not be recovered, which confirms that N-terminal phosphorylation of P is absolutely critical for viral growth and multiplication (19).…”

“…The P of the VSV Indiana serotype has been shown to be phosphorylated in two different domains: N-terminal domain I, in which phosphorylation sites were mapped to S60, T62, and S64, and C-terminal domain II, in which phosphorylation sites were mapped to S226 and S227. Since P was shown to be phosphorylated in specific domains involved in regulating transcription in vitro, phosphorylation of P and its role in transcription have been investigated intensively (11)(12)(13)(14)(15)(16)(17)(18). The conclusions derived from these investigations have demonstrated that the phosphorylation of P is important for its functionality in various ways.…”

Reversal of the measles virus-mediated increase of phosphorylating activity in persistently infected mouse neuroblastoma cells by anti-measles virus antibodies

“…Although it is well established that MV proteins P, N and V are phosphorylated, from this study it appears that PKC is not involved in this process. Another serine kinase, such as casein kinase II, shown by Barik & Banerjee (1992) to phosphorylate the P protein of vesicular stomatitis virus, is a probable candidate. The strong correlation found between the high MV protein content and both Ca2+/ lipid-dependent or -independent phosphorylating activity may indicate that MV infection can directly or indirectly mediate the activity of these kinases.…”