Cellular casein kinase II-mediated phosphorylation of rinderpest virus P protein is a prerequisite for its role in replication/transcription of the genome

Kaushik, Rajnish; Shaila, M.S.

doi:10.1099/vir.0.19702-0

Cited by 29 publications

(26 citation statements)

References 18 publications

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“…Rabbit polyclonal antibodies against RPV P (Kaushik & Shaila, 2004), N (Mitra- Kaushik et al, 2001) and L proteins were made previously in the laboratory. Horseradish peroxidase-conjugated goat anti-rabbit IgG was obtained from Sigma.…”

Section: Methodsmentioning

confidence: 99%

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

Gopinath

Shaila

2009

Journal of General Virology

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Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L-P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N 7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of c-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 59 sequence. The L protein forms a covalent enzyme-guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717-2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

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Section: Methodsmentioning

confidence: 99%

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

Gopinath

Shaila

2009

Journal of General Virology

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“…For instance, removal or inhibition of kinase activity for P protein decreases transcription/replication activity or viral growth in canine distemper virus [13], sendai virus [14], human parainfluenza virus type 3 [15,16], and vesicular stomatitis virus [17]. As a more direct approach, site-directed mutagenesis at the phosphorylation site of P protein causes a decrease in viral gene expression in rinderpest virus [18,19] and human respiratory syncytial virus [20]. These phosphorylation sites seem to be required for efficient P protein function.…”

Section: Discussionmentioning

confidence: 99%

“…T49 of MV-P protein is homologous to the major phosphorylation site of rinderpest virus (RPV) P protein, which is a closely related morbillivirus [18,19]. To further clarify the correlation between phosphorylation of P protein and viral transcription/replication activity, we generated mutants of P protein whose phosphorylation sites were substituted with alanine residues: S86A/S151A (dP) and T49A/S86A/S151A (tP).…”

Section: 3mentioning

confidence: 99%

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Phosphorylation of measles virus phosphoprotein at S86 and/or S151 downregulates viral transcriptional activity

et al. 2012

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a b s t r a c tMeasles virus phosphoprotein (P protein) is a cofactor of the viral RNA polymerase (L protein) that associates with the nucleoprotein-RNA complex to support viral transcription and replication. Here, we report a significant inverse correlation between the phosphorylation level of MV-P protein and viral transcriptional activity. Upregulation of P protein phosphorylation resulted in reduction of viral transcription. Additionally, we found that strong phosphorylation at S86 and S151 of P protein, which may be generally prevented by association with nucleoprotein, downregulates the viral transcriptional activity. These findings suggest that P protein is involved in regulation of viral transcription through changes in its phosphorylation status.

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“…However, CK2 phosphorylated P-T286A at a level similar to that for wild-type P. It is possible that there are several potential CK2 sites: mutating one site may not make a significant difference in the overall phosphorylation levels of the P protein. Previous studies suggested that CK2 phosphorylated the P protein from RSV, measles virus, rinderpest virus, and Borna disease virus (8,13,19,25). Due to numerous subunits and isoforms of CK2 and ubiquitous potential CK2 sites, work on the role of CK2 has Western blotting was performed to show the input of NP and P or P-T286K.…”

Section: Vol 85 2011mentioning

confidence: 99%

Identification of a Phosphorylation Site within the P Protein Important for mRNA Transcription and Growth of Parainfluenza Virus 5

Sun

Luthra

et al. 2011

J Virol

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The viral RNA-dependent RNA polymerase (vRdRp) of paramyxovirus consists of the large (L) protein and the phosphoprotein (P). P is heavily phosphorylated, and it is thought that the phosphorylation of P plays a role in regulating viral RNA synthesis. However, no phosphorylation site within the P protein in paramyxovirus has been identified as playing a positive role in viral RNA synthesis in virus infection. Using mass spectrometry analysis, the threonine residue at position 286 of P of parainfluenza virus 5 (PIV5) was found phosphorylated. Mutation of T286 to alanine (T286A), aspartic acid (T286D), or glutamic acid (T286E) reduced minigenome activity. Recombinant virus containing a mutation at the T286 position (rPIV5-P-T286A) grew slower than wild-type virus; viral mRNA synthesis and protein expression of rPIV5-P-T286A were delayed. Biochemical studies showed that the binding of NP or L protein with the P mutants or tetramer formation by the mutant P proteins was unaltered from that for wild-type P. While we failed to rescue rPIV5-P-T286E virus, several revertant viruses were obtained. All non-wild-type revertants had mutations at T286 and showed defects in both minigenome activity and viral growth. This is the first time that a phosphorylation site within the P protein in paramyxovirus has been found to play a positive role in viral mRNA synthesis and virus growth.Parainfluenza virus 5 (PIV5) is a prototypical member of the paramyxovirus family, which contains many human and animal pathogens, such as Sendai virus (SeV), mumps virus (MuV), respiratory syncytial virus (RSV), Hendra virus (HeV), and Nipah virus (NiV) (15). The negative-stranded RNA genome of PIV5 contains seven genes yet encodes eight proteins in the order NP-V/P-M-F-SH-HN-L (15). While V mRNA is transcribed faithfully by the V/P gene, P mRNA is produced by the V/P gene through an insertion of two nontemplate guanine residues at a specific sequence during viral RNA transcription (30). The phosphoprotein (P) and large polymerase (L) protein constitute the viral RNA-dependent RNA polymerase complex (vRdRP). The L protein has enzymatic activities capable of initiation, elongation, and termination of RNA synthesis, as well as addition of the 5Ј cap structure and 3Ј poly(A) sequence (15). The P protein does not have intrinsic enzymatic activity; however, it is an essential cofactor for the polymerase, regulating polymerase activity in viral RNA replication and transcription (15).The P proteins of all paramyxoviruses are heavily phosphorylated and hence are named phosphoproteins (15). However, the role of P protein phosphorylation in the replication of paramyxoviruses has been an enigma. It was initially suggested that phosphorylation of the P protein was essential for viral RNA synthesis. In paramyxovirus, the P proteins of SeV and RSV have been studied the most extensively. Up to 11 phosphorylation sites of SeV P protein were detected (14, 32), and S249 was identified as the major phosphorylation site (6, 7). However, mutation at S249 showed normal ...

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Cellular casein kinase II-mediated phosphorylation of rinderpest virus P protein is a prerequisite for its role in replication/transcription of the genome

Cited by 29 publications

References 18 publications

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

Phosphorylation of measles virus phosphoprotein at S86 and/or S151 downregulates viral transcriptional activity

Identification of a Phosphorylation Site within the P Protein Important for mRNA Transcription and Growth of Parainfluenza Virus 5

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