Phosphorylation status of the phosphoprotein P of rinderpest virus modulates transcription and replication of the genome

Saikia, Paramananda; Gopinath, Madhumala; Shaila, M.S.

doi:10.1007/s00705-008-0034-9

Cited by 22 publications

(17 citation statements)

References 31 publications

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“…Prime examples in the order Mononegavirales are the family of P proteins which are highly phosphorylated, and the phosphorylation of P plays a critical role in the regulation of viral RNA synthesis in complex with the polymerase L and the nucleoprotein N (or NP) (26)(27)(28)(29)(30)(31)(32). However, filoviruses represent an exception among the Mononegavirales; VP35, the P protein homologue, is only very weakly phosphorylated, and the potential role of VP35 phosphorylation for viral replication is unclear (5,33).…”

Section: Discussionmentioning

confidence: 99%

Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle

Biedenkopf

Lier

Becker

2016

J Virol

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Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus transcription and replication currently is not well understood. An important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30 phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the viral RNA. VP30 thereby is excluded from the viral transcription complex, simultaneously leading to increased viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of viral RNA synthesis, where VP30 is required to initiate primary viral transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the viral life cycle. IMPORTANCEThe current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that the situation is more complex and that primary transcription as well as the rescue of recombinant Ebola virus also requires the transient phosphorylation of VP30. VP30 encodes six N-proximal serine residues that serve as phosphorylation acceptor sites. The present study shows that the dynamic phosphorylation of serine at position 29 alone is sufficient to activate primary viral transcription. Our results indicate a series of phosphorylation/dephosphorylation events that trigger binding to and release from the nucleocapsid and transcription complex to be essential for the full activity of VP30.

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Section: Discussionmentioning

confidence: 99%

Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle

Biedenkopf

Lier

Becker

2016

J Virol

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“…N-RNA was purified by caesium chloride density-gradient centrifugation . P protein was purified from insect cells infected with recombinant baculovirus by Ni-NTA affinity chromatography (Saikia et al, 2008). Purification of the recombinant deletion mutants of RPV L protein, LD1 and LD3 is described in the Supplementary Method (available in JGV Online).…”

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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“…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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Phosphorylation status of the phosphoprotein P of rinderpest virus modulates transcription and replication of the genome

Cited by 22 publications

References 31 publications

Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle

Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle

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

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