Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription

Philipp A. Ilinykh, Bersabeh Tigabu, Andrey Ivanov, Tatiana Ammosova, Yuri Obukhov, Tania Garron, Namita Kumari, Dmytro Kovalskyy, Maxim O. Platonov, Vasiliy S. Naumchik, Alexander Freiberg, Sergei Nekhai, Alexander Bukreyev

Research output: Contribution to journalArticle

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Abstract

The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30mustbein a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr143 and Thr146 of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29-46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species.

Original languageEnglish (US)
Pages (from-to)22723-22738
Number of pages16
JournalJournal of Biological Chemistry
Volume289
Issue number33
DOIs
StatePublished - Aug 15 2014

Fingerprint

Protein Phosphatase 1
Protein Transport
Transcription
Phosphorylation
Molecules
Viral Genome
Threonine
DNA-Directed RNA Polymerases
ebola virus VP30 protein
Serine
Small Interfering RNA
Antiviral Agents
Cultured Cells
Cytoplasm
Fever
Genes
Cells
RNA
Messenger RNA
Peptides

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription. / Ilinykh, Philipp A.; Tigabu, Bersabeh; Ivanov, Andrey; Ammosova, Tatiana; Obukhov, Yuri; Garron, Tania; Kumari, Namita; Kovalskyy, Dmytro; Platonov, Maxim O.; Naumchik, Vasiliy S.; Freiberg, Alexander; Nekhai, Sergei; Bukreyev, Alexander.

In: Journal of Biological Chemistry, Vol. 289, No. 33, 15.08.2014, p. 22723-22738.

Research output: Contribution to journalArticle

Ilinykh, PA, Tigabu, B, Ivanov, A, Ammosova, T, Obukhov, Y, Garron, T, Kumari, N, Kovalskyy, D, Platonov, MO, Naumchik, VS, Freiberg, A, Nekhai, S & Bukreyev, A 2014, 'Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription', Journal of Biological Chemistry, vol. 289, no. 33, pp. 22723-22738. https://doi.org/10.1074/jbc.M114.575050
Ilinykh, Philipp A. ; Tigabu, Bersabeh ; Ivanov, Andrey ; Ammosova, Tatiana ; Obukhov, Yuri ; Garron, Tania ; Kumari, Namita ; Kovalskyy, Dmytro ; Platonov, Maxim O. ; Naumchik, Vasiliy S. ; Freiberg, Alexander ; Nekhai, Sergei ; Bukreyev, Alexander. / Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription. In: Journal of Biological Chemistry. 2014 ; Vol. 289, No. 33. pp. 22723-22738.
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AU - Ammosova, Tatiana

AU - Obukhov, Yuri

AU - Garron, Tania

AU - Kumari, Namita

AU - Kovalskyy, Dmytro

AU - Platonov, Maxim O.

AU - Naumchik, Vasiliy S.

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AU - Nekhai, Sergei

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N2 - The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30mustbein a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr143 and Thr146 of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29-46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species.

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