title: Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription.
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, VP30 must be in 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 Thr(143) and Thr(146) 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.
authors.affiliation: Departments of Pathology and University of Texas Medical Branch at Galveston, Galveston, Texas 77555; Galveston National Laboratory, Galveston, Texas 77555.
authors.forename: Philipp A
authors.initials: PA
authors.lastname: Ilinykh
authors.affiliation: Departments of Pathology and University of Texas Medical Branch at Galveston, Galveston, Texas 77555; Galveston National Laboratory, Galveston, Texas 77555.
authors.forename: Bersabeh
authors.initials: B
authors.lastname: Tigabu
authors.affiliation: Center for Sickle Cell Disease and Howard University, Washington, D. C. 20059.
authors.forename: Andrey
authors.initials: A
authors.lastname: Ivanov
authors.affiliation: Center for Sickle Cell Disease and Howard University, Washington, D. C. 20059; Departments of Medicine and Howard University, Washington, D. C. 20059.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Ammosova
authors.affiliation: Center for Sickle Cell Disease and Howard University, Washington, D. C. 20059.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Obukhov
authors.affiliation: Departments of Pathology and University of Texas Medical Branch at Galveston, Galveston, Texas 77555; Galveston National Laboratory, Galveston, Texas 77555,; Departments of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555.
authors.forename: Tania
authors.initials: T
authors.lastname: Garron
authors.affiliation: Center for Sickle Cell Disease and Howard University, Washington, D. C. 20059.
authors.forename: Namita
authors.initials: N
authors.lastname: Kumari
authors.affiliation: Kiev National Taras Shevchenko University, Kiev 01601, Ukraine, and; Enamine Ltd., Kiev 01103, Ukraine.
authors.forename: Dmytro
authors.initials: D
authors.lastname: Kovalskyy
authors.affiliation: Kiev National Taras Shevchenko University, Kiev 01601, Ukraine, and; Enamine Ltd., Kiev 01103, Ukraine.
authors.forename: Maxim O
authors.initials: MO
authors.lastname: Platonov
authors.affiliation: Kiev National Taras Shevchenko University, Kiev 01601, Ukraine, and; Enamine Ltd., Kiev 01103, Ukraine.
authors.forename: Vasiliy S
authors.initials: VS
authors.lastname: Naumchik
authors.affiliation: Departments of Pathology and University of Texas Medical Branch at Galveston, Galveston, Texas 77555; Galveston National Laboratory, Galveston, Texas 77555.
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Freiberg
authors.affiliation: Galveston National Laboratory, Galveston, Texas 77555,; Departments of Medicine and Howard University, Washington, D. C. 20059; Departments of Microbiology, Howard University, Washington, D. C. 20059,. Electronic address: snekhai@howard.edu.
authors.forename: Sergei
authors.initials: S
authors.lastname: Nekhai
authors.affiliation: Departments of Pathology and University of Texas Medical Branch at Galveston, Galveston, Texas 77555; Galveston National Laboratory, Galveston, Texas 77555,; Departments of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555,. Electronic address: alexander.bukreyev@utmb.edu.
authors.forename: Alexander
authors.initials: A
authors.lastname: Bukreyev
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D029043:Ebolavirus / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology*
chemical_list: D000998:Antiviral Agents; D012333:RNA, Messenger; D012367:RNA, Viral; D014157:Transcription Factors; C479897:VP30 protein, ebola virus; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases; D054645:Protein Phosphatase 1
keywords: Ebola Virus; Phosphoprotein Phosphatase 1 (PP1); Small Molecule; Transcription; Viral Replication