Studies thread: Ebola VP30 Gene

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J Infect Dis. 2011 Nov;204 Suppl 3:S934-40. doi: 10.1093/infdis/jir320.
Role of VP30 phosphorylation in the Ebola virus replication cycle.
Martinez MJ1, Volchkova VA, Raoul H, Alazard-Dany N, Reynard O, Volchkov VE.

Full Paper: http://www.jid.oxfordjournals.org/cg...&pmid=21987772

Abstract

Ebola virus (EBOV) transcription is dependent on the phosphoprotein VP30, a component of the viral nucleocapsid. VP30 is phosphorylated at 2 serine residue clusters located at the N-terminal part of the protein. In this report, we have investigated the role of VP30 phosphorylation in EBOV replication using a reverse genetics approach. In effect, recombinant EBOVs with the VP30 serine clusters substituted either by nonphosphorylatable alanines or phosphorylation-mimicking aspartates were generated and characterized. We show that in comparison to the wild-type EBOV the mutated viruses possess reduced infectivity. This difference is explained by alterations in the balance between the transcription and replication processes and appear to be associated with the state of VP30 phosphorylation. Here we propose a model in which dynamic phosphorylation of VP30 is an important mechanism to regulate the EBOV replication cycle.

PMID:
21987772
[PubMed - indexed for MEDLINE]

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Re: Studies thread: Ebola VP30 Gene

J Biol Chem. 2014 Aug 15;289(33):22723-38. doi: 10.1074/jbc.M114.575050. Epub 2014 Jun 16.
Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription.
Ilinykh PA1, Tigabu B1, Ivanov A2, Ammosova T3, Obukhov Y2, Garron T4, Kumari N2, Kovalskyy D5, Platonov MO5, Naumchik VS5, Freiberg AN1, Nekhai S6, Bukreyev A7.

Full Paper: http://www.jbc.org/cgi/pmidlookup?vi...&pmid=24936058

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.

? 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Re: Studies thread: Ebola VP30 Gene

J Biol Chem. 2013 Apr 19;288(16):11165-74. doi: 10.1074/jbc.M113.461285. Epub 2013 Mar 14.
Phosphorylation of Ebola virus VP30 influences the composition of the viral nucleocapsid complex: impact on viral transcription and replication.
Biedenkopf N1, Hartlieb B, Hoenen T, Becker S.

Full paper: http://www.jbc.org/content/288/16/11165.long

Abstract

Ebola virus is a non-segmented negative-sense RNA virus causing severe hemorrhagic fever with high fatality rates in humans and nonhuman primates. For transcription of the viral genome four viral proteins are essential: the nucleoprotein NP, the polymerase L, the polymerase cofactor VP35, and VP30. VP30 represents an essential Ebola virus-specific transcription factor whose activity is regulated via its phosphorylation state. In contrast to viral transcription, VP30 is not required for viral replication. Using a minigenome assay, we show that phosphorylation of VP30 inhibits viral transcription while viral replication is increased. Concurrently, phosphorylation of VP30 reciprocally regulates a newly described interaction of VP30 with VP35, and strengthens the interaction with NP. Our results indicate a critical role of VP30 phosphorylation for viral transcription and replication, suggesting a mechanism by which VP30 phosphorylation modulates the composition of the viral polymerase complex presumably forming a transcriptase in the presence of non-phosphorylated VP30 or a replicase in the presence of phosphorylated VP30.

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Re: Studies thread: Ebola VP30 Gene

Ebola Virus Transcription Activator VP30 Is a Zinc-Binding Protein
J. Virol. March 2003 vol. 77 no. 5 3334-3338

http://jvi.asm.org/content/77/5/3334.full

ABSTRACT

Ebola virus VP30 is an essential activator of viral transcription. In viral particles, VP30 is closely associated with the nucleocapsid complex. A conspicuous structural feature of VP30 is an unconventional zinc-binding Cys3-His motif comprising amino acids 68 to 95. By using a colorimetric zinc-binding assay we found that the VP30-specific Cys3-His motif stoichiometrically binds zinc ions in a one-to-one relationship. Substitution of the conserved cysteines and the histidine within the motif led to a complete loss of the capacity for zinc binding. Functional analyses revealed that none of the tested mutations of the proposed zinc-coordinating residues influenced binding of VP30 to nucleocapsid-like particles but, concerning its role in activating viral transcription, all resulted in a protein that was inactive.

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Re: Studies thread: Ebola VP30 Gene

Phosphorylation of VP30 Impairs Ebola Virus Transcription
September 6, 2002 The Journal of Biological Chemistry, 277, 33099-33104

Full Paper: http://www.jbc.org/content/277/36/33099.full.pdf+html


Abstract

Transcription of the highly pathogenic Ebola virus (EBOV) is dependent on VP30, a constituent of the viral nucleocapsid complex. Here we present evidence that phosphorylation of VP30, which takes place at six N-terminal serine residues and one threonine residue, is of functional significance. Replacement of the phosphoserines by alanines resulted in an only slightly phosphorylated VP30 (VP306A) that is still able to activate EBOV-specific transcription in a plasmid-based minigenome system. VP306A, however, did not bind to inclusions that are induced by the major nucleocapsid protein NP. Three intracellular phosphatases (PP1, PP2A, and PP2C) have been determined to dephosphorylate VP30. The presence of okadaic acid (OA), an inhibitor of PP1 and PP2A, had the same negative effect on transcription activation by VP30 as the substitution of the six phosphoserines for aspartate residues. OA, however, did not impair transcription when VP30 was replaced by VP306A. In EBOV-infected cells, OA blocked virus growth dose-dependently. The block was mediated by the extensive phosphorylation of VP30, which is evidenced by the result that expression of VP306A, in trans, led to the progression of EBOV infection in the presence of OA. In conclusion, phosphorylation of VP30 was shown to regulate negatively transcription activation and positively binding to the NP inclusions.

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Re: Studies thread: Ebola VP30 Gene

Oligomerization of Ebola virus VP30 is essential for viral transcription and can be inhibited by a synthetic peptide.

J Biol Chem 2003 Oct 11;278(43):41830-6. Epub 2003 Aug 11.
Full Paper: http://www.pubfacts.com/fulltext_fra...etic%20peptide.

Abstract

Transcription of Ebola virus (EBOV)-specific mRNA is driven by the nucleocapsid proteins NP, VP35, and L. This process is further dependent on VP30, an essential EBOV-specific transcription factor. The present study addresses the self-assembly of VP30 and the functional significance of this process for viral transcription and propagation. Essential for oligomerization of VP30 is a region spanning amino acids 94-112. Within this region a cluster of four leucine residues is of critical importance. Mutation of only one of these leucine residues resulted in oligomerization-deficient VP30 molecules that were no longer able to support EBOV-specific transcription. The essential role of homo-oligomerization for the function of VP30 was further corroborated by the finding that mixed VP30 oligomers consisting of VP30 and transcriptionally inactive VP30 mutants were impaired in their ability to support EBOV transcription. The dominant negative effect of these VP30 mutants was dependent on their ability to bind to VP30. The oligomerization of VP30 could be dose dependently inhibited by a 25-mer peptide (E30pep-wt) derived from the presumed oligomerization domain (IC50,1 mum). A control peptide (E30pep-3LA), in which three leucines were changed to alanine, had no inhibitory effect. Thus, E30pep-wt seemed to bind efficiently to VP30 and consequently blocked the oligomerization of the protein. When E30pep-wt was transfected into EBOV-infected cells, the peptide inhibited viral replication suggesting that inhibition of VP30 oligomerization represents a target for EBOV antiviral drugs.

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Re: Studies thread: Ebola VP30 Gene

Ebola Virus VP30 Is an RNA Binding Protein▿
J. Virol. September 2007 vol. 81 no. 17 8967-8976

http://jvi.asm.org/content/81/17/8967.short

ABSTRACT

The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.