Phosphorylation of VP30 impairs ebola virus transcription.
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 (VP30(6A)) that is still able to activate EBOV-specific transcription in a plasmid-based minigenome system. VP30(6A), 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 VP30(6A). 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 VP30(6A), 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.
Modrof
Jens
J
Institut für Virologie der Philipps-Universität Marburg, Robert-Koch-Strasse 17, Marburg 35037, Germany.
Mühlberger
Elke
E
Klenk
Hans-Dieter
HD
Becker
Stephan
S
D000818:Animals; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D029043:Ebolavirus / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D019590:Nucleocapsid Proteins / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012694:Serine / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D019590:Nucleocapsid Proteins; C094001:VP30 protein, Marburg virus; D014764:Viral Proteins; D019319:Okadaic Acid; D012694:Serine