Analysis of rotavirus non-structural protein NSP5 by mass spectrometry reveals a complex phosphorylation pattern.
Genomic replication and partial assembly of Rotavirus takes place in cytoplasmic viral structures called viroplasms. NSP5 is a viral phosphoprotein localized in viroplasms and its expression is imperative for viral cycle progress. During infection three isoforms of NSP5 can be observed by SDS-PAGE (26, 28 and 33-35kDa) and previous reports suggested that they differ in their phosphorylation patterns. In this study we obtained NSP5 from infected cells and by mass spectrometry we were able to identify nine phosphorylation sites. We detected that in all the isoforms the same residues can be found either phosphorylated or unmodified. Quantitative analysis showed that the 28kDa isoform has a higher phosphorylation level than the 26kDa isoform suggesting that migration properties depend on the total number of phosphorylated residues. Moreover, we identified two not previously described modifications for this protein: an N-acetylation in Serine-2 and an intramolecular disulfide bond in a highly conserved motif, CXXC which is located between two charged alpha-helix motifs.
Sotelo
Pablo H
PH
Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Av. Independencia, 1027 (Sector J) Independencia, Santiago, Chile. phsotelo@gmail.com
Schümann
Michael
M
Krause
Eberhard
E
Chnaiderman
Jonas
J
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides; D008253:Macaca mulatta; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry; D012401:Rotavirus / Q000737:chemistry / Q000502:physiology*; D016415:Sequence Alignment; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000302:isolation & purification; D014779:Virus Replication
D004220:Disulfides; C434828:NSP5 protein, rotavirus group A; D020033:Protein Isoforms; D017361:Viral Nonstructural Proteins