title: Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex.
abstract: Bamboo mosaic virus (BaMV) satellite RNA (satBaMV) depends on BaMV for its replication and encapsidation. SatBaMV-encoded P20 protein is an RNA-binding protein that facilitates satBaMV systemic movement in co-infected plants. Here, we examined phosphorylation of P20 and its regulatory functions. Recombinant P20 (rP20) was phosphorylated by host cellular kinase(s) in vitro, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and mutational analyses revealed Ser-11 as the phosphorylation site. The phosphor-mimic rP20 protein interactions with satBaMV-translated mutant P20 were affected. In overlay assay, the Asp mutation at S11 (S11D) completely abolished the self-interaction of rP20 and significantly inhibited the interaction with both the WT and S11A rP20. In chemical cross-linking assays, S11D failed to oligomerize. Electrophoretic mobility shift assay and subsequent Hill transformation analysis revealed a low affinity of the phospho-mimicking rP20 for satBaMV RNA. Substantial modulation of satBaMV RNA conformation upon interaction with nonphospho-mimic rP20 in circular dichroism analysis indicated formation of stable satBaMV ribonucleoprotein complexes. The dissimilar satBaMV translation regulation of the nonphospho- and phospho-mimic rP20 suggests that phosphorylation of P20 in the ribonucleoprotein complex converts the translation-incompetent satBaMV RNA to messenger RNA. The phospho-deficient or phospho-mimicking P20 mutant of satBaMV delayed the systemic spread of satBaMV in co-infected Nicotiana benthamiana with BaMV. Thus, satBaMV likely regulates the formation of satBaMV RNP complex during co-infection in planta.
authors.affiliation: The Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan, Republic of China.
authors.forename: Paramasivan
authors.initials: P
authors.lastname: Vijayapalani
authors.forename: Jeff Chien-Fu
authors.initials: JC
authors.lastname: Chen
authors.forename: Ming-Ru
authors.initials: MR
authors.lastname: Liou
authors.forename: Hsin-Chuan
authors.initials: HC
authors.lastname: Chen
authors.forename: Yau-Heiu
authors.initials: YH
authors.lastname: Hsu
authors.forename: Na-Sheng
authors.initials: NS
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D019281:Dimerization; D015536:Down-Regulation; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D017863:Potexvirus / Q000235:genetics*; D014176:Protein Biosynthesis; D019244:RNA, Satellite / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D012694:Serine / Q000502:physiology; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019244:RNA, Satellite; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D014764:Viral Proteins; D012694:Serine