title: RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp.
abstract: Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His(6)-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (-) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met(11) to Arg(60). Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11)PVRRPLPPQPPRNARLI(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.
authors.affiliation: CSIRO Livestock Industries, Queensland Bioscience Precinct, St. Lucia, Queensland, Australia. chumporn@biotec.or.th
authors.forename: Chumporn
authors.initials: C
authors.lastname: Soowannayan
authors.forename: Jeff A
authors.initials: JA
authors.lastname: Cowley
authors.forename: Wojtek P
authors.initials: WP
authors.lastname: Michalski
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Walker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002043:Bunyaviridae / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005880:Gills / Q000821:virology; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D033561:Penaeidae / Q000821:virology*; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D046828:Roniviridae / Q000235:genetics / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D012313:RNA