rna-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp核衣壳蛋白的rna结合域gill-associated nidovirus penaeid虾.pdfVIP

RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp 1,3,4 1 2 1,2 Chumporn Soowannayan *, Jeff A. Cowley , Wojtek P. Michalski , Peter J. Walker 1 CSIRO Livestock Industries, Queensland Bioscience Precinct, St. Lucia, Queensland, Australia, 2 CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria, Australia, 3 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand Science Park, Klong Luang, Patumthani, Thailand, 4 CENTEX SHRIMP, Faculty of Science, Mahidol University, Bangkok, Thailand 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 His6-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 ( 2) 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 Met11 to Arg60. Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine