Chumporn SoowannayanJeff A. CowleyWojtek P. MichalskiPeter J. WalkerCSIRO Livestock IndustriesThailand National Center for Genetic Engineering and BiotechnologyMahidol University2018-05-032018-05-032011-08-08PLoS ONE. Vol.6, No.8 (2011)193262032-s2.0-79961046297https://repository.li.mahidol.ac.th/handle/20.500.14594/11283Gill-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. © 2011 Soowannayan et al.Mahidol UniversityAgricultural and Biological SciencesBiochemistry, Genetics and Molecular BiologyMedicineArticleSCOPUS10.1371/journal.pone.0022156