Open AccessRNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp
Author(s) -
Chumporn Soowannayan,
Jeff A. Cowley,
Wojtek P. Michalski,
Peter Walker
Publication year - 2011
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0022156
Subject(s) - rna , biology , peptide sequence , amino acid , nucleic acid , rna binding protein , gene , microbiology and biotechnology , genetics
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.