المؤلفون: Soowannayan, Chumporn, Cowley, Jeff A., Michalski, Wojtek P., Walker, Peter J.

المصدر: PLoS ONE; 2011, Vol. 6 Issue 8, p1-9, 9p

مصطلحات موضوعية: CRUSTACEA, NUCLEOCAPSIDS, NIDOVIRUSES, SHRIMPS, PENAEIDAE, INVERTEBRATES, TRUNCATELLIDAE, RNA-protein interactions, NON-coding RNA

مستخلص: 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 sRNAs 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 sDNA. 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) Nterminal sequence spanning Met¹¹ to Arg⁶⁰. 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 (M¹¹PVRRPLPPQPPRNARLI²⁹) 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 [ABSTRACT FROM AUTHOR]

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