RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp

Chumporn Soowannayan,Jeff A Cowley,Peter J Walker,Wojtek P Michalski,David Harrich

doi:10.1371/journal.pone.0022156

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 (−) 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 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.

Highlights

Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species of the genus Okavirus in the Roniviridae, the only currently known invertebrate nidoviruses
We have examined recombinant GAV N protein constructs in electrophoretic mobility shift assays (EMSAs) to identify its nucleic acid binding specificities in vitro, and its RNA binding domain, as initial steps to understanding the process by which nucleocapsids form in okaviruses
In addition to using Coomassie brilliant blue (CBB)-stained gels (Fig. 1B), the fidelity of the recombinant His6-N protein constructs was confirmed in Western blots using horseradish peroxidase (HRP) labeled-Ni2+ to detect the His6-tag on each protein (Fig. 1C)

Summary

Introduction

Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species of the genus Okavirus in the Roniviridae, the only currently known invertebrate nidoviruses. Amongst strains of genotypes 1 (YHV), 2 (GAV), 3, 4 and 5 in the YHV complex [6], most amino acid variations (up to 17.2%) in the deduced N protein sequence occur in the highly charged Nand C-terminal domains [3,6]. As with the N proteins of coronaviruses and toroviruses [8,9], the N proteins of GAV and YHV lack cysteine residues and are highly basic [2,3]. It possesses proline-rich and basic residue-rich domains likely to facilitate RNA binding as hypothesized for similar sequences in the N protein of toroviruses [2,10]. The N protein length in okaviruses is intermediate to the corresponding proteins of arteriviruses (110– 128 aa) and toroviruses (160–167 aa), and much shorter than the N protein of coronaviruses (377–454 aa) [2,11,12,13,14]

Methods

Results

Conclusion