Establishment of a Bacterial Expression System and Immunoassay Platform for the Major Capsid Protein of HcRNAV, a Dinoflagellate-Infecting RNA Virus

Abstract

HcRNAV is a small icosahedral virus that infects the shellfish-killing marine dinoflagellate Heterocapsa circularisquama, which harbors a dicistronic linear single-stranded RNA (ssRNA) genome ca. 4.4 kb in length. Its major capsid protein (MCP) gene sequence is not expressed by various strains of Escherichia coli, possibly because of a codon usage problem. To solve this problem, a chemically modified (i.e., de novo synthesized) gene was designed and cloned into the pCold-GST expression vector, and transformed into E. coli strain C41 (DE3), in which codon usage was universally optimized to efficiently express the polypeptide having the viral MCP amino acid sequence. The bacterially expressed protein, which was purified after a procedure involving denaturation and refolding, successfully formed virus-like particles that significantly resembled native HcRNAV particles. The purified, denatured protein was used as an antigen to immunize rabbits, and the resulting antiserum was shown to be strongly reactive to not only the bacterially expressed recombinant protein, but also to native HcRNAV MCP by Western blotting and dot immunoassays, respectively. These results indicate that an antiserum recognizing native HcRNAV MCP was successfully obtained using bacterially expressed HcRNAV MCP as the antigen.

Construction of target‐specific virus‐like particles for the delivery of algicidal compounds to harmful algae

“…Although a native‐like recombinant HcRNAV34 capsid protein was obtained herein from an E. coli overexpression system by thiol‐induced cleavage of the intein fusion protein, the observed particle size of the in vitro ‐assembled VLP (∼ 23–25 nm in diameter) was substantially smaller than that of the capsid shell of the authentic HcRNAV34 virion (Miller et al ., ). The self‐assembled VLP was also smaller than the in vitro assembly products of the HcRNAV109 (Wu et al ., ) and HcRNAV34 (Wada et al ., ) capsid proteins, which are ∼ 34 nm in diameter. The difference in size may reflect various morphological forms that a virion may adopt during the maturation, in which conformational changes of the capsid protein and the rearrangement of the capsomers by altered intermolecular interaction may take place (Salunke et al ., ; Hyun et al ., ).…”

“…A recent study heterologously expressed another recombinant HcRNAV34 capsid protein in E. coli that self‐assembled in the correct size of the native HcRNAV particle (Wada et al ., ), but this capsid protein was only obtained in soluble form by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) following a refolding procedure after denaturation of the recombinant protein with 8 M urea. We likewise failed in our previous attempts to obtain a soluble HcRNAV34 capsid protein in E. coli by using several types of tagging systems, including a glutathione S‐transferase tag, a maltose‐binding protein tag and a hexaHis‐tag.…”

Virologists are “Symbionts” in Microbial Ecology

“…The DNA viruses that would be involved in retreat of red tide algaes (24, 25) and the RNA virus infecting dinoflagellates (26, 35, 38) represent excellent examples of functional environmental viruses in aquatic ecosystems. In addition, although it is not yet justified as true viruses, there are pioneering reports on intracellular genetic elements that are of great interest as a gene transfer agent (9, 10).…”