Infection competition against grouper nervous necrosis virus by virus-like particles produced in Escherichia coli Ming-Wei Lu, Wangta Liu and Chan-Shing Lin Correspondence Chan-Shing Lin shinlin@mail.nsysu.edu.tw Institute of Marine Resources 804, National Sun Yat-Sen University, Kaohsiung, Taiwan Received 11 June 2002 Accepted 14 January 2003 Dragon grouper (Epinephelus lanceolatus) nervous necrosis virus (DGNNV) comprises 180 copies of capsid protein that encapsulate a bipartite genome of single-stranded (+)-RNAs. This study reports that virus-like particles (VLPs) are formed in Escherichia coli expressing the full-length ORF encoding the DGNNV capsid protein. Two sizes of VLPs are observed. The heavier particles resemble the native piscine nodavirus in size and stain permeability, while the lighter ones are approximately two-thirds of the full size. The recombinant VLPs block attachment of native virus to the surface of cultured fish nerve cells, blocking infection by the native virus. INTRODUCTION Piscine nodaviruses infect more than 20 species of fish around Asia (Skliris et al., 2001), Europe (Grotmol et al., 1997; Starkey et al., 2001) and Australia (Munday et al., 1994). Infection causes nervous necrosis, encephalopathy and retinopathy, accompanied by abnormal swimming beha- viour, dark colouring, and massive mortality in hatchery- reared larvae and juveniles (Munday & Nakai, 1997). The piscine nodaviruses possess two positive RNA strands, ~3?1 kb RNA1 and ~1?4 kb RNA2. The nucleotide sequences of both genomic RNAs of striped jack nervous necrosis virus (SJNNV) (Nishizawa et al., 1995; Nagai & Nishizawa, 1999) and greasy grouper nervous necrosis virus (GGNNV) (Tan et al., 2001) have been determined. The sequences of the RNA2 of Dicentrarchus labrax encephalitis virus (DlEV) (Delsert et al., 1997), Atlantic halibut virus (AHV) (Grotmol et al., 2000) and two grouper viruses [malabaricus grouper (MGNNV) and dragon grouper (DGNNV)] (Lin et al., 2001) have also been determined. The larger genome strand, RNA1, presumably encodes the viral replicase, while the smaller genome strand, RNA2, has been demonstrated to encode the capsid protein (Lin et al., 2001). By using the baculovirus expression system in Spodoptera frugiperda cell line Sf21, our previous study demonstrated that MGNNV VLPs are small, nonenveloped T=3 quasi- symmetric particles (Lin et al., 2001; Tang et al., 2002). The capsid protein of MGNNV spontaneously assembles into recombinant VLPs when expressed in Sf21 cells infected with a recombinant baculovirus (Lin et al., 2001). The VLPs were indistinguishable from the native virus particles by electron microscopy. The three-dimensional structure of the VLPs has been determined to a resolution of 2?3 nm by electron cryomicroscopy reconstruction imaging (Tang et al., 2002). The VLPs and related mutants can be used to understand the structure of the virus. High purity of virus or VLPs in quantities of about 100 mg is required for determination of crystal structure by X-ray diffraction. In situ hybridization has been employed to study the infection path of the virus in fish nerve cells (Comps et al., 1996). The labelled virus or structure-analogue VLPs can be used to study the receptor on the nerve cells. Nevertheless, the quantities of native virus and VLPs from Sf21 cells are limiting for crystallization and receptor-binding experiments. In contrast, Escherichia coli is the simplest host system for gene manipulation and has also been employed to express several viral VLPs (Qi et al., 1996; Bragard et al., 2000). This report is the first attempt to express nodavirus VLPs in E. coli and characterizes the ability of the VLPs to compete against binding of and infection by native DGNNV. METHODS Constructing the full-length ORF encoding DGNNV capsid protein. Plasmid pCA3, bearing the capsid protein gene, was con- structed using the RNA template from dragon grouper (Epinephelus lanceolatus) nervous necrosis virus. The sequence of the capsid pro- tein gene has been documented in GenBank (AF245004) and described by Lin et al. (2001). The viral capsid protein ORF was inserted by PCR into the vector pQE30 (Qiagen), to give pDA8. The annealing conditions for the PCR reaction were 49 ˚ C for 2 min. The primers were NW9a (59-CAC AGA ATT CAT TAA AGA GGA GAA ATT AAC CAT GGT ACG CAA AGG T-39) and NW2 (59-TTT ATC TAG ATG GCG GTG-39), and the template was pCA3. The PCR product was digested with EcoRI and KpnI and ligated into pQE30. The NcoI site of pQE30 was filled-in prior to ligation. This resulting plasmid, pDA8, contained an E. coli ribosome-binding site (AGGAG) and a full-length DGNNV capsid protein ORF, flanked by an IPTG-inducible promoter (T5pr) and the 0001-8649 G 2003 SGM Printed in Great Britain 1577 Journal of General Virology (2003), 84, 1577–1582 DOI 10.1099/vir.0.18649-0