A C-terminal truncated hepatitis C virus core protein variant assembles in vitro into virus-like particles in the absence of structured nucleic acids

Little is known about the assembly pathway or structure of the hepatitis C virus (HCV). In this work a truncated HCcAg variant covering the first 120 aa (HCcAg.120) with a 32 aa N-terminal fusion peptide (6x Histag-Xpress epitope) was purified as a monomer under strong denaturing conditions. In addition, minor HCcAg.120 peaks exhibiting little different molecular mass by SDS-PAGE which possibly represents alternative forms harboring the N-termini of HCcAg.120 were detected. Analysis using gel filtration chromatography showed that HCcAg.120 assembled into high molecular weight structures in vitro in the absence of structured nucleic acids. The negative-stain electron microscopy analysis revealed that these structures correspond with spherical VLPs of uniform morphology and size distribution. The diameters of these particles ranged from 20 to 43nm with an average diameter of approximately 30 nm and were specifically immunolabelled with a mouse monoclonal antibody against the residues 5-35 of HCcAg. Results presented in this work showed that HCcAg.120 assembled in vitro into VLPs in the absence of structured nucleic acids with similar morphology and size distribution to those found in sera and hepatocytes from HCV-infected patients. Therefore, these VLPs would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure.     

Nucleic acid binding properties and intermediates of HCV core protein multimerization in Pichia pastoris

Little is known about the in vivo assembly pathway or structure of the hepatitis C virus nucleocapsid. In this work the intermediates of HCcAg multimerization in Pichia pastoris cells and the nucleic acid binding properties of structured nucleocapsid-like particles (NLPs) were studied. Extensive cross-linking was observed for HCcAg after glutaraldehyde treatment. Data suggest that HCcAg exists in dimeric forms probably representing P21-P21, P21-P23, and P23-P23 dimers. In addition, the presence of HCcAg species that might represent trimers and multimers was observed. After sucrose equilibrium density gradient purification and nuclease digestion, NLPs were shown to contain both RNA and DNA molecules. Finally, the analysis by electron microscopy indicated that native NLPs were resistant to nuclease treatment. These results indicated that HCcAg assembles through dimers, trimers, and multimers' intermediates into capsids in P. pastoris cells. Assembly of NLPs in its natural environment might confer stability to these particles by adopting a compact structure.     

In vitro self-assembled HCV core virus-like particles induce a strong antibody immune response in sheep.

The in vitro self-assembly properties of the entire hepatitis C virus core protein (HCcAg) obtained from Pichia pastoris cells and the induction of specific antibody immune response were studied. HCcAg was purified as a low-molecular-weight species by electroelution under denaturing conditions for confirmation of its self-assembly properties. After renaturalization, electron microscopy showed that HCcAg assembled into spherical particles of 30 nm. HCcAg also showed homogeneity and was specifically recognized by serum from a chronic HCV carrier patient. The data indicated that in vitro assembly of HCcAg, into virus-like particles resembling HCV nucleocapsid particles at a mature stage, is an intrinsic quality of this protein. Finally, HCcAg generated a strong antibody immune response in sheep, suggesting its usefulness for stimulating the host immune response against HCV.     

Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast

The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.     

Mutation of interfacial residues disrupts subunit folding and particle assembly of Physalis mottle tymovirus

Virus-like particles (VLPs) serve as excellent model systems to identify the pathways of virus assembly. To gain insights into the assembly mechanisms of the Physalis mottle tymovirus (PhMV), six interfacial residues, identified based on the crystal structure of the native and recombinant capsids, were targeted for mutagenesis. The Q37E, Y67A, R68Q, D83A, I123A, and S145A mutants of the PhMV recombinant coat protein (rCP) expressed in Escherichia coli were soluble. However, except for the S145A mutant, which assembled into VLPs similar to that of wild type rCP capsids, all the other mutants failed to assemble into VLPs. Furthermore, the purified Q37E, Y67A, R68Q, D83A, and I123A rCP mutants existed essentially as partially folded monomers as revealed by sucrose density gradient analysis, circular dichroism, fluorescence, thermal, and urea denaturation studies. The rCP mutants locked into such conformations probably lack the structural signals/features that would allow them to assemble into capsids. Thus, the mutation of residues involved in inter-subunit interactions in PhMV disrupts both subunit folding and particle assembly.     

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants.

Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.     

Large conformational changes in the maturation of a simple RNA virus, nudaurelia capensis omega virus (NomegaV)

An assembly intermediate of a small, non-enveloped RNA virus has been discovered that exhibits striking differences from the mature virion. Virus-like particles (VLPs) of Nudaurelia capensis omega virus (NomegaV), a T=4 icosahedral virus infecting Lepidoptera insects, were produced in insect cells using a baculovirus vector expressing the coat protein. A procapsid form was discovered when NomegaV VLPs were purified at neutral pH conditions. These VLPs were fragile and did not undergo the autoproteolytic maturation that occurs in the infectious virus. Electron cryo-microscopy (cryoEM) and image analysis showed that, compared with the native virion, the VLPs were 16% larger in diameter, more rounded, porous, and contained an additional internal domain. Upon lowering the pH to 5.0, the VLP capsids became structurally indistinguishable from the authentic virion and the subunits autoproteolyzed. The NomegaV protein subunit coordinates, which were previously determined crystallographically, were modelled into the 28 A resolution cryoEM map of the procapsid. The resulting pseudo-atomic model of the NomegaV procapsid demonstrated the large rearrangements in quaternary and tertiary structure needed for the maturation of the VLPs and presumably of the virus. Based on this model, we propose that electrostatically driven rearrangements of interior helical regions are responsible for the large conformational change. These results are surprising because large structural rearrangements have not been found in the maturation of any other small RNA viruses. However, similarities of this conformational change to the maturational processes of more complex DNA viruses (e.g. bacteriophages and herpesvirus) and to the swelling of simple plant viruses suggest that structural changes in icosahedral viruses, which are integral to their function, have similar strategies and perhaps mechanisms.     

Distinct roles for nucleic acid in in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins

In contrast to other retroviruses, Mason-Pfizer monkey virus (M-PMV) assembles immature capsids in the cytoplasm. We have compared the ability of minimal assembly-competent domains from M-PMV and human immunodeficiency virus type 1 (HIV-1) to assemble in vitro into virus-like particles in the presence and absence of nucleic acids. A fusion protein comprised of the capsid and nucleocapsid domains of Gag (CANC) and its N-terminally modified mutant (DeltaProCANC) were used to mimic the assembly of the viral core and immature particles, respectively. In contrast to HIV-1, where CANC assembled efficiently into cylindrical structures, the same domains of M-PMV were assembly incompetent. The addition of RNA or oligonucleotides did not complement this defect. In contrast, the M-PMV DeltaProCANC molecule was able to assemble into spherical particles, while that of HIV-1 formed both spheres and cylinders. For M-PMV, the addition of purified RNA increased the efficiency with which DeltaProCANC formed spherical particles both in terms of the overall amount and the numbers of completed spheres. The amount of RNA incorporated was determined, and for both rRNA and MS2-RNA, quantities similar to that of genomic RNA were encapsidated. Oligonucleotides also stimulated assembly; however, they were incorporated into DeltaProCANC spherical particles in trace amounts that could not serve as a stoichiometric structural component for assembly. Thus, oligonucleotides may, through a transient interaction, induce conformational changes that facilitate assembly, while longer RNAs appear to facilitate the complete assembly of spherical particles.     

Folding and particle assembly are disrupted by single-point mutations near the autocatalytic cleavage site of Nudaurelia capensis omega virus capsid protein

Protein subunits of several RNA viruses are known to undergo post-assembly, autocatalytic cleavage that is required for infectivity. Nudaurelia capensis omega virus (Nomega V) is one of the simplest viruses to undergo an autocatalytic cleavage, making it an excellent model to understand both assembly and the mechanism of autoproteolysis. Heterologous expression of the coat protein gene of Nomega V in a baculovirus system results in the spontaneous assembly of virus-like particles (VLPs) that remain uncleaved when purified at neutral pH. After acidification to pH 5.0, the VLPs autocatalytically cleave at residue 570, providing an in vitro control of the cleavage. The crystal structure of Nomega V displays three residues near the scissile bond that were candidates for participation in the reaction. These were changed by site-directed mutagenesis to conservative and nonconservative residues and the products analyzed. Even conservative changes at the three residues dramatically reduced cleavage when the subunits assembled properly. Unexpectedly, we discovered that these residues are not only critical to the kinetics of Nomega V autoproteolysis, but are also necessary for proper folding of subunits and, ultimately, assembly of Nomega V VLPs.     

Development of a microRNA delivery system based on bacteriophage MS2 virus-like particles

Recently, microRNA (miRNA)-mediated RNA interference has been developed as a useful tool in gene function analysis and gene therapy. A major obstacle in miRNA-mediated RNAi is cellular delivery, which requires an efficient and flexible delivery system. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for RNA and drug delivery. However, MS2 VLP-mediated miRNA delivery has not yet been reported. We therefore used an Escherichia coli expression system to produce the pre-miR 146a contained MS2 VLPs, and then conjugated these particles with HIV-1 Tat(47-57) peptide. The conjugated MS2 VLPs effectively transferred the packaged pre-miR146a RNA into various cells and tissues, with 0.92-14.76-fold higher expression of miR-146a in vitro and about two-fold higher expression in vivo, and subsequently suppressed its targeting gene. These findings suggest that MS2 VLPs can be used as a novel vehicle in miRNA delivery systems, and may have applications in gene therapy.     

Self-assembly of viral capsid protein and RNA molecules of different sizes: requirement for a specific high protein/RNA mass ratio

Virus-like particles can be formed by self-assembly of capsid protein (CP) with RNA molecules of increasing length. If the protein "insisted" on a single radius of curvature, the capsids would be identical in size, independent of RNA length. However, there would be a limit to length of the RNA, and one would not expect RNA much shorter than native viral RNA to be packaged unless multiple copies were packaged. On the other hand, if the protein did not favor predetermined capsid size, one would expect the capsid diameter to increase with increase in RNA length. Here we examine the self-assembly of CP from cowpea chlorotic mottle virus with RNA molecules ranging in length from 140 to 12,000 nucleotides (nt). Each of these RNAs is completely packaged if and only if the protein/RNA mass ratio is sufficiently high; this critical value is the same for all of the RNAs and corresponds to equal RNA and N-terminal-protein charges in the assembly mix. For RNAs much shorter in length than the 3,000 nt of the viral RNA, two or more molecules are assembled into 24- and 26-nm-diameter capsids, whereas for much longer RNAs (>4,500 nt), a single RNA molecule is shared/packaged by two or more capsids with diameters as large as 30 nm. For intermediate lengths, a single RNA is assembled into 26-nm-diameter capsids, the size associated with T=3 wild-type virus. The significance of these assembly results is discussed in relation to likely factors that maintain T=3 symmetry in vivo.     

Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent

The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.     

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.     

The role of arginine-rich motif and beta-annulus in the assembly and stability of Sesbania mosaic virus capsids

Sesbania mosaic virus (SeMV) capsids are stabilized by protein-protein, protein-RNA and calcium-mediated protein-protein interactions. The N-terminal random domain of SeMV coat protein (CP) controls RNA encapsidation and size of the capsids and has two important motifs, the arginine-rich motif (ARM) and the beta-annulus structure. Here, mutational analysis of the arginine residues present in the ARM to glutamic acid was carried out. Mutation of all the arginine residues in the ARM almost completely abolished RNA encapsidation, although the assembly of T=3 capsids was not affected. A minimum of three arginine residues was found to be essential for RNA encapsidation. The mutant capsids devoid of RNA were less stable to thermal denaturation when compared to wild-type capsids. The results suggest that capsid assembly is entirely mediated by CP-dependent protein-protein inter-subunit interactions and encapsidation of genomic RNA enhances the stability of the capsids. Because of the unique structural ordering of beta-annulus segment at the icosahedral 3-folds, it has been suggested as the switch that determines the pentameric and hexameric clustering of CP subunits essential for T=3 capsid assembly. Surprisingly, mutation of a conserved proline within the segment that forms the beta-annulus to alanine, or deletion of residues 48-53 involved in hydrogen bonding interactions with residues 54-58 of the 3-fold related subunit or deletion of all the residues (48-59) involved in the formation of beta-annulus did not affect capsid assembly. These results suggest that the switch for assembly into T=3 capsids is not the beta-annulus. The ordered beta-annulus observed in the structures of many viruses could be a consequence of assembly to optimize intersubunit interactions.     

Tetracycline-inducible packaging cell line for production of flavivirus replicon particles

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 x 10(9) VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 10(10) VLPs per 10(6) transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8(+)-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.     

Assembly of human papillomavirus type-16 virus-like particles: multifactorial study of assembly and competing aggregation

Pentameric capsomeres of human papillomavirus capsid protein L1 expressed in Escherichia coli self-assemble into virus-like particles (VLPs) in vitro. A multifactorial experimental design was used to explore a wide range of solution conditions to optimize the assembly process. The degree of assembly was measured using an enzyme-linked immunosorbent assay, and a high-throughput turbidity assay was developed to monitor competing aggregation. The presence of zinc ions in the assembly buffer greatly increased the incidence of aggregation and had to be excluded from the experiment for meaningful analysis. Assembly of VLPs was optimal at a pH of about 6.5, calcium and sodium ions had no measurable effect, and dithiothreitol and glutathione inhibited assembly. Tryptophan fluorescence spectroscopy demonstrated that an increase in urea concentration reduced the rate of VLP formation but had no effect on the final concentration of assembled VLPs. This study demonstrates the use of the hanging-drop vapor-diffusion crystallization method to screen for conditions that promote aggregation and the use of tryptophan fluorescence spectroscopy for real-time monitoring of the assembly process.     

Roles of carboxyl-terminal and farnesylated residues in the functions of the large hepatitis delta antigen

The large hepatitis delta antigen (HDAg-L) mediates hepatitis delta virus (HDV) assembly and inhibits HDV RNA replication. Farnesylation of the cysteine residue within the HDAg-L carboxyl terminus is required for both functions. Here, HDAg-L proteins from different HDV genotypes and genotype chimeric proteins were analyzed for their ability to incorporate into virus-like particles (VLPs). Observed differences in efficiency of VLP incorporation could be attributed to genotype-specific differences within the HDAg-L carboxyl terminus. Using a novel assay to quantify the extent of HDAg-L farnesylation, we found that genotype 3 HDAg-L was inefficiently farnesylated when expressed in the absence of the small hepatitis delta antigen (HDAg-S). However, as the intracellular ratio of HDAg-S to HDAg-L was increased, so too was the extent of HDAg-L farnesylation for all three genotypes. Single point mutations within the carboxyl terminus of HDAg-L were screened, and three mutants that severely inhibited assembly without affecting farnesylation were identified. The observed assembly defects persisted under conditions where the mutants were known to have access to the site of VLP assembly. Therefore, the corresponding residues within the wild-type protein are likely required for direct interaction with viral envelope proteins. Finally, it was observed that when HDAg-S was artificially myristoylated, it could efficiently inhibit HDV RNA replication. Hence, a general association with membranes enables HDAg to inhibit replication. In contrast, although myristoylated HDAg-S was incorporated into VLPs far more efficiently than HDAg-S or nonfarnesylated HDAg-L, it was incorporated far less efficiently than wild-type HDAg-L; thus, farnesylation was required for efficient assembly.     

Does a cdc2 Kinase-Like Recognition Motif on the Core Protein of Hepadnaviruses Regulate Assembly and Disintegration of Capsids?

Hepadnaviruses are enveloped viruses, each with a DNA genome packaged in an icosahedral nucleocapsid, which is the site of viral DNA synthesis. In the presence of envelope proteins, DNA-containing nucleocapsids are assembled into virions and secreted, but in the absence of these proteins, nucleocapsids deliver viral DNA into the cell nucleus. Presumably, this step is identical to the delivery of viral DNA during the initiation of an infection. Unfortunately, the mechanisms triggering the disintegration of subviral core particles and delivery of viral DNA into the nucleus are not yet understood. We now report the identification of a sequence motif resembling a serine- or threonine-proline kinase recognition site in the core protein at a location that is required for the assembly of core polypeptides into capsids. Using duck hepatitis B virus, we demonstrated that mutations at this sequence motif can have profound consequences for RNA packaging, DNA replication, and core protein stability. Furthermore, we found a mutant with a conditional phenotype that depended on the cell type used for virus replication. Our results support the hypothesis predicting that this motif plays a role in assembly and disassembly of viral capsids.     

Assembly of the capsid protein of red-spotted grouper nervous necrosis virus during purification,and role of calcium ions in chromatography

Currently virus-like particles (VLPs) are receiving much attention as platforms for next generation vaccines. However, chromatography-based methods for purifying VLPs remain challenging. Unlike traditional methods using density gradient for purifying VLPs, there have been few advances in explaining how assembled particles can be obtained by chromatography. Nervous necrosis virus (NNV) infects over 30 species of fish and leads to large economic losses in the farmed fish industry. Previously we developed a heparin chromatography-based method for purifying red-spotted grouper NNV (RGNNV) VLPs. However it is unclear how the assembled RGNNV VLPs are obtained by this method. It is known that assembly of NNV capsid proteins depends on calcium ions. In the present study, we found that the yield of purified RGNNV capsid protein in heparin chromatography was enhanced when calcium ions were present during binding. Also, it appears that the capsid protein of RGNNV undergoes partial disassembly and reassembly during sample preparation prior to heparin chromatography and the protein finally undergoes assembly during the chromatography. Therefore, our results indicated that heparin-binding affinity of RGNNV capsid protein is linked to its ability for VLP formation. The assembly of RGNNV capsid proteins recombinantly produced is a good model for explaining VLP formation during chromatography-based purification processes.