Packaging of Single-Stranded RNA in Viruses and Virus-Like Particles.

Porcine circovirus type 2 (PCV2) is considered to be associated with post-weaning multisystemic wasting syndrome (PMWS), which is a newly emerged economically important swine disease. The entire coding region of open reading frame 2 (ORF2), encoding the viral capsid protein (Cap), of PCV2 was cloned and sequenced from the clinical specimen obtained from PMWS-affected piglets. Six recombinant subunits, A–F, spanning the defined regions of Cap were produced by Escherichia coli (E. coli) expression system and used as antigens for testing their reactivities with swine sera in the indirect enzyme-linked immunosorbent assay (indirect ELISA). The recombinant Cap subunit-based ELISA was evaluated by examining a panel of 12 PCV2-negative and 26 PCV2-positive sera. When the positive/negative cut-off value was set at the mean value of negative sera plus 3 standard deviations, all subunits-based ELISA demonstrated 100% specificities. The N-terminal subunits, A and B, revealed poor reactivity with positive swine sera, whereas, greater immunoreactivity was observed for the C-terminal subunits of which subunits C and D demonstrated good sensitivities of 96.2% and 84.6%, respectively. The recombinant Cap subunits possessing defined antigenicity are easy to produce and the subunit-based ELISA was developed with a high specificity and sensitivity that may provide a useful method for routine serodiagnosis of PCV2 infection. Moreover, the sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using E. coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5′-end of the ORF2 gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.

RNA Homopolymers Form Higher-Curvature Virus-like Particles Than Do Normal-Composition RNAs

Keywords: : RNA virusRNA-dependent RNA polymeraseflock house virushorizontal gene transfernext-generation sequencing

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.

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.

Introduction. Viral capsid proteins can assemble into virus-like particles lacking infectivity and bearing parental virus antigens or artificially introduced antigens from other pathogens. At least some of such particles are highly immunogenic and could serve as a platform for promising vaccines. In this work, we assessed an effect of virus-like particles decorated with a SARS-CoV-2 spike protein fragment on human dendritic cell phenotype and functional properties. Materials and methods. The virus-like particles were assembled using chimeric molecules obtained by fusing genetic sequences encoding a norovirus major capsid protein VP1 fragment and a coronavirus spike protein fragment, including the receptor-binding domain. Dendritic cells were obtained from monocytes in vitro. Results. Incubation of immature dendritic cells with virus-like particles induced their phenotypic and functional maturation. The former was revealed by significantly increased expression of HLA-DR, CD80, CD86 and CD83. Dendritic cell phenotype after incubation with virus-like particles at the maximum concentration of 10 μg/ml did not differ significantly from that of mature dendritic cells in positive control. Along with phenotypic maturation, virus-like particles caused a manifold increase in the production of pro-inflammatory tumor necrosis factor-α, anti-inflammatory interleukin-10, as well as interleukin-6, which can stimulate both antibody synthesis and cellular pro-inflammatory reactions. The pronounced stimulation of dendritic cells by virus-like particles coated with coronavirus antigens evidence about successful particle recognition. Finally, we discuss plausible mechanisms for recognition of such virus-like particles by dendritic cell receptors. Conclusion. It has been shown that chimeric virus-like particles induced phenotypic and functional dendritic cell maturation, which is manifested by markedly elevated expression of functionally important membrane molecules, as well as a manifold rise in production of cytokines with a wide functional range. In our opinion, the data obtained indicate a promise of using virus-like particles based on norovirus proteins to display SARS-CoV-2 antigens.

The Assembly Pathway of an Icosahedral Single-Stranded RNA Virus Depends on the Strength of Inter-Subunit Attractions

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein–cargo and protein–protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

BackgroundProtein shells assembled from viral coat proteins are an attractive platform for development of new vaccines and other tools such as targeted bioimaging and drug delivery agents. Virus-like particles (VLPs) derived from the single-stranded RNA (ssRNA) bacteriophage coat proteins (CPs) have been important and successful contenders in the area due to their simplicity and robustness. However, only a few different VLP types are available that put certain limitations on continued developments and expanded adaptation of ssRNA phage VLP technology. Metagenomic studies have been a rich source for discovering novel viral sequences, and in recent years have unraveled numerous ssRNA phage genomes significantly different from those known before. Here, we describe the use of ssRNA CP sequences found in metagenomic data to experimentally produce and characterize novel VLPs.ResultsApproximately 150 ssRNA phage CP sequences were sourced from metagenomic sequence data and grouped into 14 different clusters based on CP sequence similarity analysis. 110 CP-encoding sequences were obtained by gene synthesis and expressed in bacteria which in 80 cases resulted in VLP assembly. Production and purification of the VLPs was straightforward and compatible with established protocols, with the only exception that a considerable proportion of the CPs had to be produced at a lower temperature to ensure VLP assembly. The VLP morphology was similar to that of the previously studied phages, although a few deviations such as elongated or smaller particles were noted in certain cases. In addition, stabilizing inter-subunit disulfide bonds were detected in six VLPs and several possible candidate RNA structures in the phage genomes were identified that might bind to the coat protein and ensure specific RNA packaging.ConclusionsCompared to the few types of ssRNA phage VLPs that were used before, several dozens of new particles representing ten distinct similarity groups are now available with a notable potential for biotechnological applications. It is believed that the novel VLPs described in this paper will provide the groundwork for future development of new vaccines and other applications based on ssRNA bacteriophage VLPs.

Nucleocapsid formation is a primary function of the rubella virus capsid protein, which also promotes viral RNA synthesis via an unknown mechanism. The present study demonstrates that in infected cells, the capsid protein is associated with the nonstructural p150 protein via the short self-interacting N-terminal region of the capsid protein. Mutational analyses indicated that hydrophobic amino acids in this N-terminal region are essential for its N-terminal self-interaction, which is critical for the capsid-p150 association. An analysis based on a subgenomic replicon system demonstrated that the self-interacting N-terminal region of the capsid protein plays a key role in promoting viral gene expression. Analyses using a virus-like particle (VLP) system also showed that the self-interacting N-terminal region of the capsid protein is not essential for VLP production but is critical for VLP infectivity. These results demonstrate that the close cooperative actions of the capsid protein and p150 require the short self-interacting N-terminal region of the capsid protein during the life cycle of the rubella virus. The capsid protein of rubella virus promotes viral RNA replication via an unknown mechanism. This protein interacts with the nonstructural protein p150, but the importance of this interaction is unclear. In this study, we demonstrate that the short N-terminal region of the capsid protein forms a homo-oligomer that is critical for the capsid-p150 interaction. These interactions are required for the viral-gene-expression-promoting activity of the capsid protein, allowing efficient viral growth. These findings provide information about the mechanisms underlying the regulation of rubella virus RNA replication via the cooperative actions of the capsid protein and p150.

The early history of life on Earth likely included a stage in which life existed as self-replicating protocells with single-stranded RNA (ssRNA) genomes. In this RNA world, genome damage from a variety of sources (spontaneous hydrolysis, UV, etc.) would have been a problem for survival. Selection pressure for dealing with genome damage would have led to adaptive strategies for mitigating the damage. In today’s world, RNA viruses with ssRNA genomes are common, and these viruses similarly need to cope with genome damage. Thus ssRNA viruses can serve as models for understanding the early evolution of genome repair. As the ssRNA protocells in the early RNA world evolved, the RNA genome likely gave rise, through a series of evolutionary stages, to the double-stranded DNA (dsDNA) genome. In ssRNA to dsDNA evolution, genome repair processes also likely evolved to accommodate this transition. Some of the basic features of ssRNA genome repair appear to have been retained in descendants with dsDNA genomes. In particular, a type of strand-switching recombination occurs when ssRNA replication is blocked by a damage in the template strand. Elements of this process appear to have a central role in recombinational repair processes during meiosis and mitosis of descendant dsDNA organisms.

JC Virus Intranuclear Inclusions Associated with PML-NBs: Analysis by Electron Microscopy and Structured Illumination Microscopy

BackgroundViruses cause significant economic losses to shrimp aquaculture worldwide. In severe cases, they can lead to 100% mortality within a matter of days, hence the aquaculture industry requires antiviral strategies to minimize economic impacts. Currently, a double-stranded RNA (dsRNA)-based platform has been proven effective at a laboratory scale. The bottleneck for its industrialization is the lack of low-cost, efficient and practical delivery approaches. In an effort to bridge the gap between laboratory and farm applications, virus-like particles (VLP) have been used as nanocarriers of dsRNA. However, the implementation of this approach still suffers from high costs and a lengthy procedure, co-expression of subunits of VLP or capsid proteins (CPs) and dsRNA can be the solution for the problem. CP and dsRNA are traditionally expressed in two different E. coli hosts: protease-deficient and RNase III-deficient strains. To condense the manufacturing of dsRNA-containing VLP, this study constructed a novel E. coli strain that is able to co-express viral capsid proteins and dsRNA in the same E. coli cell.ResultsA novel bacterial strain DualX-B15(DE3) was engineered to be both protease- and RNase III-deficiency via P1 phage transduction. The results revealed that it could simultaneously express recombinant proteins and dsRNA.ConclusionCo-expression of viral capsid proteins and dsRNA in the same cell has been shown to be feasible. Not only could this platform serve as a basis for future cost-effective and streamlined production of shrimp antiviral therapeutics, it may be applicable for other applications that requires co-expression of recombinant proteins and dsRNA.

Rabbit haemorrhagic disease virus (RHDV) capsid protein was expressed in a baculovirus system. Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in ELISA and Western blot with the antibodies raised against RHDV. Direct electron microscopy of the cell culture supernatant and the purified protein demonstrated that the capsid protein expressed in insect cells self-assembled to form empty virus-like particles (VLP) which are similar in size and morphology to that of native virus. These particles were immunoreactive with polyclonal anti-RHDV antibodies and with four monoclonal antibodies which recognise conformational epitopes of the virus. The results indicated that the VLPs were morphologically and antigenically indistinguishable from native virus. The recombinant VLPs induced high levels of RHDV-specific antibodies in rabbits and mice following immunisation. The immune response to the VLPs protected the rabbits following challenge with the virulent RHDV. In haemagglutination assays, the VLPs bound to human red blood cells similar to the native virus particles. The recombinant protein and or VLPs is suitable for the development of a rapid, sensitive and reliable test for detection of antibodies to RHDV and for use as a vaccine for domestic rabbits.

Objective To evaluate the immunopotentiating effect of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) as an adjuvant on norovirus (GⅡ.4) virus like particles (VLPs) in the development of norovirus vaccine. Methods BALB/c mice were intramuscularly immunized with norovirus (GⅡ.4) VLPs composed of capsid protein VP1 in combination with cGAMP or Al(OH)3. Norovirus VLPs-specific antibodies in serum were detected by ELISA. A synthetic histo-blood group antigen (HBGA)-VLPs blocking assay was used to analyze neutralizing antibodies against norovirus VLPs in serum samples. Results Immunization with norovirus VLPs in the presence of cGAMP induced a strong humoral immune response in BALB/c mice. Levels of specific IgG antibodies in serum induced by using cGAMP as the adjuvant were significantly higher than those induced by using Al(OH)3 adjuvant when immunization of BALB/c mice with the same dosage of VLPs. The antibody level induced by 1 μg of VLPs in combination with cGAMP was equivalent to that elicited by 10 μg of VLPs combined with Al(OH)3 adjuvant. Results of the synthetic HBGA-VLPs blocking assay showed that the blocking rate in cGAMP+ VLPs immunization group were significantly higher than that in Al(OH)3+ VLPs immunization group when using the same dosage of VLPs. No significant difference in blocking rate was observed between cGAMP+ VLPs (1 μg) and Al(OH)3+ VLPs (10 μg) immunization groups. Conclusion cGAMP significantly enhanced the specific humoral immune response induced by norovirus (GⅡ.4) VLPs in mice as compared with Al(OH)3 adjuvant. It might be used as a novel adjuvant to replace the traditional aluminum adjuvant in the development of norovirus vaccine. Key words: cGAMP; Norovirus; Virus like particles (VLPs); Aluminum adjuvant