Challenges for the production of virus-like particles in insect cells: The case of rotavirus-like particles

Virus-like particles (VLP) are formed when viral structural proteins are produced in an heterologous expression system. Such proteins assemble into structures that are morphologically similar to native viruses but lack the viral genome. VLP are complex structures with a wide variety of applications, ranging from basic research and vaccines to potential new uses in nanotechnology. Production of VLP is a challenging task, as both the synthesis and assembly of one or more recombinant proteins are required. This is the case for VLP of rotavirus (RLP), which is an RNA virus with a capsid formed by 1860 monomers of four different proteins. In addition, the production of most VLP requires the simultaneous expression and assembly of several recombinant proteins, which – for the case of RLP – needs to occur in a single host cell. The insect cell baculovirus expression vector system (IC-BEVS) has been shown to be a powerful and convenient system for rapidly and easily producing VLP, due to several convenient features, including its versatility and the short time needed for construction of recombinant baculovirus. In this review, the specific case of rotavirus-like particle (RLP) production by the IC-BEVS is discussed, with emphasis on bioprocess engineering issues that exist and their solutions. Many culture strategies discussed here can be useful for the production of other VLP.     

Intracellular distribution of rotavirus structural proteins and virus-like particles expressed in the insect cell-baculovirus system

The production of virus-like particles (VLP) is of interest to several fields. However, little is known about their assembly when they are expressed in insect cells, as it occurs in conditions different to those of native virus. Knowledge of the localization of recombinant proteins and of the site of accumulation of VLP can increase the understanding of VLP assembly and be useful for proposing production strategies. In this work, the rotavirus proteins VP6 and the fusion protein GFPVP2 were expressed in High Five insect cells. Recombinant proteins and rotavirus-like particles (RLP) were located and visualized by confocal, epifluorescence and electron microscopy. Single-layered (sl) RLP (conformed by GFPVP2) accumulated in the cytoplasm as highly ordered aggregates. In contrast, VP6 formed fibrillar structures composed of various tubes of VP6 that were not associated to microtubules. Coexpression of GFPVP2 and VP6 altered the distribution of both proteins. VP6 formed aggregates, even when all other conditions of individual protein expression remained unchanged. Double-layered (dl) RLP were observed in dense zones of the cytoplasm, but were not in ordered aggregates. It was determined that the assembly of both slRLP and dlRLP occurs intracellularly. Accordingly, strategies for the optimum assembly of dlRLP should guarantee that each cell produces both recombinant proteins.     

On the effect of thermodynamic equilibrium on the assembly efficiency of complex multi-layered virus-like particles (VLP): the case of rotavirus VLP

Previous studies have reported the production of malformed virus-like-particles (VLP) in recombinant host systems. Here we computationally investigate the case of a large triple-layered rotavirus VLP (RLP). In vitro assembly, disassembly and reassembly data provides strong evidence of microscopic reversibility of RLP assembly. Light scattering experimental data also evidences a slow and reversible assembly untypical of kinetic traps, thus further strengthening the fidelity of a thermodynamically controlled assembly. In silico analysis further reveals that under favourable conditions particles distribution is dominated by structural subunits and completely built icosahedra, while other intermediates are present only at residual concentrations. Except for harshly unfavourable conditions, assembly yield is maximised when proteins are provided in the same VLP protein mass composition. The assembly yield decreases abruptly due to thermodynamic equilibrium when the VLP protein mass composition is not obeyed. The latter effect is more pronounced the higher the Gibbs free energy of subunit association is and the more complex the particle is. Overall this study shows that the correct formation of complex multi-layered VLPs is restricted to a narrow range of association energies and protein concentrations, thus the choice of the host system is critical for successful assembly. Likewise, the dynamic control of intracellular protein expression rates becomes very important to minimize wasted proteins.     

Population kinetics during simultaneous infection of insect cells with two different recombinant baculoviruses for the production of rotavirus-like particles

Background  

The simultaneous production of various recombinant proteins in every cell of a culture is often needed for the production of virus-like particles (VLP) or vectors for gene therapy. A common approach for such a purpose is the coinfection of insect cell cultures with different recombinant baculoviruses, each containing one or more recombinant genes. However, scarce information exists regarding kinetics during multiple infections, and to our knowledge, no studies are available on the behavior of the different populations that arise during coinfections. Such information is useful for designing infection strategies that maximize VLP or vector yield. In this work, kinetics of cell populations expressing rotavirus GFPVP2 (infected with bacGFPVP2), VP6 (infected with bacVP6), or both proteins simultaneously (coinfected with both baculoviruses) were followed by flow cytometry.     

Catalase effect on cell death for the improvement of recombinant protein production in baculovirus-insect cell system

Baculovirus expression vector system (BEVS) in host insect cells is a powerful technology to produce recombinant proteins, as well as virus-like particles (VLP). However, BEVS is based on baculovirus infection, which limits the recombinant protein production by inducing insect cell death. Herein a new strategy to enhance cell life span and to increase recombinant protein production was developed. As baculovirus infection induces cellular oxidative stress, the ability of several antioxidants to inhibit cell death was tested during infection. The production of rotavirus structural proteins was used as model to analyse this new strategy. We found that only catalase is able to partially prevent cell death triggered by baculovirus infection and to inhibit lipid peroxidation. An increase in recombinant protein production was coupled with the partial cell death inhibition. In summary, the addition of catalase is a promising strategy to improve recombinant protein production in BEVS, by delaying insect cell death.     

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.     

Assembly of highly infectious rotavirus particles recoated with recombinant outer capsid proteins

Assembly of the rotavirus outer capsid is the final step of a complex pathway. In vivo, the later steps include a maturational membrane penetration that is dependent on the scaffolding activity of a viral nonstructural protein. In vitro, simply adding the recombinant outer capsid proteins VP4 and VP7 to authentic double-layered rotavirus subviral particles (DLPs) in the presence of calcium and acidic pH increases infectivity by a factor of up to 10(7), yielding particles as infectious as authentic purified virions. VP4 must be added before VP7 for high-level infectivity. Steep dependence of infectious recoating on VP4 concentration suggests that VP4-VP4 interactions, probably oligomerization, precede VP4 binding to particles. Trypsin sensitivity analysis identifies two populations of VP4 associated with recoated particles: properly mounted VP4 that can be specifically primed by trypsin, and nonspecifically associated VP4 that is degraded by trypsin. A full complement of properly assembled VP4 is not required for efficient infectivity. Minimal dependence of recoating on VP7 concentration suggests that VP7 binds DLPs with high affinity. The parameters for efficient recoating and the characterization of recoated particles suggest a model in which, after a relatively weak interaction between oligomeric VP4 and DLPs, VP7 binds the particles and locks VP4 in place. Recoating will allow the use of infectious modified rotavirus particles to explore rotavirus assembly and cell entry and could lead to practical applications in novel immunization strategies.     

Display of whole proteins on inner and outer surfaces of grapevine fanleaf virus‐like particles

Virus‐like particles (VLPs) derived from nonenveloped viruses result from the self‐assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self‐assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N‐ and C‐terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant‐based production of nucleic acid‐free VLPs. Remarkably, expression of N‐ or C‐terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.     

The concentration of Ca2+ that solubilizes outer capsid proteins from rotavirus particles is dependent on the strain.

It has been previously shown that rotavirus maturation and stability of the outer capsid are calcium-dependent processes. More recently, it has been hypothesized that penetration of the cell membrane is also affected by conformational changes of the capsid induced by Ca2+. In this study, we determined quantitatively the critical concentration of calcium ion that leads to solubilization of the outer capsid proteins VP4 and VP7. Since this critical concentration is below or close to trace levels of Ca2+, we have used buffered solutions based on ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca-EGTA. This method allowed us to show a very high variability of the free [Ca2+] needed to stabilize, at room temperature, the outer capsid of several rotavirus strains. This concentration is about 600 nM for the two bovine strains tested (RF and UK), 100 nM for the porcine strain OSU, and only 10 to 20 nM for the simian strain SA11. Titration of viral infectivity after incubation in buffer of defined [Ca2+] confirmed that the loss of infectivity occurs at different [Ca2+] for these three strains. For the bovine strain, the cleavage of VP4 by trypsin has no significant effect on the [Ca2+] that solubilizes outer shell proteins. The outer layer (VP7) of virus-like particles (VLP) made of recombinant proteins VP2, VP6, and VP7 (VLP2/6/7) was also solubilized by lowering the [Ca2+]. The critical concentration of Ca2+ needed to solubilize VP7 from VLP2/6/7 made of protein from the bovine strain is close to the concentration needed for the corresponding virus. Genetic analysis of this phenotype in a set of reassortant viruses from two parental strains having the phenotypes of strains OSU (porcine) and UK (bovine) confirmed that this property of viral particles is probably associated with the gene coding for VP7. The analysis of VLP by reverse genetics might allow the identification of the region(s) essential for calcium binding.     

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.     

Role of ubiquitin in parainfluenza virus 5 particle formation

Ubiquitin is important for the budding of many retroviruses and other enveloped viruses, but the precise role of ubiquitin in virus budding remains unclear. Here, we characterized the ubiquitination of the matrix (M) protein of a paramyxovirus, parainfluenza virus 5 (PIV5). The PIV5 M protein (but not the PIV5 nucleocapsid protein) was found to be targeted for monoubiquitination in transfected mammalian cells. Major sites of ubiquitin attachment identified by mass spectrometry analysis were lysine residues at amino acid positions 79/80, 130, and 247. The cumulative mutation of lysine residues 79, 80, and 130 to arginines led to an altered pattern of M protein ubiquitination and impaired viruslike particle (VLP) production. However, the cumulative mutation of lysine residues 79, 80, 130, and 247 to arginines restored M protein ubiquitination and VLP production, suggesting that ubiquitin is attached to alternative sites on the M protein when the primary ones have been removed. Additional lysine residues were targeted for mutagenesis based on the UbiPred algorithm. An M protein with seven lysine residues changed to arginines exhibited altered ubiquitination and poor VLP production. A recombinant virus encoding an M protein with seven lysines mutated was generated, and this virus exhibited a 6-fold-reduced maximum titer, with the defect being attributed mainly to the budding of noninfectious particles. The recombinant virus was assembly deficient, as judged by the redistribution of viral M and hemagglutinin-neuraminidase proteins in infected cells. Similar assembly defects were observed for the wild-type (wt) virus after treatment with a proteasome inhibitor. Collectively, these findings suggest that the monoubiquitination of the PIV5 M protein is important for proper virus assembly and for the budding of infectious particles.     

Influenza virus matrix protein is the major driving force in virus budding

To get insights into the role played by each of the influenza A virus polypeptides in morphogenesis and virus particle assembly, the generation of virus-like particles (VLPs) has been examined in COS-1 cell cultures expressing, from recombinant plasmids, different combinations of the viral structural proteins. The presence of VLPs was examined biochemically, following centrifugation of the supernatants collected from transfected cells through sucrose cushions and immunoblotting, and by electron-microscopic analysis. It is demonstrated that the matrix (M1) protein is the only viral component which is essential for VLP formation and that the viral ribonucleoproteins are not required for virus particle formation. It is also shown that the M1 protein, when expressed alone, assembles into virus-like budding particles, which are released in the culture medium, and that the recombinant M1 protein accumulates intracellularly, forming tubular structures. All these results are discussed with regard to the roles played by the virus polypeptides during virus assembly.     

Different applications of virus‐like particles in biology and medicine: Vaccination and delivery systems

Virus‐like particles (VLPs) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. VLPs can elicit efficient protective immunity as direct immunogens compared to soluble antigens co‐administered with adjuvants in several booster injections. Up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and E. coli were used to express recombinant proteins, especially for VLP production. Recent studies are also generating VLPs in plants using different transient expression vectors for edible vaccines. VLPs and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. Herein, we describe VLP production in different systems as well as its applications in biology and medicine. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 113–132, 2016.     

Triple layered rotavirus VLP production: kinetics of vector replication, mRNA stability and recombinant protein production

Rotavirus infection causes diarrhoeal disease in infants, killing more than half million children each year. Virus-like particles (VLP) seem to be excellent vaccine candidates, since they are cheaper to produce than attenuated viral vaccines and safer, as they do not contain genetic material. The present work focus on a triple layered particle composed by three rotavirus structural proteins: VP2, VP6 and VP7, produced in an insect cell/baculovirus expressing system. Two strategies were evaluated for 2/6/7 VLP production: co-infection with three monocistronic baculovirus vectors or single-infection with a tricistronic multi-gene baculovirus vector; these strategies were followed at different levels: baculovirus DNA replication kinetics, mRNA stability, protein production and VLP formation. This study highlights some of the reasons why the tricistronic baculovirus strategy is more efficient for production of triple layered rotavirus 2/6/7 VLP than monocistronic co-infection, in particular: (i) the tricistronic vector presents higher DNA replication rates than the monocistronic vectors, (ii) the mRNA stability is invariant for all mRNAs corresponding to VP2, VP6 and VP7 and (iii) the tricistronic baculovirus strategy produces an excess of VP7 over VP6 when compared to the VP7/VP6 stoichiometric ratio in the native rotavirus. Although the co-infection strategy leads to protein production akin to the rotavirus VP7/VP6 stoichiometric ratio, the tricistronic vector strategy yields higher amounts of rotavirus-like particles.     

The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles

The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.     

Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells.

Rotaviruses are triple-layered particles that contain four major capsid proteins, VP2, VP4, VP6, and VP7, and two minor proteins, VP1 and VP3. We have cloned each of the rotavirus genes coding for a major capsid protein into the baculovirus expression system and expressed each protein in insect cells. Coexpression of different combinations of the rotavirus major structural proteins resulted in the formation of stable virus-like particles (VLPs). The coexpression of VP2 and VP6 alone or with VP4 resulted in the production of VP2/6 or VP2/4/6 VLPs, which were similar to double-layered rotavirus particles. Coexpression of VP2, VP6, and VP7, with or without VP4, produced triple-layered VP2/6/7 or VP2/4/6/7 VLPs, which were similar to native infectious rotavirus particles. The VLPs maintained the structural and functional characteristics of native particles, as determined by electron microscopic examination of the particles, the presence of nonneutralizing and neutralizing epitopes on VP4 and VP7, and hemagglutination activity of the VP2/4/6/7 VLPs. The production of VP2/4/6 particles indicated that VP4 interacts with VP6. Cell binding assays performed with each of the VLPs indicated that VP4 is the viral attachment protein. Chimeric particles containing VP7 from two different G serotypes also were obtained. The ability to express individual proteins or to coexpress different subsets of proteins provides a system with which to examine the interactions of the rotavirus structural proteins, the role of individual proteins in virus morphogenesis, and the feasibility of a subunit vaccine.     

Baculovirus-mediated production of HDV-like particles in BHK cells using a novel oscillating bioreactor

We have recently demonstrated the assembly of hepatitis delta virus-like particles (HDV VLP) by co-transducing hepatoma cells using two recombinant baculoviruses, one encoding hepatitis B surface antigen (HBsAg), and one encoding large delta antigen (L-HDAg). In this study, we further demonstrated the assembly and secretion of VLP in other mammalian cells. The assembly efficiency varied depending on cell lines, the baculovirus constructs and the relative dosage of both recombinant viruses. The co-transduction of BHK cells led to the formation of VLPs resembling authentic virions in size and appearance. The production process was transferred to a novel oscillating packed bed bioreactor, BelloCell, in which the transduction efficiency was up to approximately 90% for a high cell density of 1.5 x 10(7) cells/cm(3) bed and a total yield of 427 microg based on HBsAg in the VLP (harvested from 940 ml medium) was obtained. The particle yield corresponded to an average volumetric yield of 454 ngml(-1) and a specific yield of 285 microg/10(9) cells, and is significantly superior to that can be obtained by the commonly employed transfection method. The combination of baculovirus transduction and BelloCell reactor, thus, may represent a simple and efficient approach for the production of HDV VLP and viral vectors.     

Accelerating HIV-1 VLP production using stable High Five insect cell pools

Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.     

Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli

Virus-like particles (VLPs) are formed by the self-assembly of envelope and/or capsid proteins from many viruses. Some VLPs have been proven successful as vaccines, and others have recently found applications as carriers for foreign antigens or as scaffolds in nanoparticle biotechnology. However, production of VLP was usually impeded due to low water-solubility of recombinant virus capsid proteins. Previous studies revealed that virus capsid and envelope proteins were often posttranslationally modified by SUMO in vivo, leading into a hypothesis that SUMO modification might be a common mechanism for virus proteins to retain water-solubility or prevent improper self-aggregation before virus assembly. We then propose a simple approach to produce VLPs of viruses, e.g., foot-and-mouth disease virus (FMDV). An improved SUMO fusion protein system we developed recently was applied to the simultaneous expression of three capsid proteins of FMDV in E. coli. The three SUMO fusion proteins formed a stable heterotrimeric complex. Proteolytic removal of SUMO moieties from the ternary complexes resulted in VLPs with size and shape resembling the authentic FMDV. The method described here can also apply to produce capsid/envelope protein complexes or VLPs of other disease-causing viruses.