Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells

Kosukegawa, Akinobu; Arisaka, Fumio; Takayama, Makoto; Yajima, Hiroaki; Kaidow, Akihiro; Handa, Hiroshi

doi:10.1016/0304-4165(95)00184-0

Cited by 63 publications

(53 citation statements)

References 29 publications

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“…The baculovirus expression system has extensively been used to express capsid protein virus genes and to produce selfassembled VLPs which represent a good tool for the study of viral morphogenesis (7,11,15,17,39). In the present work, it has been shown that the expression of the full ORF2 of the HAstV-1 genome in the baculovirus expression system leads to the formation of VLPs, as it has been recently described for the vaccinia system (8).…”

Section: Discussionmentioning

confidence: 99%

Structural Requirements of Astrovirus Virus-Like Particles Assembled in Insect Cells

Caballero¹,

Guix²,

Ribes

et al. 2004

J Virol

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Expression of the complete ORF2 of human astrovirus serotype 1 (HAstV-1) in the baculovirus system led to the formation of virus-like particles (VLPs) of around 38 nm. The same kind of VLPs were also obtained either with the expression of a truncated form of ORF2 lacking the first 70 amino acids (aa), or with the same truncated form in which those 70 aa were replaced by the green fluorescent protein. All three kinds of VLPs were equally recognized by an anti-HAstV-1 polyclonal antibody and by two monoclonal antibodies (MAbs; 8E7 and 5B7), indicating a nonessential role of those amino acids neither in the capsid assembly nor in the antigen structure. A second type of structure consisting of 16-nm ring-like units was observed in all of the cases, mostly after disassembling the 38-nm VLPs through the addition of EDTA. The removal of the EDTA and the addition of Mg 2؉ ions promoted the reassembly of the 38-nm VLPs. The nature of these 16-nm ring-like structures, capsomers or T ‫؍‬ 1 VLPs, still remains unclear. Biochemical analysis revealed no differences between the 38-nm VLPs and the 16-nm structures, whereas antigenically, they shared the 8E7 MAb epitope but differed in the 5B7 MAb epitope, with the latter structures being more readily recognized.Human astroviruses (HAstV) are a frequent causal agent of gastroenteritis in children worldwide (5,10,12,14,24,27,28,35,38), although they have also been associated with the elderly (4, 29, 34). Eight serotypes have been described, with serotype 1 (HAstV-1) being the most globally prevalent (12,14,18,27,28,33,36). Astroviruses are nonenveloped viruses whose capsid is around 28 to 41 nm in diameter and contains a plus-sense single-stranded RNA of around 6.9 kb organized in three open reading frames (ORFs) (20,32,37). ORF1a and ORF1b encode the nonstructural proteins (19,21,22), whereas ORF2 encodes the structural proteins through a subgenomic RNA (25). ORF2 of HAstV-1 encodes a polyprotein of 787 amino acids (aa) in length, with a molecular mass of around 87 kDa (26), which is the precursor of the smaller 24-to 26-kDa, 29-to 31-kDa, and 32-to 34-kDa structural proteins (1,3,26). The proteolytical processing from the precursor to the mature structural proteins is still very controversial, and to date, three different models have been proposed (1,13,23). In the first model, Bass and Qiu (1) proposed an intracellular processing of the precursor polyprotein at the amino terminus between residues 70(R) and 71(K) before capsid assembly, with this capsid being further processed extracellularly by the action of trypsin and giving the above-mentioned mature proteins. In subsequent studies, the intracellular processing of this model was refused (13), with the complete precursor being the assembly unit. Later on, Méndez and colleagues (23) proposed a third model in which the structural precursor of HAstV-8 is intracellularly processed at the carboxy terminus prior to its assembly into the capsid, although the cleavage site has not yet been identified.The expression of the genomes en...

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Section: Discussionmentioning

confidence: 99%

Structural Requirements of Astrovirus Virus-Like Particles Assembled in Insect Cells

Caballero¹,

Guix²,

Ribes

et al. 2004

J Virol

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“…Expression and Purification of Capsid Proteins-The construction of the SV40 VP1-expressing baculovirus and expression of the VP1 protein in Spodoptera frugiperda (Sf9) cells have been described (9,25,26). Briefly, VP1 protein assembled into particles (VP1-VLPs) in Sf9 cells was purified in two successive fractionations by cesium chloride density gradient centrifugation.…”

Section: Methodsmentioning

confidence: 99%

“…Particles-We have established a system to evaluate conditions for the assembly of highly purified recombinant VP1 pentamers into particles by dialyzing a VP1-containing solution against various buffers, with subsequent EM observation (9,25,26). As we showed previously, VP1 pentamers do not assemble into particles under physiological salt and pH conditions (150 mM NaCl, pH 7.0) (Fig.…”

Section: Minor Capsid Protein Vp2 Promotes Assembly Of Vp1 Into Virusmentioning

confidence: 99%

“…The properties of VP1-VLPs are very similar to those of wild type virions in that they dissociate into pentamers following treatment with a calcium-chelating agent (EGTA) under reducing conditions in vitro (22)(23)(24). Previously, we prepared highly purified SV40 VP1 pentamers (9,25,26), which assembled into morphologically heterogeneous particles under various nonphysiological conditions (26). Such particles include T ϭ 7d (triangulation number 7 dextro) spherical particles, small T ϭ 1 (triangulation number 1) particle composed of 12 pentamers, and long tubular structures of 40 -45 nm in diameter (26).…”

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confidence: 99%

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The VP2/VP3 Minor Capsid Protein of Simian Virus 40 Promotes the in Vitro Assembly of the Major Capsid Protein VP1 into Particles

Kawano

Inoue

Tsukamoto

et al. 2006

Journal of Biological Chemistry

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The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119 -272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors. Viral capsids are highly organized protein complexes that assemble and disassemble depending on environmental conditions during the viral life cycle. An elucidation of the mechanisms of assembly and disassembly of viral capsids may greatly help in understanding these highly organized protein complexes.SV40 is a small, nonenveloped DNA tumor virus that belongs to the family Polyomaviridae. Its capsid is formed by 72 copies of pentamers of the VP1 major capsid protein (360 molecules in total) and 72 molecules of the VP2 or VP3 minor capsid proteins. VP1 pentamers are arranged in a T ϭ 7d icosahedral lattice, and the carboxyl-terminal arm of VP1 mediates inter-pentameric contacts that hold the capsid together (1-4). VP3 is an amino-terminally truncated form of VP2. One molecule of either minor capsid protein binds to the center of a VP1 pentamer through their common carboxyl-terminal region inside the virion (5-7).However, the precise molecular mechanism for virion assembly is not known.We and others have shown that the VP1 proteins of SV40 (8 -12) and of closely related viruses such as JC virus (13, 14), murine polyomavirus (8,(15)(16)(17)(18)(19), and papillomavirus (20, 21) form virus-like particles (VLPs) 2 when expressed in insect Sf9 cells from baculoviral vectors. The properties of VP1-VLPs are very similar to those of wild type virions in that they dissociate into pentamers following treatment with a calcium-chelating agent (EGTA) under reducing conditions in vitro (22-24). Previously, we prepared highly purified SV40 VP1 pentamers (9, 25, 26), which assembled into morphologically heterogeneous particles under various nonphysiological conditions (26). Such particles include T ϭ 7d (triangulation number 7 dextro) spherical particles, small T ϭ 1 (triangulation number 1) particle composed of 12 pentamers, and long tubu...

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“…Of these, HbsAg has a well-established scale-up platform in different hosts (Hardy et al 2000;James et al 2007;Shah et al 2015;Valenzuela et al 1982), while large scale platforms for AdDd in insect cells (Szurgot et al 2015), and SV40 in insect cells (Kosukegawa et al 1996) are still in development.…”

Section: ) They All Havementioning

confidence: 99%

Murine polyomavirus VLPs as a platform for cytotoxic T cell epitope-based influenza vaccine candidates

Abidin¹

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This research examined the ability of virus-like particles (VLP) assembled from the coat proteins of Murine polyomavirus (MPyV) to carry cytotoxic T cell (Tc) epitopes. MPyV VLPs can be assembled in vitro from purified subunits composed of pentamers of the major coat protein VP1 called capsomeres; and this approach provides fine control over VLP composition and structure. Tc-epitopes are often highly hydrophobic, which poses a significant bioengineering challenge and two distinct approaches were pursued to determine the optimal delivery strategy of Influenza Tc-epitopes by MPyV VLP. Firstly, identified epitopes were externally presented using established sites for peptide insertion on the MPyV major coat protein, VP1. Secondly, polypeptides possessing multiple epitopes were encapsidated within VP1 VLP using precise elements of the minor coat protein VP2/3 that interact with the interior cavity of capsomeres. Hydrophobicity-related capsomere aggregation arising from single Tc epitope presentation was successfully circumvented by engineering charged amino acids (DD) flanking the epitope displayed on a surface-exposed loop of VP1 and by use of an optimised buffer condition. A multiparametric, high-throughput buffer screen was implemented to optimise pH and buffer additives during affinity tag removal. Stable modified capsomere recovered from this reaction were assembly competent in the presence of L-Arginine, and VLP yield was high when modular capsomeres were co-assembled with unmodified VP1 capsomeres forming stable mosaic VLPs. The ability of the MPyV VLP to encapsidate foreign protein was first evaluated and optimised with green fluorescent protein (GFP) as a model protein to enable monitoring and analysis.A minimal VP1-interacting sequence was defined from the carboxy-terminus of VP2/3 (VP2C) and fused to the amino-terminus of GFP, ensuring internalisation of the reporter protein. VP1:VP2C-GFP capsomere complexes were successfully recovered when VP1 was co-expressed with VP2C-GFP, whereas complex formation was not observed when VP1 and VP2C-GFP were mixed in vitro.Recovered capsomere complexes were assembly competent and amount of encapsidated GFP was controllable when VP1:VP2C-GFP capsomere complexes were co-assembled with unmodified VP1 capsomeres in a defined molar ratio. The encapsidation approach was then applied to a subdomain of the Influenza matrix protein M1 by co-expressing VP1 with VP2C-M1-I. M1-I capsomere complexes were assembly competent as indicated by gel electrophoresis, dynamic light scattering, and transmission electron microscopy. To enable recovery of VLPs for analysis and characterisation as well as to confirm encapsidation and the formation of mosaic VLPs, a semi-preparative size exclusion chromatography method was developed. This research was able to address bioengineering challenges posed by hydrophobic epitope presentation and developed MPyV

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Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells

Cited by 63 publications

References 29 publications

Structural Requirements of Astrovirus Virus-Like Particles Assembled in Insect Cells

Structural Requirements of Astrovirus Virus-Like Particles Assembled in Insect Cells

The VP2/VP3 Minor Capsid Protein of Simian Virus 40 Promotes the in Vitro Assembly of the Major Capsid Protein VP1 into Particles

Murine polyomavirus VLPs as a platform for cytotoxic T cell epitope-based influenza vaccine candidates

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