Site-directed mutation affecting polyomavirus capsid self-assembly in vitro

Garcea, Robert L.; Salunke, Dinakar M.; Caspar, D. L. D.

doi:10.1038/329086a0

Cited by 78 publications

(44 citation statements)

References 10 publications

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“…In vitro assembly of polyomavirus VP1 into virus-like particles is induced by adding CaCl 2 and inhibited by EDTA (15, 16). Capsomere contacts within the virus shell are accomplished by VP1's flexible C-terminal arm, which protrudes into a neighboring pentamer (10,20). Although intrapentamer disulfide bonds are observed in the crystal structure between residues C19 and C114Ј of neighboring subunits (18,20), it is not clear whether they are sufficient or essential for capsid stabilization, or if there is also involvement of intercapsomere disulfide bonds which are not resolved in the crystal structure.…”

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confidence: 95%

Mechanism of Assembly of Recombinant Murine Polyomavirus-Like Particles

Schmidt

Rudolph

Böhm

2000

J Virol

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VP1 is the major viral coat protein of murine polyomavirus and can be used for the generation of virus-like particles in vitro. Here, we demonstrate that capsid assembly is an equilibrium reaction followed by oxidation of intracapsomere disulfide bonds, which are not essential for the formation of virus-like particles but enable complete particle assembly and prevent capsid dissembly.Murine polyomavirus is a nonenveloped, double-stranded DNA virus with a circular genome 5.3 kb in size. The icosahedral polyomavirus shell has a diameter of approximately 45 nm and consists of 72 pentameric VP1 proteins and the minor core proteins VP2 and VP3 (9). Virion disruption experiments indicated the importance of disulfide bonds and bound calcium in capsid stability for polyomavirus shells (1, 2, 8). In vitro assembly of polyomavirus VP1 into virus-like particles is induced by adding CaCl 2 and inhibited by EDTA (15, 16). Capsomere contacts within the virus shell are accomplished by VP1's flexible C-terminal arm, which protrudes into a neighboring pentamer (10,20). Although intrapentamer disulfide bonds are observed in the crystal structure between residues C19 and C114Ј of neighboring subunits (18,20), it is not clear whether they are sufficient or essential for capsid stabilization, or if there is also involvement of intercapsomere disulfide bonds which are not resolved in the crystal structure.

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confidence: 95%

Mechanism of Assembly of Recombinant Murine Polyomavirus-Like Particles

Schmidt

Rudolph

Böhm

2000

J Virol

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“…These VP1 pentamers fail to assemble into shells due to a deletion of 63 aas at the C terminus that are involved in interpentamer contacts. They nevertheless retain the ability to bind to cells via sialic acid-containing receptors (17).…”

Section: Py Vlps Elicit Host-specific Cytokine Responses From Apcs Inmentioning

confidence: 99%

“…Pentamers of a VP1 mutant truncated at the C terminus were purified as described (17) and further adsorbed against polymyxin B Sepharose to remove any residual endotoxin. Both protein preparations had equivalent trace endotoxin levels (Յ125 IU/ml) measured by the limulus assay (Sigma-Aldrich).…”

Section: Generation Of Virus-like Particles (Vlps) and Vp1 Pentamersmentioning

confidence: 99%

Polyoma Virus-Like Particles Elicit Polarized Cytokine Responses in APCs from Tumor-Susceptible and -Resistant Mice

Venkatesan

Garcea

Benjamin

2006

The Journal of Immunology

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PERA/Ei (PE) mice are highly susceptible to tumor induction by polyoma virus, whereas C57BR/cdj (BR) mice are highly resistant. PE mice respond to viral infection with a type 2 (IL-10) and BR mice with a type 1 (IL-12) cytokine response, underlining the importance of a sustained T cell response for effective antitumor immunity. PE and BR mice showed comparable Ab responses to the virus, indicating that a Th1 response is fully compatible with strong humoral immunity. Tumor susceptibility is dominant, and a type 2 response prevails in F1 mice derived from these strains. In this study, we show that the different cytokine responses of virus-infected hosts are recapitulated in vitro by exposure of APCs from uninfected PE, BR, and F1 animals to the virus. Importantly, virus-like particles formed from recombinant VP1, the major viral capsid protein, elicited the same host-specific cytokine responses as infectious virus. Assembly of VP1 pentamers into capsid shells is required because unassembled VP1 pentamers were ineffective. Binding of virus-like particles to sialic acid is required because pretreatment of APCs with neuraminidase prevented the response. Expression of TLR2 and TLR4 differed among different subpopulations of APCs and also between resistant and susceptible mice. Evidence is presented indicating that these TLRs play a role in mediating the host-specific cytokine responses to the virus.

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“…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)(2)(3)(4). VP3 is an amino-terminally truncated form of VP2.…”

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confidence: 99%

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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Site-directed mutation affecting polyomavirus capsid self-assembly in vitro

Cited by 78 publications

References 10 publications

Mechanism of Assembly of Recombinant Murine Polyomavirus-Like Particles

Mechanism of Assembly of Recombinant Murine Polyomavirus-Like Particles

Polyoma Virus-Like Particles Elicit Polarized Cytokine Responses in APCs from Tumor-Susceptible and -Resistant Mice

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

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