Self-Assembly and Protein–Protein Interactions between the SV40 Capsid Proteins Produced in Insect Cells (original) (raw)
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Biochimica et Biophysica Acta (BBA) - General Subjects, 1996
Three capsid proteins of SV40 (VP1, VP2, and VP3) were expressed in insect cells using recombinant baculoviruses. When the VPl capsid protein was expressed alone or co-expressed with VP2 and VP3, virus-like particles (VLP) were produced. In the latter case, the minor capsid proteins, VP2 and VP3, were incorporated into the VLP. VLPs with and without VP2 and VP3, and the wild type SV40 virions were indistinguishable under electron microscope. The sedimentation coefficient, s20.w, obtained for the VLP consisting of VP1 alone (VPI-VLP) was 170 S, and that for the VLP consisting of all of the capsid proteins (VP1/2/3-VLP) was 174 S. Treatment of the VP1-VLP with a calcium ion chelating agent and a reducing agent caused dissociation of the VPI-VLP. The dissociated and purified VPl proteins were identified as pentamers of VP1 based on the molecular weight determination by sedimentation equilibrium. The pentamers were shown to possess the ability to reassemble into VLP which had the Sz0.w of 141S. The results are discussed in relation to the morphogenesis of SV40.
High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly
PLOS One, 2007
SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ,6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility. Citation: Mukherjee S, Abd-El-Latif M, Bronstein M, Ben-nun-Shaul O, Kler S, et al (2007) High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly. PLoS ONE 2(8): e765.
SV40 Assembly In Vivo and In Vitro
Computational and Mathematical Methods in Medicine, 2008
The Simian virus 40 (SV40) capsid is aT = 7dicosahedral lattice ∼45 nm in diameter surrounding the ∼5 kb circular minichromosome. The outer shell is composed of 360 monomers of the major capsid protein VP1, tightly bound in 72 pentamers. VP1 is a jellyroll β-barrel, with extending N- and C-terminal arms. The N-terminal arms bind DNA and face the interior of the capsid. The flexible C-arms tie together the 72 pentamers in three distinct kinds of interactions, thus facilitating the formation of aT = 7 icosahedron from identical pentameric building blocks. Assemblyin vivowas shown to occur by addition of capsomers around the DNA. We apply a combination of biochemical and genetic approaches to study SV40 assembly. Ourin vivoandin vitrostudies suggest the following model: one or two capsomers bind at a high affinity toses, the viral DNA encapsidation signal, forming the nucleation centre for assembly. Next, multiple capsomers attach concomitantly, at lower affinity, around the minichromo...
Genes to Cells, 2007
The simian virus 40 (SV40) particle is mainly composed of the major capsid protein termed VP1. VP1 self‐assembles into virus‐like particles (VLPs) of approximately 40 nm in diameter when over‐expressed in bacteria or in insect cells, but purified VP1 does not form such a structure under physiological conditions, and thus, the mechanism of VP1 assembly is not well understood. Using a highly purified VP1 assembly/disassembly system in vitro, here we provide evidence that DNA is a factor that contributes to VP1 assembly into 40‐nm spherical particles. At pH 5, for example, VP1 preferentially assembles into 40‐nm particles in the presence of DNA, whereas VP1 assembles into tubular structures in the absence of DNA. Electron microscopic observations revealed that the concentration of DNA and its length are important for the formation of 40‐nm particles. In addition, sucrose gradient sedimentation analysis and DNase I‐sensitivity assays indicated that DNA of up to 2000 bp is packaged into ...
Journal of …, 1995
Recently, recombinant baculoviruses have been used to show that expression of six herpes simplex virus type 1 genes results in the formation of capsid-like particles. We have applied cryoelectron microscopy and three-dimensional image reconstruction to establish their structural authenticity to a resolution of ϳ2.7 nm. By comparing capsids assembled with and without the expression of gene UL35, we have confirmed the presence of six copies of its product, VP26 (12 kDa), around each hexon tip. However, VP26 is not present on pentons, indicating that the conformational differences between the hexon and penton states of the major capsid protein, VP5, extend to the VP26 binding site.
Virus Research, 1999
The baculovirus system was used to construct and isolate AcMNPV-VP1, AcMNPV-VP2 and AcMNPV-VP3 recombinant viruses which express the respective avian polyomavirus (APV) structural proteins in Sf9 insect cells. These recombinant AcMNPVs containing APV structural protein genes were utilized to investigate protein–protein interactions between the structural proteins. Immunofluorescence studies utilizing Sf9 cells infected with the AcMNPV-VP1 revealed that the VP1 protein was expressed and localized in the cytoplasm and not transported into the nucleus. When the cells were co-infected with the VP1 and either VP2 or VP3 recombinant viruses, immunofluorescence of the VP1 protein was localized in the nucleus, indicating that the VP1 protein was transported to the nucleus by both the VP2 and VP3 minor proteins. This observation was suggestive of a protein–protein interaction between the expressed proteins. This protein–protein interaction was substantiated by laser scanning confocal microscopy of Sf9 cells that were co-infected with VP1, VP2 and VP3 recombinant viruses. However, the minor proteins could not be co-isolated with VP1 protein by immunoaffinity chromatography using a monoclonal anti-VP1 serum. In addition, capsid-like particles could not be purified either by CsC1 density gradient centrifugation or by immunoaffinity chromatography. VP1 capsomeres were isolated by immunoaffinity chromatography from Sf9 cells infected with AcMNPV-VP1, with or without the minor protein(s), and these capsomeres could assemble in vitro into capsid-like particles. Electron microscopic observation of thin-sectioned Sf9 cells, which were co-infected with VP1, VP2 and VP3 recombinant viruses, demonstrated capsomere-like structures in the nucleus, but capsid-like particles were not observed.
Journal of Virology, 2001
The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indic...
Assemblages of simian virus 40 capsid proteins and viral DNA visualized by electron microscopy
Biochemical and Biophysical Research Communications, 2007
SV40 assembles in the nucleus by addition of capsid proteins to the minichromosome. The VP1 5 VP2/3 capsomer is composed of a pentamer of the major protein VP1 complexed with a monomer of a minor protein, VP2 or VP3. In the capsid, the capsomers are bound together via their flexible carboxy-terminal arms. Our previous studies suggested that the capsomers are recruited to the packaging signal ses via avid interaction with Sp1. During assembly Sp1 is displaced, allowing chromatin compaction. Here we investigated the interactions in vitro of VP1 5 VP2/3 capsomers with the entire SV40 genome, using mutant VP1 deleted in the carboxy-arm that cannot assemble, but retains DNA-binding capacity. EM revealed that VP1 5 VP2/3 complexes bind non-specifically at random locations around the DNA. Sp1 was absent from mature virions. The findings suggest that multiple capsomers attach simultaneously to the viral genome, increasing their local concentration, facilitating rapid, concerted assembly reaction and removal of Sp1.
Journal of Biological Chemistry, 2006
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. * This work was supported by a grant-in-aid for scientific research, the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science, and Technology in Japan, and a grant for research and development projects in cooperation with academic institutions from the New Energy and Industrial Technology Development Organization.
Simian virus 40 VP1 capsid protein forms polymorphic assemblies in vitro
Journal of General Virology, 2003
The simian virus 40 (SV40) capsid is composed of 72 pentamers of VP1, the major protein of SV40. These pentamers are arranged in a T=7d icosahedral surface lattice, which is maintained by three types of appropriately arranged, non-equivalent interactions between the pentamers. However, it remains unclear how these interactions are achieved. In this study, the in vitro assembly of recombinant VP1 was analysed. Electron microscopy observations revealed that these recombinant VP1 proteins assembled into structurally polymorphic particles depending on environmental conditions. VP1 pentamers assembled efficiently into virus-like particles (VLPs) when high concentrations of ammonium sulfate were present. However, in the presence of 1 M NaCl and 2 mM CaCl 2 at neutral pH, VP1 pentamers formed not only VLPs but also produced tiny T=1 icosahedral particles and tubular structures. The exclusion of CaCl 2 resulted in the exclusive formation of tiny particles. In contrast, in the presence of 150 mM NaCl at pH 5, the VP1 pentamers produced only extraordinarily long tubular structures. VP1 is thus quite unique in that it can assemble into such diverse structures. These observations provide clues that will help elucidate the mechanisms underlying SV40 capsid formation.