Self-assembly of purified polyomavirus capsid protein VP1

The organization of the major (L1) and minor (L2) proteins in the human papillomavirus capsid is still largely unknown. In this study we analysed the disulphide bonding between L1 proteins and the association of L2 proteins with capsomers using virus-like particles obtained in insect cells by co-expression of the L1 and L2 genes of human papillomavirus type 33. About 50% of the L1 protein molecules in these particles (1.29 g/cm z) formed disulphide-bonded trimers. Reduction of the intermolecular disulphide bonds by dithiothreitol (DTT) treatment caused disassembly of virus-like particles into capsomers. This indicates that disulphide bonds between capsomers at the threefold symmetry positions of the capsid are essential for the assembly of the papiUomavirus capsid. In contrast, the L2 protein was not engaged in intermolecular disulphide bonding. The L2 protein remained associated with capsomers on disassembly by treatment with DTT. When the disassembly was carried out in 0"65 u-NaC1, complete L2 protein molecules bound preferentially to capsomer oligomers, whereas truncated L2 protein molecules bound only to monomers. In 0.15 u-NaCl only complete L2 protein molecules remained bound to capsomers. This indicates that different regions of the L2 protein molecule are differentially involved in the association of the papillomavirus capsid.Papillomaviruses are a subfamily of the papovaviruses. The properties shared by these viruses include a circular dsDNA genome, a nonenveloped virion and an icosahedral capsid. Papillomaviruses have a specific tropism for epithelial cells and have been found in a great variety of species. More than 70 types of human papillomavirus (HPV) have been recognized but only a few, e.g. HPV-I (which induces cutaneous warts), can be isolated as virus particles. The other HPVs, e.g. HPV-16 or HPV-33 (which are associated with genital carcinoma), have only been identified by cloning their genomes from epithelial lesions. Moreover, no system exists for the efficient propagation of HPVs in vitro.

Section: J M M W a L B O O M E R S And R E S T R E E C K U Nsupporting

confidence: 83%

Organization of the major and minor capsid proteins in human papillomavirus type 33 virus-like particles

Sapp¹,

Volpers²,

Müller

et al. 1995

“…It has been demonstrated that bacterially expressed recombinant VP1 can self-assemble into capsid-like particles in vitro in the presence of calcium ions (Haynes et al, 1993 ;Salunke et al, 1986Salunke et al, , 1989 and that eukaryotic baculovirus-expressed polyomavirus VP1 can also produce capsid-like particles in the nuclei of insect cells (Delos et al, 1993 ;Forstova! et al, 1993 ;Li et al, 1995 ;Montross et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…This protein possesses the receptor-binding domain Stehle et al, 1994), a nuclear localization signal domain, a DNA-binding domain and a calcium-ion-binding domain Haynes et al, 1993 ;Moreland et al, 1991), which makes it crucial in virus attachment and virion assembly. When expressed in the bacterial system, polyomavirus VP1 could assemble into empty capsid-like structures spontaneously in the presence of calcium ions, with a size and morphology similar to those of the native polyomavirus capsids (Leavitt et al, 1985 ;Salunke et al, 1986 ;Haynes et al, 1993). The bacterial expression systems have proven to be an invaluable resource for the production and purification of large quantities of all the polyomavirus structural proteins (Cai et al, 1994 ;Chang et al, a, b, 1993Delos et al, 1993 ;Haynes et al, 1993 ;Moreland et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

Use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles.

Gillock

Sweat

et al. 1999

The genes encoding the structural proteins (VP1, VP2 and VP3) of murine polyomavirus were cloned into the p2Bac dual multiple cloning site vector, individually or jointly, and the corresponding proteins were expressed in Spodoptera frugiperda (Sf9) insect cells by cotransfecting Sf9 cells with the constructed vector and the linear DNA of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Recombinant capsid-like particles could be purified 5 days post-infection from Sf9 cells infected with AcMNPV-VP1, with or without the involvement of minor protein (VP2 or VP3). Although VP2 and VP3 alone could not generate recombinant particles, they became incorporated into these particles when expressed with VP1 in Sf9 cells. Recombinant particles with different polyomavirus structural protein(s) were obtained by using different combined expression of these proteins in Sf9 cells. Cellular DNA of 5 kbp in size was packaged in all of the recombinant particles, which showed the same diameter as that of native virions. Agarose gel electrophoresis indicated that DNA packaged in these recombinant particles had a different pattern than that of native virions. Two-dimensional gel electrophoresis of the VP1 species of recombinant particles showed more VP1 species than those of the native virions from mouse cells, and an additional species of VP1 when VP2 was co-expressed with VP1. The recombinant particles were also compared for their ability to compete for polyomavirus infection. The competition assay indicated that the recombinant particles containing VP2 were the most efficient in inhibiting the native polyomavirus infection of 3T6 cells.

“…The prokaryote-generated capsid-like particles can cause HA of human O-type red blood cells and this HA activity can be inhibited by JCV-positive human serum (Table 1) and rabbit anti-JCV VP1 antiserum (data not shown), indicating a native conformation. Previously, E. coli-expressed murine polyomavirus VP1 (Haynes et al, 1993 ;Salunke et al, 1986) and human papillomavirus major capsid protein L1 (Li et al, 1997) have been isolated as pentameric capsomeres. The pentameric polyomavirus VP1 (Haynes et al, 1993 ;Salunke et al, 1986) and papillomavirus L1 (Li et al, 1997) are able to form capsidlike structures in the presence of calcium ions or high concentrations of NaCl.…”

Section: Discussionmentioning

confidence: 99%

“…These studies have shown that the major capsid protein, VP1, of polyomaviruses is able to self-assemble into capsid-like particles when expressed in insect cells (Chang et al, 1997 ;Forstova et al, 1993 ;Gillock et al, 1997 ;Kosukegawa et al, 1996 ;Montross et al, 1991 ;Pawlita et al, 1996). Furthermore, VP1 from murine polyomavirus (Salunke et al, 1986), avian polyomavirus, budgerigar fledgling disease virus (BFDV) (Rodgers et al, 1994) and the L1 capsid protein of papillomavirus (Li et al, 1997) have also been expressed in E. coli and isolated as pentameric capsomeres. Although capsomeres isolated from E. coli could be assembled into a capsid-like structure in the presence of calcium ions in vitro, self-assembly in E. coli of capsid-like particles made up of major capsid proteins has not been reported.…”

Section: Introductionmentioning

confidence: 99%

The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells.

Wang

Fung

et al. 1999

The full-length major capsid protein, VP1, of the human polyomavirus JC virus was cloned and expressed in Escherichia coli. VP1 protein expressed in E. coli self-assembled into capsid-like particles and caused haemagglutination of human O-type red blood cells. Caesium chloride density-gradient centrifugation analysis revealed that the capsid-like particles consisted of virionlike pseudovirion and empty capsid-like pseudocapsid populations. The morphology of pseudovirion and pseudocapsid particles was observed under the electron microscope. The pseudovirions contained DNA and RNA molecules but the pseudocapsids did not contain any nucleic acid, as analysed by DNA extraction. DNA-binding activity of VP1 was also demonstrated by the SouthWestern probing method in vitro. Furthermore, pseudocapsids were able to deliver exogenous DNA into human foetal kidney epithelial cells. These results indicate that recombinant JC virus VP1 is able to self-assemble into capsid-like particles and to package DNA in the absence of the minor capsid proteins, VP2 and VP3. This prokaryotic assembly system may facilitate the investigation of maturation mechanism(s) of polyomaviruses. Furthermore, capsid-like particles of JC virus VP1 generated in E. coli potentially could be used as a human gene transfer vector.