Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1

Moreland, Robert B.; Montross, Lynne K.; Garcea, Robert L.

doi:10.1128/jvi.65.3.1168-1176.1991

Cited by 82 publications

(55 citation statements)

References 49 publications

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“…We did not observe any preference for the SV40 genomic DNA. These data are consistent with previous reports showing that VP1, VP2 and VP3 all possess sequence nonspecific DNA-binding activity (Soussi 1986;Moreland et al 1991;Clever et al 1993;Li et al 2001). According to a series of studies (Dalyot-Herman et al 1996), however, the SV40 genome contains a DNA element called ses that is important for viral genome packaging.…”

Section: Time-course Of the Dna-mediated Particle Formationsupporting

confidence: 93%

Evidence that SV40 VP1–DNA interactions contribute to the assembly of 40‐nm spherical viral particles

et al. 2007

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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 the 40-nm particles under the conditions examined. We propose that DNA may facilitate the formation of 40-nm spherical particles by acting as a scaffold that increases the local concentration of VP1 and/or by acting as an allosteric effector that alters the structure of VP1.

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Section: Time-course Of the Dna-mediated Particle Formationsupporting

confidence: 93%

Evidence that SV40 VP1–DNA interactions contribute to the assembly of 40‐nm spherical viral particles

et al. 2007

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“…The South-Western assay, which allows the detection of DNA^protein interactions, was based on previously published procedures with some modi¢cations [4,18]. Brie£y, puri¢ed VLPs were boiled in the presence of 1% SDS and the denatured L1 proteins were separated on 10% SDS-PAGE and transferred to BA 83 nitrocellulose by electroblotting (Hoefer Semiphor, Pharmacia).…”

Section: Detection Of Dna Bindingmentioning

confidence: 99%

The nine C-terminal amino acids of the major capsid protein of the human papillomavirus type 16 are essential for DNA binding and gene transfer capacity

Touzé¹

2000

FEMS Microbiology Letters

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Four C-terminal deletion mutants of the human papillomavirus 16 L1 protein were expressed in the baculovirus expression system. They consist of the deletion of amino acids 497^505, 477^505, 403^505 and 302^505 (vC9, vC31, vC103 and vC204 respectively). Only two of the C-terminally deleted proteins, vC9 and vC31, retained the ability to form virus-like particles (VLPs) resembling those obtained with the full length L1 protein. Analysis of deleted L1 proteins and corresponding VLPs indicated that the C-terminus was necessary both for DNA binding and DNA packaging. These results were corroborated by the loss of the gene transfer capacities of C-terminal deleted VLPs. ß

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“…Bacterial strains. E. coli RB791 (W3110 lacI q L8) (1) and SG935 [F Ϫ lac(Am) trp(Am) pho(Am) mal(Am) htpR(Am) rpsL Sup(Ts) tsx::Tn10 lon-100] (14,27) were used for protein expression.…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of antibodies. The methods used for preparation of the rabbit polyclonal antibody I58 and the antipeptide antibody R4 against the common C terminus of VP2 and VP3, and antipeptide antibodies DE and BC against the BC1 and DE loops of VP1, respectively, have been described previously (7,21,26,27). Peptides MGAALTILVDLIEGLAEVSTLTGC (corresponding to VP2 residues 1 to 23), SGEALAALDGEITALTLEGVMSSETC (VP2 residues 31 to 55), and QGASTISLGIQRYLHNEEVPTVNRNC (VP2 residues 91 to 115), each containing an additional C-terminal cysteine residue, were also synthesized (Dana-Farber Molecular Biology Core Facility; Applied Biosystems 430A peptide synthesizer).…”

Section: Methodsmentioning

confidence: 99%

“…The major capsid protein VP1 has been purified, after expression in Escherichia coli, as pentamers which self-assemble in vitro into capsid-like aggregates (30). Attempts to encapsi-date viral minichromosomes with recombinant VP1 in the absence of VP2 and VP3 have been unsuccessful, suggesting that VP2 and VP3 may be important for packaging of the viral genome (27). To study the biochemical interactions between the capsid proteins in more detail, we have expressed VP2 and VP3 in E. coli.…”

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confidence: 99%

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Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres

Delos

Cripe

Leavitt

et al. 1995

J Virol

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The polyomavirus VP2 and VP3 capsid proteins were expressed in Escherichia coli. The majority of the expressed proteins were in an insoluble fraction, and they were extracted and initially purified in 8 M urea before renaturation. Soluble VP2 and VP3 were mixed with purified recombinant VP1 capsomeres, and their interactions were assayed by immunoprecipitation and ion-exchange chromatography. Coimmunoprecipitation could be demonstrated with antibodies to either VP1 or VP2/VP3. Mixing recombinant VP1 with VP2 and VP3 modified the recognition of VP1 by domain-specific antipeptide antibodies and altered the chromatographic behavior of the individual proteins. Similar results were observed when a truncated VP1 protein, ⌬NCOVP1, with 62 amino acids deleted from the carboxy terminus was mixed with VP2/VP3. After the mixing, equilibrium dissociation constants for their binding to either VP1 or ⌬NCOVP1 were determined to be 0.37 ؎ 0.23 M for VP2 and 0.18 ؎ 0.21 M for VP3. These studies demonstrate that the recombinant VP2 and VP3 proteins interact with VP1 to affect the biochemical properties of VP1 capsomeres and to change the epitope accessibility of VP1 pentamers. These changes may reflect conformational alterations in VP1 capsomeres which are necessary for viral genome encapsidation.

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Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1

Abstract: The major capsid protein of polyomavirus, VP1, has been expression cloned in Escherichia coli, and the recombinant VP1 protein has been purified to near homogeneity (A

Cited by 82 publications

References 49 publications

Evidence that SV40 VP1–DNA interactions contribute to the assembly of 40‐nm spherical viral particles

Evidence that SV40 VP1–DNA interactions contribute to the assembly of 40‐nm spherical viral particles

The nine C-terminal amino acids of the major capsid protein of the human papillomavirus type 16 are essential for DNA binding and gene transfer capacity

Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres

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