Functional complementation of nuclear targeting-defective mutants of simian virus 40 structural proteins

Ishii, Naoya; Nakanishi, Akira; Yamada, Masao; Macalalad, M H; Kasamatsu, Harumi

doi:10.1128/jvi.68.12.8209-8216.1994

Cited by 53 publications

(21 citation statements)

References 31 publications

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“…We found that MPyV VP1 is efficiently transported into the cell nucleus when coexpressed with VP2 or VP3. The interchangeability of the NLS of polyomaviral structural proteins was described previously [31,32]; however, our results indicate that the NLS of the minor protein (VP2 or VP3) is absolutely indispensable for efficient VP1 nuclear localization. During infection, the complex of VP1 pentamer with one of the minor proteins is formed in the cytoplasm and then transported to the cell nucleus, where the virion assembly takes place.…”

Section: Discussionsupporting

confidence: 61%

VP1, the major capsid protein of the mouse polyomavirus, binds microtubules, promotes their acetylation and blocks the host cell cycle

et al. 2017

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VP1, the major structural protein of the mouse polyomavirus (MPyV), is the major architectural component of the viral capsid. Its pentamers are able to self-assemble into capsid-like particles and to non-specifically bind DNA. Surface loops of the protein interact with sialic acid of ganglioside receptors. Although the replication cycle of the virus, including virion morphogenesis, proceeds in the cell nucleus, a substantial fraction of the protein is detected in the cytoplasm of late-phase MPyV-infected cells. In this work, we detected VP1 mainly in the cytoplasm of mammalian cells transfected with plasmid expressing VP1. In the cytoplasm, VP1-bound microtubules, including the mitotic spindle, and the interaction of VP1 with microtubules resulted in cell cycle block at the G2/M phase. Furthermore, in the late phase of MPyV infection and in cells expressing VP1, microtubules were found to be hyperacetylated. We then sought to understand how VP1 interacts with microtubules. Dynein is not responsible for the VP1-microtubule association, as neither overexpression of p53/dynamitin nor treatment with ciliobrevin-D (an inhibitor of dynein activity) prevented binding of VP1 to microtubules. A pull-down assay for VP1-interacting proteins identified the heat shock protein 90 (Hsp90) chaperone, and Hsp90 was also detected in the VP1-microtubule complexes. Although Hsp90 is known to be associated with acetylated microtubules, it does not mediate the interaction between VP1 and microtubules. Our study provides insight into the role of the major structural protein in MPyV replication, indicating that VP1 is a multifunctional protein that participates in the regulation of cell cycle progression in MPyV-infected cells.

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Section: Discussionsupporting

confidence: 61%

VP1, the major capsid protein of the mouse polyomavirus, binds microtubules, promotes their acetylation and blocks the host cell cycle

et al. 2017

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“…pBS-SK(+), pEGFP-N3 (Clontech, Otsu, Japan), pFastBac1 (Invitrogen, Carlsbad, CA) and hSpt6WT/pFB (Endoh et al 2004) were used as 3000-, 4729-, 5000-and 10 000-bp circular DNAs, respectively. pSV40, containing the complete SV40 genome (Ishii et al 1994), and pLAPSN (Clontech) were also used in some experiments. These circular plasmid DNAs were linearized with appropriate restriction enzymes to obtain linear DNAs ranging in size from 2000 to 10 000 bp.…”

Section: Formation Of Various Atypical Structuresmentioning

confidence: 99%

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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“…The exposure of the R4 domain, which contains the nuclear localization signal of VP2/VP3 (5), is consistent with the ability of VP2 to aid in transporting a nuclear localization signal-defective VP1 to the nucleus (3). A similar observation has been made for the SV40 VP3 protein, in which the nuclear localization signal and VP1 interaction domain could be functionally separated (12,17). lon-exchange chromatography was used as an independent method to identify an interaction between VP1 and VP2/VP3, as well as to determine if biochemical properties of VP1 capsomeres changed with this interaction.…”

Section: Discussionmentioning

confidence: 70%

“…In vivo experiments with SV40 suggest that VP1, VP2, and VP3 interact prior to assembly to ensure the proper stoichiometry of capsid proteins within the nucleus (18,23,24). One hypothesis proposes that VP1 pentamers interact with VP2 and/or VP3 in the cytoplasm for cotransport into the nucleus (7,17). For example, temperature-sensitive tsB, tsC, or tsBC SV40 mutants, which have mutations in VP1, exhibit defects in both VP1 and VP2/VP3 subcellular localization at the nonpermissive temperature (18).…”

mentioning

confidence: 99%

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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Functional complementation of nuclear targeting-defective mutants of simian virus 40 structural proteins

Cited by 53 publications

References 31 publications

VP1, the major capsid protein of the mouse polyomavirus, binds microtubules, promotes their acetylation and blocks the host cell cycle

VP1, the major capsid protein of the mouse polyomavirus, binds microtubules, promotes their acetylation and blocks the host cell cycle

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

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

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