Generation of Recombinant Virus-Like Particles of Human and Non-Human Polyomaviruses in Yeast &lt;i&gt;Saccharomyces cerevisiae&lt;/i&gt;

Sasnauskas, Kęstutis; Bulavaitė, Aistė; Hale, Antony; Jin, Li; Knowles, Wendy A.; Gedvilaitė, Alma; Dargeviciute, Ausra; Bartkeviciūte, D; Žvirblienė, Aurelija; Staniulis, J.; Brown, David; Ulrich, Rainer G.

doi:10.1159/000067922

Cited by 76 publications

(58 citation statements)

References 32 publications

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“…Compared to mammalian polyomaviruses, however, available data indicate marked differences in the structure and assembly of the APV capsid due to (i) the incorporation of a fourth structural protein, VP4, in addition to VP1, VP2, and VP3 (12) and (ii) the missing or inefficient formation of VLP following VP1 expression in insect cells (1) or yeast (35), respectively. In order to investigate the requirements for APV capsid assembly, the APV structural proteins were expressed in CE cells permissive for APV replication, the proteins were localized within the cell, and the conditions for VLP formation were analyzed.…”

Section: Discussionmentioning

confidence: 99%

“…In the case of APV, however, expression of VP1, either alone or after coexpression with VP2 and/or VP3, by recombinant-baculovirus-infected insect cells did not lead to VLP formation (1). Recently, VLP formation has been reported after recombinant expression of VP1 proteins of several polyomaviruses, including APV, in the yeast Saccharomyces cerevisiae (35). In this system, VLP formed by APV VP1 were generated; it was observed, however, that the efficacy of VLP formation was ϳ10-fold lower than that of the mammalian polyomaviruses.…”

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confidence: 99%

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Nuclear Localization of Avian Polyomavirus Structural Protein VP1 Is a Prerequisite for the Formation of Virus-Like Particles

Johne

Müller

2004

J Virol

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Virions of polyomaviruses consist of the major structural protein VP1, the minor structural proteins VP2 and VP3, and the viral genome associated with histones. An additional structural protein, VP4, is present in avian polyomavirus (APV) particles. As it had been reported that expression of APV VP1 in insect cells did not result in the formation of virus-like particles (VLP), the prerequisites for particle formation were analyzed. To this end, recombinant influenza viruses were created to (co)express the structural proteins of APV in chicken embryo cells, permissive for APV replication. VP1 expressed individually or coexpressed with VP4 did not result in VLP formation; both proteins (co)localized in the cytoplasm. Transport of VP1, or the VP1-VP4 complex, into the nucleus was facilitated by the coexpression of VP3 and resulted in the formation of VLP. Accordingly, a mutant APV VP1 carrying the N-terminal nuclear localization signal of simian virus 40 VP1 was transported to the nucleus and assembled into VLP. These results support a model of APV capsid assembly in which complexes of the structural proteins VP1, VP3 (or VP2), and VP4, formed within the cytoplasm, are transported to the nucleus using the nuclear localization signal of VP3 (or VP2); there, capsid formation is induced by the nuclear environment.Polyomaviruses are nonenveloped icosahedral particles with a diameter of ϳ45 nm containing a circular double-stranded DNA genome complexed with cellular histones (41). The capsid is composed of the major capsid protein VP1 and the two minor capsid proteins VP2 and VP3, which are expressed late in the viral replication cycle. VP3 is identical to the C-terminal region of VP2; translation is initiated by an internal initiation codon within the VP2-encoding region. Five molecules of VP1 assemble into a pentameric complex which interacts with one molecule of either VP2 or VP3, thus representing a capsomer of the polyomavirus particle. Infectious viral particles, consisting of 72 capsomers and the viral genome, are assembled within the nucleus of the infected cell. Although the structure of the polyomavirus particle has been well characterized by X-ray crystallography (18, 38), the detailed mechanisms of capsid assembly and the process of DNA packaging into the capsid are still subjects of intensive study (7,15,16).Several polyomaviruses infecting different mammalian species have been described, including the human polyomaviruses JC virus (JCPyV) and BK virus (BKPyV), simian virus 40 (SV40), and the murine polyomavirus (MPyV) (41). Compared to these viruses, avian polyomavirus (APV) exhibits unique biological and structural characteristics: whereas mammalian polyomaviruses usually cause innocuous infections in their natural nonimmunocompromised hosts, APV is the causative agent of a fatal multisystemic disease in several species of birds (11,14,29,34), observed all over the world. Recently, an additional structural protein, VP4, was identified within APV particles, a feature which is unique among the polyomaviruses...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Nuclear Localization of Avian Polyomavirus Structural Protein VP1 Is a Prerequisite for the Formation of Virus-Like Particles

Johne

Müller

2004

J Virol

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“…After 6 days, a third passage was performed analogously. The cells were harvested and subjected to immunoblotting using a rabbit antiserum directed against APV particles (49) and cross-reacting with VP1 proteins of several other polyomaviruses (44).…”

Section: Methodsmentioning

confidence: 99%

Characterization of Two Novel Polyomaviruses of Birds by Using Multiply Primed Rolling-Circle Amplification of Their Genomes

Johne

Wittig²,

Fernández-de-Luco

et al. 2006

J Virol

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“…The assembly of Vp1 pentamers into icosahedral shells is an intrinsic property of Vp1, since Vp1 alone of SV40 and several other polyomaviruses is sufficient for the formation of capsidlike structures in vitro (53), in Escherichia coli (45), in Saccharomyces cerevisiae (46,55), in insect cells (5,22,36,47,54), and in mammalian cells (15,34). Furthermore, the capsid proteins of polyomaviruses have been implicated, by numerous studies using different experimental systems, in other important functions of viral infection.…”

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confidence: 99%

Minor Capsid Proteins of Simian Virus 40 Are Dispensable for Nucleocapsid Assembly and Cell Entry but Are Required for Nuclear Entry of the Viral Genome

Nakanishi

Itoh

et al. 2007

J Virol

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We investigated the roles of simian virus 40 capsid proteins in the viral life cycle by analyzing point mutants in Vp1 and Vp2/3, as well as a deletion mutant lacking the Vp2/3 coding sequence. The Vp1 mutants (V243E and L245E) and the Vp2/3 mutants (F157E-I158E and P164R-G165E-G166R) were previously shown to be defective in Vp1-Vp2/3 interaction and to be noninfectious or poorly infectious, respectively. Here, we show that all these point mutants form stable particles following DNA transfection into cells. The Vp2/3-mutant particles contained very low levels of Vp2/3, whereas the Vp1 mutant particles contained no detectable Vp2/3. As expected, the deletion mutant also formed particles that were noninfectious. We further characterized the two Vp1 point mutants and the deletion mutant. All three mutant particles comprised Vp1 and histone-associated viral DNA, and all were able to enter cells. However, the mutant complexes failed to associate with host importins (owing to the loss of the Vp2/3 nuclear localization signal), and the mutant viral DNAs prematurely dissociated from the Vp1s, suggesting that the nucleocapsids did not enter the nucleus. Consistently, all three mutant particles failed to express large T antigen. Together, our results demonstrate unequivocally that Vp2/3 is dispensable for the formation of nucleocapsids. Further, the nucleocapsids' ability to enter cells implies that Vp1 contains the major determinants for cell attachment and entry. We propose that the major role of Vp2/3 in infectivity is to mediate the nuclear entry of viral DNA.

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Generation of Recombinant Virus-Like Particles of Human and Non-Human Polyomaviruses in Yeast <i>Saccharomyces cerevisiae</i>

Cited by 76 publications

References 32 publications

Nuclear Localization of Avian Polyomavirus Structural Protein VP1 Is a Prerequisite for the Formation of Virus-Like Particles

Nuclear Localization of Avian Polyomavirus Structural Protein VP1 Is a Prerequisite for the Formation of Virus-Like Particles

Characterization of Two Novel Polyomaviruses of Birds by Using Multiply Primed Rolling-Circle Amplification of Their Genomes

Minor Capsid Proteins of Simian Virus 40 Are Dispensable for Nucleocapsid Assembly and Cell Entry but Are Required for Nuclear Entry of the Viral Genome

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