Yeast Cells Allow High-Level Expression and Formation of Polyomavirus-Like Particles

Sasnauskas, Kęstutis; Buzaitė, Odeta; Vogel, Frank; Jandrig, Burkhard; Ražanskas, Raimundas; Staniulis, J.; Scherneck, Siegfried; Krüger, Detlev H.; Ulrich, Rainer G.

doi:10.1515/bc.1999.050

Cited by 90 publications

(82 citation statements)

References 31 publications

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“…Yeast-derived recombinant VLPs offer many advantages over other expression systems in terms of protein yield, cost and ease of protein expression (Hale et al, 2002;Sasnauskas et al, 1999). In this study, we found such VLPs to be highly serologically reactive, with an optimum amount of VLPs as low as 6 ng per well.…”

Section: Discussionsupporting

confidence: 52%

“…The polyoma VLPs used in this study were based on the major capsid protein, VP1, and produced in yeast cells from S. cerevisiae (Gedvilaite et al, 2000;Sasnauskas et al, 1999). VLPs of JCV have also been expressed in Escherichia coli and polyomavirus VP1 will also assemble into capsid-like particles in the nucleus of insect cells when expressed in the baculovirus system Ou et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

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Seroepidemiology of the human polyomaviruses

Stolt

Sasnauskas²,

Koskela³

et al. 2003

Journal of General Virology

271

225

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To assess the stability of polyomavirus antibodies in serial samples over time and the incidence and age-specific prevalence of polyomavirus infections, we established enzyme immunoassays (EIAs) using purified yeast-expressed virus-like particles (VLPs) containing the VP1 major capsid proteins of JC virus (JCV) and the AS and SB strains of BK virus (BKV). A random subsample of 150 Finnish women who had serum samples taken during the first trimester of pregnancy and had a second pregnancy during a 5 year follow-up period was selected, grouped by age of first pregnancy. The polyomavirus antibody levels were similar in samples taken during the first and second pregnancies (correlation coefficient 0?93 for BKV SB and 0?94 for JCV). Analysis of serum samples from 290 Swedish children aged 1-13 years, grouped by age in 2 year intervals, demonstrated that BKV seropositivity increased rapidly with increasing age of the children, reaching 98 % seroprevalence at 7-9 years of age, followed by a minor decrease. JCV seroprevalence increased only slowly with increasing age and reaching 72 % positivity among mothers >25 years of age. The age-specific seroprevalence of the human polyomaviruses measured using this VLPbased EIA was similar to previous serosurveys by other methods. The stability of the antibodies over time indicates that polyomavirus seropositivity is a valid marker of cumulative virus exposure, and polyoma VLP-based EIAs may therefore be useful for epidemiological studies of these viruses.

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Section: Discussionsupporting

confidence: 52%

Section: Discussionmentioning

confidence: 99%

Seroepidemiology of the human polyomaviruses

Stolt

Sasnauskas²,

Koskela³

et al. 2003

Journal of General Virology

271

225

View full text Add to dashboard Cite

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“…14,15 The VLPs are empty capsids that consist of the major capsid protein, VP1. The VP1 gene was inserted into the yeast expression vector, pFX7.…”

Section: Polyoma Virus Like Particlesmentioning

confidence: 99%

“…14,15 The mouse monoclonal antibody against JCV and BKV was added in dilution 1/10,000 and incubated for 90 min. 17 Goat anti-Mouse IgG peroxidase conjugate (Southern Biotechnology), diluted 1/1200 in RS-PBS, were rotated for 1 hr before addition to the wells and then reacted for 60 min at room temperature.…”

Section: Igm Confirmation Testmentioning

confidence: 99%

Maternal human polyomavirus infection and risk of neuroblastoma in the child

Stolt

Kjellin

Sasnauskas³

et al. 2004

Intl Journal of Cancer

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To investigate if polyomavirus infection during pregnancy is linked to development of neuroblastoma in the child, serum samples of 115 index mothers from the pregnancy where the child eventually developed neuroblastoma were identified and matched with serum samples from 8 control mothers per index mother. The samples were tested for specific IgG and IgM antibodies to BK and JC virus using enzyme immunoassays based on purified yeastexpressed virus-like particles (VLPs). The serum samples as well as 10 neuroblastoma cell lines were also analyzed using Real Time (TaqMan) PCR for detection and quantification of BK virus DNA. The BK virus IgG seroprevalence was similar among index mothers (80%) and control mothers (83%) [OR 0.8; 95% confidence interval (95% CI): 0.5-1.3]. BK virus IgM was also not associated with neuroblastoma risk (OR was OR ‫؍‬ 0.6 ; 95% with CI, 0.2-1.9). Also JC virus had no association, neither for IgG (OR ‫؍‬ 0.9; 95% CI, 0.6 -1.4) nor for IgM (OR ‫؍‬ 0.9 ; 95% CI, 0.4 -1.9). All serum samples and all neuroblastoma cell lines were negative for BKV DNA. In summary, a comprehensive cohort using both serology and polyomavirus DNA detection found no evidence for association between BKV or JCV polyomaviruses and neuroblastoma.Key words: neuroblastoma; polyomaviruses; BK virus; JC virus; seroprevalence; maternal infection; virus like particles (VLPs); enzyme immunoassay; Real Time PCR The human polyomaviruses BK and JC can transform human cells in vitro. 1 The virally encoded T antigens of both BKV and JCV are essential for transformation and have been shown to bind the p53 and pRb tumor suppressor proteins. 2,3 BKV infection has also been reported to induce chromosomal aberrations in human cells. 4 The DNA of JCV has been detected in certain brain tumours: in particular oligoastrocytoma. 5 There are also reports of presence of the simian polyomavirus SV40 in several human tumours, including choroid plexus tumours and ependymomas. 5-8 BKV has been detected in a variety of tumours, the most thorough studies being those on neuroblastoma. 9 Neuroblastomas account for 7-10% of all childhood cancers and it is the most common cancer diagnosed during infancy. Neuroblastoma probably derives from primitive sympathetic neural precursors and about half of all neuroblastomas arise in the adrenal medulla. 10 Flaegstad et al. 9 found BKV DNA in 18/18 neuroblastomas by PCR but in 0/5 control adrenals; 16/18 neuroblastomas were also found to contain the virus DNA in the tumor cells by in situ hybridization, and the viral large T antigen was demonstrated by both immunoprecipitation and immunohistochemistry of the tumor cells. JCV and SV40 were not found and all 9 PCR amplimers that were sequenced were confirmed to be BKV DNA. 4,9 The BK-encoded large T antigen was found to bind p53, leading to an aberrant cytoplasmic localization of p53, suggesting a possible transforming mechanism for BKV in neuroblastomas in children. 11 Both BK and JC virus infect a large proportion of children all over the world. Following primar...

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“…The PCR amplification product was digested with BcuI and inserted into XbaIlinearized and dephosphorylated yeast expression plasmid pFX7 under control of yeast GAL1-10 promoter [30] and confirmed by PCR and subsequent DNA sequence analysis. The nucleotide sequence of the amplified PPV VP2 was compared with those in GenBank using the Basic Local Alignment Search Tool (BLAST).…”

Section: Serum Samplesmentioning

confidence: 99%

Generation of recombinant porcine parvovirus virus-like particles in Saccharomyces cerevisiae and development of virus-specific monoclonal antibodies

Burneikiene

Tamošiūnas

Akatov

et al. 2014

Journal of Biotechnology

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Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the selfassembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.

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Yeast Cells Allow High-Level Expression and Formation of Polyomavirus-Like Particles

Cited by 90 publications

References 31 publications

Seroepidemiology of the human polyomaviruses

Seroepidemiology of the human polyomaviruses

Maternal human polyomavirus infection and risk of neuroblastoma in the child

Generation of recombinant porcine parvovirus virus-like particles in Saccharomyces cerevisiae and development of virus-specific monoclonal antibodies

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