Production of recombinant VP1-derived virus-like particles from novel human polyomaviruses in yeast

Background: Eleven new human polyomaviruses (HPyVs) have been identified in the last decade. Serological studies show that these novel HPyVs sub-clinically infect humans at an early age. The routes of infection, entry pathways, and cell tropism of new HPyVs remain unknown. VP1 proteins of polyomaviruses can assembly into virus-like particles (VLPs). As cell culturing systems for HPyV are currently not available, VP1-derived VLPs may be useful tools in basic research and biotechnological applications. Results: Recombinant VP1-derived VLPs from 11 newly identified HPyVs were efficiently expressed in yeast. VP1 proteins derived from Merkel cell polyomavirus (MCPyV), trichodysplasia spinulosa-associated polyomavirus (TSPyV), and New Jersey polyomavirus (NJPyV) self-assembled into homogeneous similarly-sized VLPs. Karolinska Institutet polyomavirus (KIPyV), HPyV7, HPyV9, HPyV10, and St. Louis polyomavirus (STLPyV) VP1 proteins formed VLPs that varied in size with diameters ranging from 20 to 60 nm. Smaller-sized VLPs (25–35 nm in diameter) predominated in preparations from Washington University polyomavirus (WUPyV) and HPyV6. Attempts to express recombinant HPyV12 VP1-derived VLPs in yeast indicate that translation of VP1 might start at the second of two potential translation initiation sites in the VP1-encoding open reading frame (ORF). This translation resulted in a 364-amino acid-long VP1 protein, which efficiently self-assembled into typical PyV VLPs. MCPyV-, KIPyV-, TSPyV-, HPyV9-, HPyV10-, and HPyV12-derived VLPs showed hemagglutination (HA) assay activity in guinea pig erythrocytes, whereas WUPyV-, HPyV6-, HPyV7-, STLPyV- and NJPyV-derived VP1 VLPs did not. Conclusions: The yeast expression system was successfully utilized for high-throughput production of recombinant VP1-derived VLPs from 11 newly identified HPyVs. HPyV12 VP1-derived VLPs were generated from the second of two potential translation initiation sites in the VP1-encoding ORF. Recombinant VLPs produced in yeast originated from different HPyVs demonstrated distinct HA activities and may be useful in virus diagnostics, capsid structure studies, or investigation of entry pathways and cell tropism of HPyVs until cell culture systems for new HPyVs are developed

Profiling of external metabolites during production of hantavirus nucleocapsid protein with recombinant Saccharomyces cerevisiae

Recombinant strains of Saccharomyces cerevisiae, producing hantavirus Puumala nucleocapsid protein for diagnostics and as a candidate vaccine were analyzed for uptake and excretion of intermediary metabolites during process optimization studies of fed-batch bioreactor cultures. Concentrations of glucose, maltose, galactose, pyruvate, acetaldehyde, ethanol, acetate, succinate and formaldehyde (used as a selection agent) were measured in the culture medium in order to find a metabolite pattern, indicative for the physiological state of the producer culture. When the inducer galactose was employed as a growth substrate, the metabolite profile of recombinant yeast cells was different from those of the non-recombinant original strain which excreted considerable amounts of metabolites with this substrate. In contrast, galactose-induced heterologous gene expression was indicated by the absence of excreted intermediary metabolites, except succinate. A model strain expressing a GFP fusion of hantavirus nucleocapsid protein differed in the excretion of metabolites from strains without GFP. In addition, the influence of alkali ions, employed for pH control is also demonstrated

Virus-like particles derived from major capsid protein VP1 of different polyomaviruses differ in their ability to induce maturation in human dendritic cells

AbstractAs polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic, we studied their interaction with human dendritic cells (hDCs). Exposure of hDCs to VLPs originating from murine (MPyV) or hamster polyomavirus (HaPyV) induced hDC maturation. In contrast, exposure of hDCs to VLPs derived from human polyomaviruses (BK and JC) and simian virus 40 (SV40) only marginally induced DC maturation. The hDCs stimulated by HaPyV- or MPyV-derived VLPs readily produced interleukin-12 and stimulated CD8-positive T-cell responses in vitro. The highest frequencies of activated T cells were again observed after pulsing with HaPyV- and MPyV-derived VLPs. Monocyte-derived hDCs both bound and internalized the various tested polyomavirus VP1-derived VLPs with different levels of efficiency, partially explaining their individual maturation potentials. In conclusion, our data suggest a high variability in uptake of polyomavirus-derived VLPs and potency to induce hDC maturation

Klonirovanie i analiz genov drozhzhej, konstruirovanie transformatsionnykh sistem i produtsentov geterologicheskikh belkov na drozhzhakh

Summary in Russian, English, Latvian, LithuanianAvailable from Latvian Academic Library / LAL - Latvian Academic LibrarySIGLELVLatvi

Seroepidemiology of the human polyomaviruses.

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 VLP-based 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

Maternal human polyomavirus infection and risk of neuroblastoma in the child.

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 yeast-expressed 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; 956k 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