BACKGROUND
polyomaviruses  are small, non-enveloped viruses with a circular, double-stranded genome comprised of approximately  <dig> base pairs surrounded by an icosahedral capsid  <cit> .
pyvs have been found to infect both mammalian  and avian  species  <cit> .
mammalian pyv infections are usually asymptomatic, although pyvs were originally identified and named for their ability to cause multiple types of tumors  <cit> .
bk polyomavirus  and jc polyomavirus  were the first two human pyvs  isolated 40 years ago  <cit> .
as a result of improved molecular diagnostic techniques, combined with strategies in sample preparation to enrich viral dna content through reduction of host genomic dna,  <dig> new hpyvs have been identified in the last decade.
of these novel hpyvs, karolinska institutet polyomavirus , washington university polyomavirus , and merkel cell polyomavirus  were among the first to be discovered .
later, hpyv <dig>  hpyv <dig>  trichodysplasia spinulosa-associated polyomavirus , and hpyv <dig> were identified .
by  <dig>  three more hpyvs , and hpyv12) were discovered .
two other pyvs detected in human stool samples, malawi polyomavirus  and mexico polyomavirus , may represent strain variants of hpyv <dig> because their genomes are 95–99 % identical  <cit> .
the latest virus, new jersey polyomavirus , was discovered in endothelial cells, sites of myositis, and sites of cutaneous necrosis in a pancreatic transplant recipient  <cit> .
discoveries of new hpyvs have revived interest in this expanding family of human pathogens.
there are several reasons why hpyvs may be important in human pathology.
first, according to serological studies, hpyvs sub-clinically infect humans at an early age, with rates ranging from  <dig> to 90 %  <cit> .
secondly, while hpyv infection is generally asymptomatic, it can cause serious illness in immunocompromised individuals  <cit> .
accordingly, the involvement of some hpyvs in the pathology of various diseases has been reported.
infection by and replication of bkpyv in kidney epithelial cells can lead to pyv-associated nephropathy  <cit> .
progressive multifocal leukoencephalopathy is caused by uncontrolled jcpyv replication in oligodendrocytes of the brain in immunosuppressed patients, including patients with advanced acquired immune deficiency syndrome   <cit> .
trichodysplasia spinulosa, a rare skin dysplasia, occurs in immunocompromised individuals as a result of tspyv replication in the inner root sheath cells of hair follicles  <cit> .
hpyv <dig> has also been associated with novel pathogenicity: lower back rash in immunosuppressed patients  <cit> .
thirdly, several pyvs may be associated with human cancer.
mcpyv has gained the most attention due to its link with a rare and aggressive malignant human skin cancer, merkel cell carcinoma, in the elderly and chronically immunosuppressed individuals  <cit> .
it has been shown that hamster polyomavirus  is responsible for the high incidence of lymphomas in a colony of genetic audiogenic seizure hamsters   <cit> .
moreover, proteins encoded by some pyvs have transforming ability in cell culture and tumorigenic activity in animals  <cit> .
the connection with disease of other new hpyvs has yet to be determined.
the late gene region of mammalian pyvs encode three structural proteins, vp <dig>  vp <dig>  and vp <dig>  which together make up the viral capsid; the capsid of avian pyvs may additionally contain a unique vp <dig> protein  <cit> .
however, capsid-like structures can assemble of the major capsid protein vp <dig> alone .
this unique property of vp <dig> makes it an ideal protein for generating virus-like particles .
vlps resemble the native virions they are derived from in structure, immunogenicity, and tropism, but they do not contain any viral genetic material.
vlps are particularly valuable in various research applications because many pyvs are not easily cultured, which may be related to the finding that pyvs infect only a very limited number of species and cell types in nature  <cit> .
pyv vp1-derived vlps play an important role in the study of mechanisms underlying protein self-assembly, attachment to host cell receptors, virus entry, immune response, intracellular trafficking, and in the epidemiologic study and diagnosis of pyv infection  <cit> .
vp1-derived vlps may also have potential use in vaccine development, gene delivery in gene therapy, and other biomedical purposes  <cit> .
sequence alignment of vp <dig> from different hpyvs shows high amino acid  sequence identity between some hpyvs, resulting in occasional antibody cross-reactivity  <cit> .
cross-reactivity has been observed between hpyv <dig> and hpyv <dig>  or bkpyv and jcpyv , but not between mcpyv, tspyv, hpyv <dig>  or mwpyv  <cit> .
we have previously demonstrated that simian virus  <dig> -, hapyv-, bkpyv-, jcpyv-, and murine polyomavirus -derived vp <dig> vlps can be efficiently produced in the yeast  strain, ah22- <dig>  using the pfx <dig> expression plasmid  <cit> .
in this study, we showed that the applied yeast system is also suitable for efficient expression and self-assembly of vp1-derived vlps from  <dig> novel hpyvs: mcpyv, kipyv, wupyv, hpyv <dig>  hpyv <dig>  tspyv, hpyv <dig>  mwpyv, stlpyv, hpyv <dig>  and njpyv.
yeast-expressed recombinant vlps were subjected to a hemagglutination  assay, which our findings suggest might be useful in developing new hpyv diagnostic tools and in other applications.
RESULTS
production of vp1-derived vlps from mcpyv, kipyv, wupyv, hpyv <dig>  hpyv <dig>  tspyv, hpyv <dig>  hpyv <dig>  stlpyv and njpyv in yeast
for the production of kipyv-, wupyv-, hpyv6-, hpyv7-, tspyv-, hpyv9-, stlpyv-, and njpyv-derived vlps in yeast, codons in the vp1-encoding open reading frames  from corresponding hpyv genomes were optimized for expression in s. cerevisiae.
a few deviations from strict, high-frequency codon usage were allowed for the insertion or removal of restriction endonuclease recognition sites.
hpyv <dig> vp <dig> gene was synthesized according to the native virus gene sequence without codon optimization.
mcpyv vp <dig> gene was amplified by polymerase-chain reaction  from the mcpyv virus genome cloned from a healthy volunteer skin swab.
all  <dig> vp1-encoding genes were inserted in the same site in the expression vector, pfx <dig>  <cit> , under the control of a galactose-inducible promoter.
to determine whether recombinant vp <dig> protein was expressed in yeast transformed with pfx <dig> carrying hpyv vp1-encoding orfs, yeast lysates were analyzed using sodium dodecyl sulfate-polyacrylamide gels  .
the expression of all  <dig> constructs was detected in yeast lysates, and the expression level of all vp <dig> proteins derived from different hpyvs did not vary significantly.
all  <dig> recombinant vp <dig> proteins stayed in the soluble fraction after the centrifugation of yeast lysates .
the solubility of vp <dig> allowed for the efficient purification by sucrose and cscl gradient centrifugation .
the yield of purified vp1-derived vlps ranged from  <dig>  to  <dig>  mg per 1 g of wet yeast biomass .
the lower yields of purified vp <dig> from hpyv <dig>  hpyv <dig>  hpyv <dig>  and njpyv may have been the consequence of minor differences in expression level and solubility.
on the other hand, it is known that codon usage might influence the efficiency of heterologous gene expression  <cit> .
thus, the lower yield of hpyv10-derived vp <dig> could be a result of the hpyv <dig> vp1-encoding gene sequence, as it was not optimized for yeast expression.
during purification, kipyv-, hpyv6-, and hpyv7-derived vp <dig> tended to degrade.
therefore, additional bands in the sds-page gel were observed .
highly-soluble tspyv- and hpyv9-derived vp <dig> were purified with yields as high as 1 mg per 1 g of wet yeast biomass .fig.
<dig> analysis of the production of novel hpyv-derived vp <dig> proteins in yeast using coomassie blue-stained sds-page.
in lanes: 1- in all gels, negative control sample of whole crude lysate of yeast transformed with pfx <dig> plasmid;  <dig> - whole lysate of yeast transformed with pfx7-kipyv-vp <dig> , pfx7-wupyv-vp <dig> , pfx7-mcpyv-vp <dig> , pfx7-hpyv6-vp <dig> , pfx7-hpyv7-vp <dig> , pfx7-tspyv-vp <dig> , pfx7-hpyv9-vp <dig> , pfx7-hpyv10-vp <dig> , pfx7-stlpyv-vp <dig> , or pfx7-njpyv-vp <dig>  plasmid;  <dig> - the soluble fraction recovered after centrifugation of whole lysate of yeast transformed with pfx7-kipyv-vp <dig> , pfx7-wupyv-vp <dig> , pfx7-mcpyv-vp <dig> , pfx7-hpyv6-vp <dig> , pfx7-hpyv7-vp <dig> , pfx7-tspyv-vp <dig> , pfx7-hpyv9-vp <dig> , pfx7-hpyv10-vp <dig> , pfx7-stlpyv-vp <dig> , or pfx7-njpyv-vp <dig>  plasmid;  <dig> - purified kipyv vp <dig> , wupyv vp <dig> , mcpyv vp <dig> , hpyv <dig> vp <dig> , hpyv <dig> vp <dig> , tspyv vp <dig> , hpyv <dig> vp <dig> , hpyv <dig> vp <dig> , stlpyv vp <dig> , or njpyv vp <dig>  protein.
in all gels, pre-stained protein weight marker  was loaded into lane m
athe formation of vlps was confirmed by negative stain electron microscopy
bthe values for the yield of vlps are the average at least from three independent experiments
the ability of vp <dig> proteins to self-assemble into vlps was examined using negative staining electron microscopy.
all  <dig> purified vp <dig> proteins formed vlps .
mcpyv-, tspyv-, and njpyv-derived vlps were found to be the most similar in size  with diameters of 45–50 nm .
the particles formed by seven other vp <dig> proteins were more variable in size with diameters ranging from  <dig> to 55 nm.
although the majority of vlps in preparations from kipyv, hpyv <dig>  hpyv <dig>  hpyv <dig>  and stlpyv were 40–50 nm in diameter , fractions of smaller vlps, 25–35 nm in diameter, predominated in samples from wupyv and hpyv <dig> .fig.
<dig> detection of purified hpyv-derived vp <dig> vlps using negative staining electron microscopy.
electron micrographs of:  - kipyv vp <dig> vlps,  - wupyv vp <dig> vlps,  - mcpyv vp <dig> vlps,  - hpyv <dig> vp <dig> vlps,  - hpyv <dig> vp <dig> vlps,  - tspyv vp <dig> vlps,  - hpyv <dig> vp <dig> vlps,  - hpyv <dig> vp <dig> vlps,  - stlpyv vp <dig> vlps,  - hpyv <dig> vp <dig> vlps,  - hpyv <dig> vp <dig> vlps, and  - njpyv vp <dig> vlps produced in yeast.
bars, 100 nm
production of hpyv <dig> vp1-derived vlps in yeast
hpyv <dig> vp <dig> gene  encodes a 380-aa-long protein.
for production of hpyv12-derived vp <dig> vlps in yeast, we used both the native virus gene sequence  and a version that was codon-optimized for s. cerevisiae expression , synthesized by genscript.
both vp1-encoding genes were cloned into the expression vector, pfx <dig>  <cit> , and transformed into the yeast strain, ah22- <dig>  expression of hpyv <dig> vp <dig>  analyzed using sds-page, was low using both versions of the gene .
our attempts to purify hpyv <dig> vp <dig> using sucrose and cscl gradient centrifugation did not achieve complete purification .
impurities could not be removed with additional centrifugation in sucrose or cscl, and they remained in the same fractions as the vp <dig> protein.
the yield of partially-purified vp <dig> was very low: only  <dig>  mg per 1 g of wet yeast biomass.
examination of hpyv <dig> vp <dig> assembly into vlps using negative staining electron microscopy demonstrated that some spherical structures could be found, which were approximately 40 nm in diameter and similar to the typical capsomer-structured surface.
however, the majority of the recombinant vp <dig> formed atypical, small  particles or aggregates .fig.
<dig> analysis of hpyv12-derived vp <dig> vlp production in yeast.
a - analysis of hpyv <dig> vp <dig> vlp production in yeast using coomassie blue-stained sds-page.
production of hpyv <dig> vp <dig> protein was analyzed in two yeast clones transformed with pfx7-hpyv12-vp1n <dig> plasmid  and two clones transformed with pfx7-hpyv12-vp1s <dig> plasmid .
in lanes:  <dig>   <dig> - whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1n <dig> plasmid;  <dig>   <dig> - the soluble fraction recovered after centrifugation of whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1n <dig> plasmid;  <dig>   <dig> - whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1s <dig> plasmid;  <dig>   <dig> - the soluble fraction recovered after the centrifugation of whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1s <dig> plasmid;  <dig> - purified hpyv12-vp <dig> protein; m - pre-stained protein weight marker.
b - analysis of hpyv <dig> vp <dig> production in yeast using coomassie blue-stained sds-page.
production of hpyv <dig> vp <dig> protein was analyzed in two yeast clones transformed with pfx7-hpyv12-vp1n <dig> plasmid  and two clones transformed with pfx7-hpyv12-vp1s <dig> plasmid .
in lanes:  <dig>   <dig> - whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1n <dig> plasmid;  <dig>   <dig> - the soluble fraction recovered after centrifugation of whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1n <dig> plasmid;  <dig>   <dig> - whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1s <dig> plasmid;  <dig>   <dig> - the soluble fraction recovered after the centrifugation of whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1s364;  <dig> - purified hpyv12-vp <dig> protein; m - pre-stained protein weight marker
in determining why we could not produce hpyv12-derived vp <dig> vlps in yeast, we noticed that the n-terminal end of the hpyv <dig> vp <dig> protein sequence overhang in the alignment with other vp <dig> protein sequences derived from different pyvs .
on the other hand, the n-terminal arm encoded within a sequence starting from a second potential translation initiation site in the vp <dig> gene appeared to be typical of pyv vp <dig> .
based on these observations, we decided to express hpyv <dig> vp <dig> using a sequence that did not encode an atypical, hydrophobic, 16-aa-long n-terminal peptide.
we used the native virus gene sequence  and one that was codon-optimized for s.cerevisiae expression , starting at the second translation initiation site.
these were cloned in the vector, pfx <dig>  and transformed into the yeast strain, ah22- <dig>  production of the 364-aa-long hpyv <dig> vp <dig> protein was analyzed using sds-page.
the expression level of vp <dig> was greatly improved using these sequences compared with using the longer version of the gene .
hpyv <dig> vp <dig> was purified using sucrose and cscl gradient centrifugation.
the yields of vp <dig> were approximately  <dig>  mg and 1 mg per 1 g of wet yeast biomass using the native gene and the codon-optimized gene, respectively.
the purity and yield of hpyv <dig> vp <dig> were similar to those of other hpyv vp <dig> proteins .
thus, using codon-optimized hpyv <dig> vp <dig> clearly increased expression and yield of the protein.
analysis of the purified hpyv <dig> vp <dig> lacking the 16-aa-long n-terminal using negative staining electron microscopy showed that this protein efficiently assembled into vlps that were 45–50 nm in diameter .fig.
<dig> alignment of vp <dig> n-terminal aa sequences encoded in the genomes of different pyvs.
the aa residues encoded in the first of two potential translation initiation sites are typed in blue; the identical actual n-terminal aa residues are shown in red
collectively, these data suggest that the translation initiation site of vp <dig> was predicted incorrectly by the hpyv <dig> genome sequence annotated in genbank.
the second translation initiation site in the putative orf encoding vp <dig> is more likely the true start site, resulting in efficient translation of a 364-aa-long vp <dig> protein and it’s self-assembly into vlps.
detection of purified hpyvs derived vp <dig> proteins by western blot
the sequence alignment of vp <dig> proteins derived from different hpyvs showed 25–78 % degree of identity in amino acid sequence  <cit> , thus it is not likely that all purified vp <dig> proteins could be recognized by the same antibody in one western blot.
nevertheless, polyclonal antibodies of some pyvs demonstrated cross-reactivity in western blots  <cit> .
therefore, attempts were made to detect the purified vp <dig> proteins derived from hpyvs based on their cross-reactivities with anti-jcpyv, anti-hapyv, anti-mcpyv, and anti-wupyv vp <dig> polyclonal antibodies.
polyclonal antibody raised against jcpyv-vp <dig> protein cross-reacted with previously developed bkpyv vp <dig> protein   <cit>  as well as with tspyv , hpyv <dig> , and hpyv <dig>  vp <dig> proteins in western blot .
a high degree of cross-reactivity of hpyv vp <dig> proteins was observed with rabbit antiserum raised against hapyv vp <dig> protein.
this antiserum failed to detect only kipyv , wupyv , hpyv <dig> , and stlpyv  vp <dig> proteins .
interestingly, vp <dig> proteins derived from hpyv <dig> and tspyv were recognized by all four tested polyclonal antibodies despite low similarity of both proteins to wupyv vp <dig>  .
in contrast, stlpyv vp <dig> protein was hardly detected by only one antiserum raised against mcpyv vp <dig> protein  .
anti-mcpyv vp <dig> polyclonal antibody additionally cross-reacted with tspyv  and hpyv <dig>  vp <dig> proteins .
anti-wupyv vp <dig> polyclonal antibody showed a very strong cross-reactivity with kipyv vp <dig>  but also detected hpyv <dig>  hpyv <dig>  and hpyv <dig> vp <dig> proteins identical to wupyv vp <dig> only by  <dig>   <dig>  and 48 %, accordingly .
thus, the cross-reactivity allowed detection of all purified hpyv vp <dig> proteins by western blot using only four polyclonal antibodies.fig.
<dig> detection of purified hpyv-derived vp <dig> vlps in western blot with polyclonal antibodies.
a, coomassie blue-stained sds-page; , western blot with anti-jcpyv vp <dig> , anti-hapyv vp <dig> , anti-mcpyv vp <dig> , and anti-wupyv vp <dig>  polyclonal antibodies.
the same samples of purified proteins were run on each gel.
in lanes:  <dig> - hpyv <dig> l <dig> protein;  <dig> - bkpyv vp <dig> protein;  <dig> - jcpyv vp <dig> protein;  <dig> - kipyv vp <dig> protein;  <dig> - wupyv vp <dig> protein;  <dig> - mcpyv vp <dig> protein;  <dig> - hpyv <dig> vp <dig> protein;  <dig> - hpyv <dig> vp <dig> protein;  <dig> - tspyv vp <dig> protein;  <dig> - hpyv <dig> vp <dig> protein;  <dig> - hpyv <dig> vp <dig> protein;  <dig> - stlpyv vp <dig> protein;  <dig> - hpyv <dig> vp <dig> protein;  <dig> - njpyv vp <dig> protein;  <dig>  protein weight marker ;  <dig> - hapyv vp <dig> protein
ha activity of vp1-derived vlps from novel pyvs: mcpyv, kipyv, wupyv, hpyv <dig>  hpyv <dig>  tspyv, hpyv <dig>  hpyv <dig>  stlpyv, hpyv <dig>  and njpyv
all  <dig> purified vlps were subjected to ha testing using guinea pig erythrocytes.
jcpyv-, bkpyv-, and sv40-derived vp <dig> vlps were used as controls.
as shown in fig.
<dig>  the ha titer of purified preparations of jcpyv- and bkpyv-derived vp <dig> vlps with known ha activity  <cit>  was down to a concentration of  <dig>  μg ml− <dig>  jcpyv- and bkpyv-derived vp <dig> vlps served as positive controls.
the preparation of sv40-derived vp <dig> vlps did not show any ha activity using guinea pig erythrocytes and so served as a negative control  <cit> .
ha activity was also confirmed for mcpyv-derived vp <dig> vlps with the ha titer down to a concentration of  <dig>  μg ml− <dig>  <cit> .
kipyv-, hpyv10-, and hpyv12-derived vp <dig> vlps demonstrated strong ha activity, down to a concentration of  <dig>  μg ml− <dig>  tspyv- and hpyv9-derived vp <dig> vlps showed ha activity down to a concentration of  <dig>  μg ml− <dig> and  <dig>  μg ml− <dig>  respectively.
wupyv-, hpyv6-, hpyv7-, stlpyv-, and njpyv-derived vp <dig> vlps did not show any ha activity .fig.
<dig> ha activity of hpyv-derived vp <dig> vlps.
a 2-fold dilution series of purified kipyv-, wupyv-, mcpyv-, hpyv6-, hpyv7-, tspyv-, hpyv9-, hpyv10-, stlpyv-, hpyv12-, and njpyv-derived vp <dig> vlps expressed in s. cerevisiae were subjected to a ha assay with 1 % guinea pig erythrocytes.
purified jcpyv- and bkpyv-derived vp <dig> vlps were used as positive controls, and sv40-derived vp <dig> vlps were used as negative controls.
the concentrations of vp1-derived vlps in μg ml− <dig> are shown on the top.
the bar indicates the highest vlp concentration at which ha of guinea pig erythrocytes was observed
discussion
recently,  <dig> new hpyvs have been identified.
in contrast to the better-studied pyvs, jcpyv, bkpyv, and to a degree, mcpyv, the routes of infection, entry pathways, and cell tropism of these new hpyvs remain unknown.
the variety and number of hpyvs indicate that they are capable of self-propagating at low enough levels that do not cause disease, except in immunosuppressed individuals.
to advance research on new hpyvs, development of cell culture systems will be important.
however, in some applications, vlps may be used instead, particularly in cases where the wild-type virus is not easily cultivated using cell cultures.
recombinant vp <dig> proteins from previously discovered pyvs were successfully produced in escherichia coli, yeast or baculovirus/insect cell expression systems  <cit> .
yeast or baculovirus/insect cell expression systems were used for efficient production of self-assembled vp <dig> vlps derived from bkpyv, jcpyv, hapyv and other pyvs .
however, the recombinant vp <dig> proteins produced in e. coli were found mainly as pentamers which self-assembled into vlps during purification  <cit> .
only jcpyv- and hapyv-derived vp <dig> were shown to form vlps inside bacteria  <cit> .
recently large-scale and high-yield production yielding gram-per-liter levels of mpyv vp <dig> fused with glutathione s-transferase  in e. coli was reported  <cit> .
vp <dig> protein was purified as pentamer and vlps were reassembled in vitro after removal of the gst-tag.
it should be noted that the assembly of purified pentamers into vlps always has a negative impact on the final yield  <cit> , though for mpyv vp <dig> the vlp reassembly yield was improved and reached 42–56 %  <cit> .
nevertheless, other authors have shown that yeast-produced mpyv derived vp <dig> vlps were more stable as compared to vlps assembled from pentamers purified from e. coli  <cit> .
similarly, recombinant gst or 6his-tagged vp <dig> proteins or vp <dig> pentamers but not vlps derived from novel hpyvs were produced in bacterial expression systems  <cit> .
to synthesize hpyv-derived vp <dig> vlps, mammalian and/or insect cells have been used.
mcpyv-derived vp <dig> vlps have been generated in mammalian and insect cells .
hpyv6-, hpyv7-, tspyv-, hpyv9-, and mwpyv/hpyv10-derived vp <dig> vlps have also been successfully produced in insect cells  <cit> .
however, vp1-derived vlps from stlpyv, hpyv <dig>  and njpyv have not been generated at all until now.
in this study, we demonstrated that vp1-derived vlps from  <dig> recently discovered hpyvs can be efficiently produced in the yeast, s. cerevisiae.
the synthesis of vlps using the yeast expression system has several advantages.
s. cerevisiae is a gras  microorganism, free of toxins hazardous to human health  <cit> .
the preparation of products produced in yeast for human use is well established.
additionally, recombinant vp1-derived vlp expression in yeast is relatively cheaper than in insect or mammalian cell cultures, and it results in high yields of vlp  <cit> .
currently, s. cerevisiae is used in the production of vaccines against the recombinant hepatitis b virus  and the human papillomavirus   <cit> .
in this study, to generate  <dig> new hpyv-derived vp <dig> proteins in yeast, both native vp1-encoding genes and genes optimized for expression in s. cerevisiae were used.
the lowest yield  of purified hpyv10- and hpyv12-derived vp <dig> protein was obtained using the native gene sequence and njpyv-derived vp <dig> protein using gene optimized for expression in s. cerevisiae .
mcpyv-derived vp <dig> was also expressed using the native gene sequence, but we obtained a high yield of this protein , demonstrating that gene sequence is not the only factor influencing the yield of recombinant protein.
the properties of recombinant proteins, stability, resistance to proteases, and solubility, in particular, may have also had an impact on purification efficiency and the final yield.
therefore, the optimization of genes for use in s. cerevisiae resulted in only slightly better yields of vp <dig> from kipyv, wupyv, hpyv <dig>  hpyv <dig>  and stlpyv  compared to hpyv10-derived vp <dig>  the yield of highly-soluble tspyv-, hpyv9-, and hpyv12-derived vp <dig> vlps, produced using optimized genes, was almost two times greater .
on the other hand, the difference between the optimized versus native genes was obvious when a shorter hpyv <dig> vp <dig> gene version was used.
in this case, utilization of optimized vp <dig> gene improved the yield of vp1s <dig> by a factor of nearly  <dig> .
the yeast system supported self-assembly of vp1-derived vlps from all  <dig> recently discovered hpyvs.
however, hpyv6- and wupyv-derived vp <dig> vlp preparations contained mainly smaller-sized capsids.
in the case of hpyv6-derived vp <dig> vlps , small particles were also observed in other hosts.
electron micrographs of hpyv6- and hpyv7-derived vp <dig> vlps purified from mammalian 293tt cells show particles smaller than 45–50 nm in diameter  <cit> .
the proportion of small hpyv6-derived vp <dig> vlps produced in insect cells was similar to that purified in yeast.
purified hpyv7-derived vp <dig> vlps produced both in insect and yeast cells self-assemble into typical pyv-derived vlps  <cit> .
in contrast, small particles predominate in preparations of hpyv9-dervied vp <dig> vlps purified from insect cells  <cit> .
however, in our study, the yeast-generated hpyv9-derived vp <dig> vlps assembled into mostly typical pyv-like particles with diameters of approximately 45 nm .
an explanation for different efficiencies in forming normally-sized vlps from different pyvs remains elusive.
it has been proposed that variation in particle size in sv <dig> actually indicates the presence of mosaic vlps, which form from a combination of different geometries of pentamer interactions  <cit> .
due to numerous possible interactions, sv <dig> pentamers can self-assemble into a broad array of shapes; this assembly can be redirected by different nucleic acids serving as a scaffold and as a nucleating factor  <cit> .
similarly, mpyv-derived vp <dig> self-assembles into vlps that are  <dig>   <dig>  and 50 nm in diameter  <cit> .
it has been suggested that the n-terminal arm of vp <dig> forms a clamp to hold the c-terminal protrusions from neighboring capsomeres in place  <cit> , connecting the n-terminal arms of vp <dig> from different capsomeres via the intercapsomeric disulphide bridges  <cit> .
flexibility in this clamping mechanism allows capsomeres to exist in both the pentavalent and hexavalent state  <cit>  and permits the assembly of polymorphic particles in pyvs  <cit> .
on the other hand, sometimes even a single change in an aa residue can influence the size of vp1-derived vlps.
this has been shown in the hapyv, wherein a single a336g aa exchange in the c-terminal arm of vp <dig> results in smaller vp1-derived vlps that are 20 nm in diameter  <cit> .
the hpyv <dig> and hpyv <dig> genomes that have been sequenced so far reveal naturally-occurring vp <dig> aa sequence variation  <cit> ; it is not known how such variation influences vlp assembly.
because smaller particles predominate among hpyv6-derived vp <dig> vlps produced in yeast, insect, and mammalian cells, it is possible that some aa variants can support self-assembly of normal-sized hpyv6-derived vp <dig> vlps.
as a result of our attempts to express hpyv12-derived vp <dig> vlps in yeast and alignment of various pyv-derived vp <dig> n-terminal sequences, our data suggest that the true translation initiation site predicted by the vp1-encoding orf is the second translation initiation site  <cit> .
removal of the 16-aa-long n-terminal peptide greatly improved vp <dig> expression and vlp formation.
there are only a few such cases known in other pyv genomes.
hapyv-, jcpyv-, and sv40-derived vp <dig> is synthesized starting from the second of two potential translation initiation sites in their respective genes   <cit> .
the development of cell culture systems for the production of hpyv <dig> and determination of the n-terminal sequence of vp <dig> isolated from the virus capsid could be useful in confirming the true translation initiation start site.
ha activity using guinea pig erythrocytes can expand the number of potential applications for vp1-derived vlps.
they can be used as antigens, not only in enzyme immunoassays, but also in more specific ha inhibition assays to detect corresponding hpyv-specific antibodies in human sera  <cit> .
on the other hand, the ha activity of isolated hpyv-derived vp <dig> vlps could serve as an initial index of virus cell-binding properties, which are related to viral spreading, cell tropism, and pathogenicity  <cit> .
the ha activity of jcpyv is associated with the binding of vp <dig> to sialic acid residues .
in contrast, sv <dig> has no ha activity and uses other receptor molecules, e.g., major histocompatibility complex class i molecules  <cit> .
it has been reported that sv <dig> might bind to sialic acid residues present on ganglioside gm <dig>  but the steric arrangement of the binding amino acids differs from that of hemagglutinating viruses, such as mpyv .
consistent with these observations, it has been shown that mutations in the sialic acid binding site impair ha of mcpyv-derived vp <dig> capsids  <cit> .
the variation in ha activity observed in this study among vp1-derived vlps from different pyvs might reflect diversity in receptor recognition among pyvs.
ha of guinea pig erythrocytes by kipyv-, tspyv-, hpyv9-, hpyv10-, and hpyv12-derived vp <dig> vlps suggests that these viruses might use sialic acid residues to bind the virus particle to the host cell.
in contrast, wupyv-, hpyv6-, hpyv7-, stlpyv-, and njpyv-derived vp <dig> vlps showed no ha activity and thus more likely use other receptor molecules.
indeed, according to the solved high-resolution crystal structure of hpyv <dig> vp <dig> in complex with the three putative oligosaccharide receptors identified by glycan microarray screening, hpyv <dig> engages short sialylated oligosaccharides  <cit> .
consistent with these observations, hpyv9-derived vp <dig> vlps showed ha of guinea pig erythrocytes in our study.
protein structure analysis of hpyv6- and hpyv7-derived vp <dig> reveals an obstructed sialic acid binding site  <cit> .
additionally, vp <dig> derived from hpyv <dig> and hpyv <dig> does not interact with sialylated compounds in solution or in cultured human cells  <cit> .
stroh et al.
suggest that it is still possible that hpyv <dig> and hpyv <dig> bind sialic acids at a site in the fully assembled virus, but not in the free vp <dig> pentamer  <cit> .
in our study, the yeast-produced hpyv6- and hpyv7-derived vp <dig> vlps had no ha activity, supporting the recent observations that these two viruses bind non-sialylated receptors.
the vp1-derived vlps from two other members of the genus wukipolyomavirus  differed in their ability to hemagglutinate guinea pig erythrocytes, despite the fact that none of the sialic acid binding residues found in sv <dig>  jcpyv, or mpyv are conserved in the wupyv and kipyv vp <dig> structures  <cit> .
kipyv-derived vp <dig> vlps efficiently hemagglutinated guinea pig erythrocytes, whereas wupyv-derived vp <dig> vlps did not show any ha activity.
the data suggest that these two similar viruses might use different receptor molecules to bind host cells.
CONCLUSIONS
the yeast expression system was successfully adapted for high-throughput production of recombinant vp1-derived vlps from  <dig> newly identified hpyvs.
although hpyv <dig> vp <dig> was predicted to encode a 380-aa-long protein, our findings suggest that translation starts not at the first, but rather at the second, of two potential initiation sites in the vp1-encoding orf, resulting in a 364-aa-long protein.
yeast-generated recombinant vp1-derived vlps from different hpyvs demonstrated distinct ha activities and may be useful in virus diagnostics, capsid structure studies, or the investigation of entry pathways and cell tropism of new hpyvs until cell culture systems for new hpyvs are developed.

 polyomaviruses are small, non-enveloped viruses with a circular, double-stranded genome comprised of approximately base pairs surrounded by an icosahedral capsid. pyvs have been found to infect both mammalian and avian species. mammalian pyv infections are usually asymptomatic, although pyvs were originally identified and named for their ability to cause multiple types of tumors. bk polyomavirus and jc polyomavirus were the first two human pyvs isolated 40 years ago. as a result of improved molecular diagnostic techniques, combined with strategies in sample preparation to enrich viral dna content through reduction of host genomic dna, new hpyvs have been identified in the last decade. of these novel hpyvs, karolinska institutet polyomavirus, washington university polyomavirus, and merkel cell polyomavirus were among the first to be discovered. later, hpyv hpyv trichodysplasia spinulosa-associated polyomavirus, and hpyv were identified. by three more hpyvs, and hpyv12) were discovered. two other pyvs detected in human stool samples, malawi polyomavirus and mexico polyomavirus, may represent strain variants of hpyv because their genomes are 95–99 % identical. the latest virus, new jersey polyomavirus, was discovered in endothelial cells, sites of myositis, and sites of cutaneous necrosis in a pancreatic transplant recipient. discoveries of new hpyvs have revived interest in this expanding family of human pathogens. there are several reasons why hpyvs may be important in human pathology. first, according to serological studies, hpyvs sub-clinically infect humans at an early age, with rates ranging from to 90 %. secondly, while hpyv infection is generally asymptomatic, it can cause serious illness in immunocompromised individuals. accordingly, the involvement of some hpyvs in the pathology of various diseases has been reported. infection by and replication of bkpyv in kidney epithelial cells can lead to pyv-associated nephropathy. progressive multifocal leukoencephalopathy is caused by uncontrolled jcpyv replication in oligodendrocytes of the brain in immunosuppressed patients, including patients with advanced acquired immune deficiency syndrome. trichodysplasia spinulosa, a rare skin dysplasia, occurs in immunocompromised individuals as a result of tspyv replication in the inner root sheath cells of hair follicles. hpyv has also been associated with novel pathogenicity: lower back rash in immunosuppressed patients. thirdly, several pyvs may be associated with human cancer. mcpyv has gained the most attention due to its link with a rare and aggressive malignant human skin cancer, merkel cell carcinoma, in the elderly and chronically immunosuppressed individuals. it has been shown that hamster polyomavirus is responsible for the high incidence of lymphomas in a colony of genetic audiogenic seizure hamsters. moreover, proteins encoded by some pyvs have transforming ability in cell culture and tumorigenic activity in animals. the connection with disease of other new hpyvs has yet to be determined. the late gene region of mammalian pyvs encode three structural proteins, vp vp and vp which together make up the viral capsid; the capsid of avian pyvs may additionally contain a unique vp protein. however, capsid-like structures can assemble of the major capsid protein vp alone. this unique property of vp makes it an ideal protein for generating virus-like particles. vlps resemble the native virions they are derived from in structure, immunogenicity, and tropism, but they do not contain any viral genetic material. vlps are particularly valuable in various research applications because many pyvs are not easily cultured, which may be related to the finding that pyvs infect only a very limited number of species and cell types in nature. pyv vp1-derived vlps play an important role in the study of mechanisms underlying protein self-assembly, attachment to host cell receptors, virus entry, immune response, intracellular trafficking, and in the epidemiologic study and diagnosis of pyv infection. vp1-derived vlps may also have potential use in vaccine development, gene delivery in gene therapy, and other biomedical purposes. sequence alignment of vp from different hpyvs shows high amino acid sequence identity between some hpyvs, resulting in occasional antibody cross-reactivity. cross-reactivity has been observed between hpyv and hpyv or bkpyv and jcpyv, but not between mcpyv, tspyv, hpyv or mwpyv. we have previously demonstrated that simian virus -, hapyv-, bkpyv-, jcpyv-, and murine polyomavirus -derived vp vlps can be efficiently produced in the yeast strain, ah22- using the pfx expression plasmid. in this study, we showed that the applied yeast system is also suitable for efficient expression and self-assembly of vp1-derived vlps from novel hpyvs: mcpyv, kipyv, wupyv, hpyv hpyv tspyv, hpyv mwpyv, stlpyv, hpyv and njpyv. yeast-expressed recombinant vlps were subjected to a hemagglutination assay, which our findings suggest might be useful in developing new hpyv diagnostic tools and in other applications.  production of vp1-derived vlps from mcpyv, kipyv, wupyv, hpyv hpyv tspyv, hpyv hpyv stlpyv and njpyv in yeast for the production of kipyv-, wupyv-, hpyv6-, hpyv7-, tspyv-, hpyv9-, stlpyv-, and njpyv-derived vlps in yeast, codons in the vp1-encoding open reading frames from corresponding hpyv genomes were optimized for expression in s. cerevisiae. a few deviations from strict, high-frequency codon usage were allowed for the insertion or removal of restriction endonuclease recognition sites. hpyv vp gene was synthesized according to the native virus gene sequence without codon optimization. mcpyv vp gene was amplified by polymerase-chain reaction from the mcpyv virus genome cloned from a healthy volunteer skin swab. all vp1-encoding genes were inserted in the same site in the expression vector, pfx, under the control of a galactose-inducible promoter. to determine whether recombinant vp protein was expressed in yeast transformed with pfx carrying hpyv vp1-encoding orfs, yeast lysates were analyzed using sodium dodecyl sulfate-polyacrylamide gels. the expression of all constructs was detected in yeast lysates, and the expression level of all vp proteins derived from different hpyvs did not vary significantly. all recombinant vp proteins stayed in the soluble fraction after the centrifugation of yeast lysates. the solubility of vp allowed for the efficient purification by sucrose and cscl gradient centrifugation. the yield of purified vp1-derived vlps ranged from to  mg per 1 g of wet yeast biomass. the lower yields of purified vp from hpyv hpyv hpyv and njpyv may have been the consequence of minor differences in expression level and solubility. on the other hand, it is known that codon usage might influence the efficiency of heterologous gene expression. thus, the lower yield of hpyv10-derived vp could be a result of the hpyv vp1-encoding gene sequence, as it was not optimized for yeast expression. during purification, kipyv-, hpyv6-, and hpyv7-derived vp tended to degrade. therefore, additional bands in the sds-page gel were observed. highly-soluble tspyv- and hpyv9-derived vp were purified with yields as high as 1 mg per 1 g of wet yeast biomass.fig. analysis of the production of novel hpyv-derived vp proteins in yeast using coomassie blue-stained sds-page. in lanes: 1- in all gels, negative control sample of whole crude lysate of yeast transformed with pfx plasmid; - whole lysate of yeast transformed with pfx7-kipyv-vp, pfx7-wupyv-vp, pfx7-mcpyv-vp, pfx7-hpyv6-vp, pfx7-hpyv7-vp, pfx7-tspyv-vp, pfx7-hpyv9-vp, pfx7-hpyv10-vp, pfx7-stlpyv-vp, or pfx7-njpyv-vp plasmid; - the soluble fraction recovered after centrifugation of whole lysate of yeast transformed with pfx7-kipyv-vp, pfx7-wupyv-vp, pfx7-mcpyv-vp, pfx7-hpyv6-vp, pfx7-hpyv7-vp, pfx7-tspyv-vp, pfx7-hpyv9-vp, pfx7-hpyv10-vp, pfx7-stlpyv-vp, or pfx7-njpyv-vp plasmid; - purified kipyv vp, wupyv vp, mcpyv vp, hpyv vp, hpyv vp, tspyv vp, hpyv vp, hpyv vp, stlpyv vp, or njpyv vp protein. in all gels, pre-stained protein weight marker was loaded into lane m athe formation of vlps was confirmed by negative stain electron microscopy bthe values for the yield of vlps are the average at least from three independent experiments the ability of vp proteins to self-assemble into vlps was examined using negative staining electron microscopy. all purified vp proteins formed vlps. mcpyv-, tspyv-, and njpyv-derived vlps were found to be the most similar in size with diameters of 45–50 nm. the particles formed by seven other vp proteins were more variable in size with diameters ranging from to 55 nm. although the majority of vlps in preparations from kipyv, hpyv hpyv hpyv and stlpyv were 40–50 nm in diameter, fractions of smaller vlps, 25–35 nm in diameter, predominated in samples from wupyv and hpyv.fig. detection of purified hpyv-derived vp vlps using negative staining electron microscopy. electron micrographs of: - kipyv vp vlps, - wupyv vp vlps, - mcpyv vp vlps, - hpyv vp vlps, - hpyv vp vlps, - tspyv vp vlps, - hpyv vp vlps, - hpyv vp vlps, - stlpyv vp vlps, - hpyv vp vlps, - hpyv vp vlps, and - njpyv vp vlps produced in yeast. bars, 100 nm production of hpyv vp1-derived vlps in yeast hpyv vp gene encodes a 380-aa-long protein. for production of hpyv12-derived vp vlps in yeast, we used both the native virus gene sequence and a version that was codon-optimized for s. cerevisiae expression, synthesized by genscript. both vp1-encoding genes were cloned into the expression vector, pfx, and transformed into the yeast strain, ah22- expression of hpyv vp analyzed using sds-page, was low using both versions of the gene. our attempts to purify hpyv vp using sucrose and cscl gradient centrifugation did not achieve complete purification. impurities could not be removed with additional centrifugation in sucrose or cscl, and they remained in the same fractions as the vp protein. the yield of partially-purified vp was very low: only  mg per 1 g of wet yeast biomass. examination of hpyv vp assembly into vlps using negative staining electron microscopy demonstrated that some spherical structures could be found, which were approximately 40 nm in diameter and similar to the typical capsomer-structured surface. however, the majority of the recombinant vp formed atypical, small particles or aggregates.fig. analysis of hpyv12-derived vp vlp production in yeast. a - analysis of hpyv vp vlp production in yeast using coomassie blue-stained sds-page. production of hpyv vp protein was analyzed in two yeast clones transformed with pfx7-hpyv12-vp1n plasmid and two clones transformed with pfx7-hpyv12-vp1s plasmid. in lanes: - whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1n plasmid; - the soluble fraction recovered after centrifugation of whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1n plasmid; - whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1s plasmid; - the soluble fraction recovered after the centrifugation of whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1s plasmid; - purified hpyv12-vp protein; m - pre-stained protein weight marker. b - analysis of hpyv vp production in yeast using coomassie blue-stained sds-page. production of hpyv vp protein was analyzed in two yeast clones transformed with pfx7-hpyv12-vp1n plasmid and two clones transformed with pfx7-hpyv12-vp1s plasmid. in lanes: - whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1n plasmid; - the soluble fraction recovered after centrifugation of whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1n plasmid; - whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1s plasmid; - the soluble fraction recovered after the centrifugation of whole lysate of both yeast clones transformed with pfx7-hpyv12-vp1s364; - purified hpyv12-vp protein; m - pre-stained protein weight marker in determining why we could not produce hpyv12-derived vp vlps in yeast, we noticed that the n-terminal end of the hpyv vp protein sequence overhang in the alignment with other vp protein sequences derived from different pyvs. on the other hand, the n-terminal arm encoded within a sequence starting from a second potential translation initiation site in the vp gene appeared to be typical of pyv vp. based on these observations, we decided to express hpyv vp using a sequence that did not encode an atypical, hydrophobic, 16-aa-long n-terminal peptide. we used the native virus gene sequence and one that was codon-optimized for s.cerevisiae expression, starting at the second translation initiation site. these were cloned in the vector, pfx and transformed into the yeast strain, ah22- production of the 364-aa-long hpyv vp protein was analyzed using sds-page. the expression level of vp was greatly improved using these sequences compared with using the longer version of the gene. hpyv vp was purified using sucrose and cscl gradient centrifugation. the yields of vp were approximately  mg and 1 mg per 1 g of wet yeast biomass using the native gene and the codon-optimized gene, respectively. the purity and yield of hpyv vp were similar to those of other hpyv vp proteins. thus, using codon-optimized hpyv vp clearly increased expression and yield of the protein. analysis of the purified hpyv vp lacking the 16-aa-long n-terminal using negative staining electron microscopy showed that this protein efficiently assembled into vlps that were 45–50 nm in diameter.fig. alignment of vp n-terminal aa sequences encoded in the genomes of different pyvs. the aa residues encoded in the first of two potential translation initiation sites are typed in blue; the identical actual n-terminal aa residues are shown in red collectively, these data suggest that the translation initiation site of vp was predicted incorrectly by the hpyv genome sequence annotated in genbank. the second translation initiation site in the putative orf encoding vp is more likely the true start site, resulting in efficient translation of a 364-aa-long vp protein and it’s self-assembly into vlps. detection of purified hpyvs derived vp proteins by western blot the sequence alignment of vp proteins derived from different hpyvs showed 25–78 % degree of identity in amino acid sequence, thus it is not likely that all purified vp proteins could be recognized by the same antibody in one western blot. nevertheless, polyclonal antibodies of some pyvs demonstrated cross-reactivity in western blots. therefore, attempts were made to detect the purified vp proteins derived from hpyvs based on their cross-reactivities with anti-jcpyv, anti-hapyv, anti-mcpyv, and anti-wupyv vp polyclonal antibodies. polyclonal antibody raised against jcpyv-vp protein cross-reacted with previously developed bkpyv vp protein as well as with tspyv, hpyv, and hpyv vp proteins in western blot. a high degree of cross-reactivity of hpyv vp proteins was observed with rabbit antiserum raised against hapyv vp protein. this antiserum failed to detect only kipyv, wupyv, hpyv, and stlpyv vp proteins. interestingly, vp proteins derived from hpyv and tspyv were recognized by all four tested polyclonal antibodies despite low similarity of both proteins to wupyv vp. in contrast, stlpyv vp protein was hardly detected by only one antiserum raised against mcpyv vp protein. anti-mcpyv vp polyclonal antibody additionally cross-reacted with tspyv and hpyv vp proteins. anti-wupyv vp polyclonal antibody showed a very strong cross-reactivity with kipyv vp but also detected hpyv hpyv and hpyv vp proteins identical to wupyv vp only by and 48 %, accordingly. thus, the cross-reactivity allowed detection of all purified hpyv vp proteins by western blot using only four polyclonal antibodies.fig. detection of purified hpyv-derived vp vlps in western blot with polyclonal antibodies. a, coomassie blue-stained sds-page;, western blot with anti-jcpyv vp, anti-hapyv vp, anti-mcpyv vp, and anti-wupyv vp polyclonal antibodies. the same samples of purified proteins were run on each gel. in lanes: - hpyv l protein; - bkpyv vp protein; - jcpyv vp protein; - kipyv vp protein; - wupyv vp protein; - mcpyv vp protein; - hpyv vp protein; - hpyv vp protein; - tspyv vp protein; - hpyv vp protein; - hpyv vp protein; - stlpyv vp protein; - hpyv vp protein; - njpyv vp protein; protein weight marker; - hapyv vp protein ha activity of vp1-derived vlps from novel pyvs: mcpyv, kipyv, wupyv, hpyv hpyv tspyv, hpyv hpyv stlpyv, hpyv and njpyv all purified vlps were subjected to ha testing using guinea pig erythrocytes. jcpyv-, bkpyv-, and sv40-derived vp vlps were used as controls. as shown in fig.  the ha titer of purified preparations of jcpyv- and bkpyv-derived vp vlps with known ha activity was down to a concentration of  μg ml− jcpyv- and bkpyv-derived vp vlps served as positive controls. the preparation of sv40-derived vp vlps did not show any ha activity using guinea pig erythrocytes and so served as a negative control. ha activity was also confirmed for mcpyv-derived vp vlps with the ha titer down to a concentration of  μg ml−. kipyv-, hpyv10-, and hpyv12-derived vp vlps demonstrated strong ha activity, down to a concentration of  μg ml− tspyv- and hpyv9-derived vp vlps showed ha activity down to a concentration of  μg ml− and  μg ml− respectively. wupyv-, hpyv6-, hpyv7-, stlpyv-, and njpyv-derived vp vlps did not show any ha activity.fig. ha activity of hpyv-derived vp vlps. a 2-fold dilution series of purified kipyv-, wupyv-, mcpyv-, hpyv6-, hpyv7-, tspyv-, hpyv9-, hpyv10-, stlpyv-, hpyv12-, and njpyv-derived vp vlps expressed in s. cerevisiae were subjected to a ha assay with 1 % guinea pig erythrocytes. purified jcpyv- and bkpyv-derived vp vlps were used as positive controls, and sv40-derived vp vlps were used as negative controls. the concentrations of vp1-derived vlps in μg ml− are shown on the top. the bar indicates the highest vlp concentration at which ha of guinea pig erythrocytes was observed discussion recently, new hpyvs have been identified. in contrast to the better-studied pyvs, jcpyv, bkpyv, and to a degree, mcpyv, the routes of infection, entry pathways, and cell tropism of these new hpyvs remain unknown. the variety and number of hpyvs indicate that they are capable of self-propagating at low enough levels that do not cause disease, except in immunosuppressed individuals. to advance research on new hpyvs, development of cell culture systems will be important. however, in some applications, vlps may be used instead, particularly in cases where the wild-type virus is not easily cultivated using cell cultures. recombinant vp proteins from previously discovered pyvs were successfully produced in escherichia coli, yeast or baculovirus/insect cell expression systems. yeast or baculovirus/insect cell expression systems were used for efficient production of self-assembled vp vlps derived from bkpyv, jcpyv, hapyv and other pyvs. however, the recombinant vp proteins produced in e. coli were found mainly as pentamers which self-assembled into vlps during purification. only jcpyv- and hapyv-derived vp were shown to form vlps inside bacteria. recently large-scale and high-yield production yielding gram-per-liter levels of mpyv vp fused with glutathione s-transferase in e. coli was reported. vp protein was purified as pentamer and vlps were reassembled in vitro after removal of the gst-tag. it should be noted that the assembly of purified pentamers into vlps always has a negative impact on the final yield, though for mpyv vp the vlp reassembly yield was improved and reached 42–56 %. nevertheless, other authors have shown that yeast-produced mpyv derived vp vlps were more stable as compared to vlps assembled from pentamers purified from e. coli. similarly, recombinant gst or 6his-tagged vp proteins or vp pentamers but not vlps derived from novel hpyvs were produced in bacterial expression systems. to synthesize hpyv-derived vp vlps, mammalian and/or insect cells have been used. mcpyv-derived vp vlps have been generated in mammalian and insect cells. hpyv6-, hpyv7-, tspyv-, hpyv9-, and mwpyv/hpyv10-derived vp vlps have also been successfully produced in insect cells. however, vp1-derived vlps from stlpyv, hpyv and njpyv have not been generated at all until now. in this study, we demonstrated that vp1-derived vlps from recently discovered hpyvs can be efficiently produced in the yeast, s. cerevisiae. the synthesis of vlps using the yeast expression system has several advantages. s. cerevisiae is a gras microorganism, free of toxins hazardous to human health. the preparation of products produced in yeast for human use is well established. additionally, recombinant vp1-derived vlp expression in yeast is relatively cheaper than in insect or mammalian cell cultures, and it results in high yields of vlp. currently, s. cerevisiae is used in the production of vaccines against the recombinant hepatitis b virus and the human papillomavirus. in this study, to generate new hpyv-derived vp proteins in yeast, both native vp1-encoding genes and genes optimized for expression in s. cerevisiae were used. the lowest yield of purified hpyv10- and hpyv12-derived vp protein was obtained using the native gene sequence and njpyv-derived vp protein using gene optimized for expression in s. cerevisiae. mcpyv-derived vp was also expressed using the native gene sequence, but we obtained a high yield of this protein, demonstrating that gene sequence is not the only factor influencing the yield of recombinant protein. the properties of recombinant proteins, stability, resistance to proteases, and solubility, in particular, may have also had an impact on purification efficiency and the final yield. therefore, the optimization of genes for use in s. cerevisiae resulted in only slightly better yields of vp from kipyv, wupyv, hpyv hpyv and stlpyv compared to hpyv10-derived vp the yield of highly-soluble tspyv-, hpyv9-, and hpyv12-derived vp vlps, produced using optimized genes, was almost two times greater. on the other hand, the difference between the optimized versus native genes was obvious when a shorter hpyv vp gene version was used. in this case, utilization of optimized vp gene improved the yield of vp1s by a factor of nearly. the yeast system supported self-assembly of vp1-derived vlps from all recently discovered hpyvs. however, hpyv6- and wupyv-derived vp vlp preparations contained mainly smaller-sized capsids. in the case of hpyv6-derived vp vlps, small particles were also observed in other hosts. electron micrographs of hpyv6- and hpyv7-derived vp vlps purified from mammalian 293tt cells show particles smaller than 45–50 nm in diameter. the proportion of small hpyv6-derived vp vlps produced in insect cells was similar to that purified in yeast. purified hpyv7-derived vp vlps produced both in insect and yeast cells self-assemble into typical pyv-derived vlps. in contrast, small particles predominate in preparations of hpyv9-dervied vp vlps purified from insect cells. however, in our study, the yeast-generated hpyv9-derived vp vlps assembled into mostly typical pyv-like particles with diameters of approximately 45 nm. an explanation for different efficiencies in forming normally-sized vlps from different pyvs remains elusive. it has been proposed that variation in particle size in sv actually indicates the presence of mosaic vlps, which form from a combination of different geometries of pentamer interactions. due to numerous possible interactions, sv pentamers can self-assemble into a broad array of shapes; this assembly can be redirected by different nucleic acids serving as a scaffold and as a nucleating factor. similarly, mpyv-derived vp self-assembles into vlps that are and 50 nm in diameter. it has been suggested that the n-terminal arm of vp forms a clamp to hold the c-terminal protrusions from neighboring capsomeres in place, connecting the n-terminal arms of vp from different capsomeres via the intercapsomeric disulphide bridges. flexibility in this clamping mechanism allows capsomeres to exist in both the pentavalent and hexavalent state and permits the assembly of polymorphic particles in pyvs. on the other hand, sometimes even a single change in an aa residue can influence the size of vp1-derived vlps. this has been shown in the hapyv, wherein a single a336g aa exchange in the c-terminal arm of vp results in smaller vp1-derived vlps that are 20 nm in diameter. the hpyv and hpyv genomes that have been sequenced so far reveal naturally-occurring vp aa sequence variation; it is not known how such variation influences vlp assembly. because smaller particles predominate among hpyv6-derived vp vlps produced in yeast, insect, and mammalian cells, it is possible that some aa variants can support self-assembly of normal-sized hpyv6-derived vp vlps. as a result of our attempts to express hpyv12-derived vp vlps in yeast and alignment of various pyv-derived vp n-terminal sequences, our data suggest that the true translation initiation site predicted by the vp1-encoding orf is the second translation initiation site. removal of the 16-aa-long n-terminal peptide greatly improved vp expression and vlp formation. there are only a few such cases known in other pyv genomes. hapyv-, jcpyv-, and sv40-derived vp is synthesized starting from the second of two potential translation initiation sites in their respective genes. the development of cell culture systems for the production of hpyv and determination of the n-terminal sequence of vp isolated from the virus capsid could be useful in confirming the true translation initiation start site. ha activity using guinea pig erythrocytes can expand the number of potential applications for vp1-derived vlps. they can be used as antigens, not only in enzyme immunoassays, but also in more specific ha inhibition assays to detect corresponding hpyv-specific antibodies in human sera. on the other hand, the ha activity of isolated hpyv-derived vp vlps could serve as an initial index of virus cell-binding properties, which are related to viral spreading, cell tropism, and pathogenicity. the ha activity of jcpyv is associated with the binding of vp to sialic acid residues. in contrast, sv has no ha activity and uses other receptor molecules, e.g., major histocompatibility complex class i molecules. it has been reported that sv might bind to sialic acid residues present on ganglioside gm but the steric arrangement of the binding amino acids differs from that of hemagglutinating viruses, such as mpyv. consistent with these observations, it has been shown that mutations in the sialic acid binding site impair ha of mcpyv-derived vp capsids. the variation in ha activity observed in this study among vp1-derived vlps from different pyvs might reflect diversity in receptor recognition among pyvs. ha of guinea pig erythrocytes by kipyv-, tspyv-, hpyv9-, hpyv10-, and hpyv12-derived vp vlps suggests that these viruses might use sialic acid residues to bind the virus particle to the host cell. in contrast, wupyv-, hpyv6-, hpyv7-, stlpyv-, and njpyv-derived vp vlps showed no ha activity and thus more likely use other receptor molecules. indeed, according to the solved high-resolution crystal structure of hpyv vp in complex with the three putative oligosaccharide receptors identified by glycan microarray screening, hpyv engages short sialylated oligosaccharides. consistent with these observations, hpyv9-derived vp vlps showed ha of guinea pig erythrocytes in our study. protein structure analysis of hpyv6- and hpyv7-derived vp reveals an obstructed sialic acid binding site. additionally, vp derived from hpyv and hpyv does not interact with sialylated compounds in solution or in cultured human cells. stroh et al. suggest that it is still possible that hpyv and hpyv bind sialic acids at a site in the fully assembled virus, but not in the free vp pentamer. in our study, the yeast-produced hpyv6- and hpyv7-derived vp vlps had no ha activity, supporting the recent observations that these two viruses bind non-sialylated receptors. the vp1-derived vlps from two other members of the genus wukipolyomavirus differed in their ability to hemagglutinate guinea pig erythrocytes, despite the fact that none of the sialic acid binding residues found in sv jcpyv, or mpyv are conserved in the wupyv and kipyv vp structures. kipyv-derived vp vlps efficiently hemagglutinated guinea pig erythrocytes, whereas wupyv-derived vp vlps did not show any ha activity. the data suggest that these two similar viruses might use different receptor molecules to bind host cells.  the yeast expression system was successfully adapted for high-throughput production of recombinant vp1-derived vlps from newly identified hpyvs. although hpyv vp was predicted to encode a 380-aa-long protein, our findings suggest that translation starts not at the first, but rather at the second, of two potential initiation sites in the vp1-encoding orf, resulting in a 364-aa-long protein. yeast-generated recombinant vp1-derived vlps from different hpyvs demonstrated distinct ha activities and may be useful in virus diagnostics, capsid structure studies, or the investigation of entry pathways and cell tropism of new hpyvs until cell culture systems for new hpyvs are developed. 
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. vp proteins of polyomaviruses can assembly into virus-like particles. human polyomavirusvp1virus-like particlesyeasthemagglutination assayissue-copyright-statement© the author 2015 serological studies show that these novel hpyvs sub-clinically infect humans at an early age. smaller-sized vlps predominated in preparations from washington university polyomavirus and hpyv attempts to express recombinant hpyv vp1-derived vlps in yeast indicate that translation of vp might start at the second of two potential translation initiation sites in the vp1-encoding open reading frame. vp proteins derived from merkel cell polyomavirus, trichodysplasia spinulosa-associated polyomavirus, and new jersey polyomavirus self-assembled into homogeneous similarly-sized vlps. the routes of infection, entry pathways, and cell tropism of new hpyvs remain unknown. hpyv vp1-derived vlps were generated from the second of two potential translation initiation sites in the vp1-encoding orf. karolinska institutet polyomavirus, hpyv hpyv hpyv and st. louis polyomavirus vp proteins formed vlps that varied in size with diameters ranging from to 60 nm. the yeast expression system was successfully utilized for high-throughput production of recombinant vp1-derived vlps from newly identified hpyvs. mcpyv-, kipyv-, tspyv-, hpyv9-, hpyv10-, and hpyv12-derived vlps showed hemagglutination assay activity in guinea pig erythrocytes, whereas wupyv-, hpyv6-, hpyv7-, stlpyv- and njpyv-derived vp vlps did not.
 eleven new human polyomaviruses 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. vp proteins of polyomaviruses can assembly into virus-like particles. as cell culturing systems for hpyv are currently not available, vp1-derived vlps may be useful tools in basic research and biotechnological applications.  recombinant vp1-derived vlps from newly identified hpyvs were efficiently expressed in yeast. vp proteins derived from merkel cell polyomavirus, trichodysplasia spinulosa-associated polyomavirus, and new jersey polyomavirus self-assembled into homogeneous similarly-sized vlps. karolinska institutet polyomavirus, hpyv hpyv hpyv and st. louis polyomavirus vp proteins formed vlps that varied in size with diameters ranging from to 60 nm. smaller-sized vlps predominated in preparations from washington university polyomavirus and hpyv attempts to express recombinant hpyv vp1-derived vlps in yeast indicate that translation of vp might start at the second of two potential translation initiation sites in the vp1-encoding open reading frame. this translation resulted in a 364-amino acid-long vp protein, which efficiently self-assembled into typical pyv vlps. mcpyv-, kipyv-, tspyv-, hpyv9-, hpyv10-, and hpyv12-derived vlps showed hemagglutination assay activity in guinea pig erythrocytes, whereas wupyv-, hpyv6-, hpyv7-, stlpyv- and njpyv-derived vp vlps did not.  the yeast expression system was successfully utilized for high-throughput production of recombinant vp1-derived vlps from newly identified hpyvs. hpyv 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. keywords human polyomavirusvp1virus-like particlesyeasthemagglutination assayissue-copyright-statement© the author 2015 