cells : hek293 t , human embryonic kidney 293 cell line expressing sv40 tag , 
 cells were maintained under an atmosphere of 5% co2 at 37c in dulbecco s minimum 
 essential medium supplemented with 10% fbs ( fetal bovine serum ) , 2 mm l - glutamine , 
 penicillin ( 100 u ml ) and streptomycin ( 0.1 mg 
 ml ) . all experiments using hek293 t cells were performed in 
 collagen - coated dishes ( iwaki , chiba , japan ) . plasmids and transfection : for expression of vmpyv vp1 , the full - length 
 vmpyv vp1 gene was amplified from the vmpyv genomic dna ( genbank accession number : ab767298 ) 
 with xhoi and noti restriction sites added to the 5 and 
 3 ends , respectively . the pcr product was cloned into the 
 xhoi - noti restriction sites of a pcmv - flag vector , so 
 that a flag tag was added to the n - terminal of expressed vp1 . the c - terminal deletion mutant 
 of vmpyv vp1 was also cloned into the vector . these plasmids were transfected into hek293 t 
 cells individually using the lipofectamine 2000 according to the manufacturer s instructions 
 ( invitrogen , carlsbad , ca , u.s.a . ) . immunocytochemical and immunoblot analyses : hek293 t cells transfected with 
 the plasmids were collected at 48 hr post transfection . the cells were washed with pbs , 
 fixed in 100% methanol for 5 min at 30c and blocked with 1% bsa ( bovine serum albumin ) in 
 pbs containing 0.5% triton x-100 , followed by incubation with an anti - sv40 vp1 antibody 
 overnight at 4c . the cells were visualized with 
 secondary antibodies ( alexa fluor 488-conjugated goat anti - rabbit igg ; invitrogen ) and 4 , 
 6-diamidino-2-phenylindole dihydrochloride ( dapi ; invitrogen ) for 1 hr at room temperature . all the fluorescent images were captured and analyzed using a microscope ( ix70 ; olympus , 
 tokyo , japan ) , a charge - coupled device camera ( dp30bw ; olympus ) and dp controller software 
 ( olympus ) . for immunoblot analysis , the cells were harvested in ripa buffer [ 10 mm tris - hcl ( ph 7.5 ) , 
 5 mm edta , 150 mm nacl , 10% glycerol , 1% triton x-100 , 1% deoxycholic acid , 0.1% sds and 50 
 mm naf ] , supplemented with complete protease inhibitor cocktail ( roche diagnostics , 
 indianapolis , in , u.s.a . ) . cell lysates were 
 centrifuged at 20,400  g for 15 min at 4c , and the resulting supernatants 
 were subjected to sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ) and 
 immunoblotting with the following primary antibodies overnight at 4c ; an anti - sv40 vp1 
 antibody   and an anti - actin antibody ( mab1501 ; 
 millipore , bedford , ma , u.s.a . ) . actin was used as a loading control . after washing the 
 membrane with tbst ( tris - buffered saline containing 0.05% tween 20 ) , the membrane was 
 incubated with the following secondary antibodies for 1 hr at room temperature ; a 
 horseradish peroxidase ( hrp)-conjugated anti - rabbit igg ( biosource international , camarillo , 
 ca , u.s.a . ) and hrp - conjugated anti - mouse igg ( biosource international ) . the 
 chemiluminescence signals were visualized using a versadoc 5000mp ( bio - rad , hercules , ca , 
 u.s.a . ) , and images were analyzed using quantity one software ( bio - rad ) . electron microscopy : ultra - thin - section electron microscopy was performed 
 as described previously . in brief , 48 hr post 
 transfection cells were fixed with 2.5% glutaraldehyde in 0.1 m cacodylate buffer ( ph 7.3 ) 
 for 20 min at 4c . the cells were scraped from the dish and fixed with 2% osmium tetroxide 
 in the same buffer for 1 hr at 4c . pellets were dehydrated with a series of ethanol 
 gradients ( 50% , 70% , 90% and 99.5% ) followed by propylene oxide , embedded in epon 812 resin 
 mixture ( taab laboratories equipment , berkshire , u.k . ) and polymerized for 3 days at 60c . thin - sections ( 70 nm ) were stained with uranyl acetate and lead citrate . for negative staining , purified fractions fixed with 0.25% glutaraldehyde were adsorbed to 
 collodion - carbon - coated copper grids ( nisshin em corporation , tokyo , japan ) and negatively 
 stained with 2% phosphotungstic acid solution ( ph 5.8 ) . all samples were examined with an 
 h-7650 electron microscope at 80 kv ( hitachi , kyoto , japan ) . sucrose gradient sedimentation analysis : sucrose gradient sedimentation 
 analysis was performed as described previously . briefly , 72 hr post transfection cells were harvested in 10 mm tris - hcl ( ph 7.5 ) , 2 mm 
 mgcl2 and 0.25% brij 58 ( sigma , st . the cellular lysates 
 were subjected to three cycles of freezing and thawing , cellular debris was removed via 
 centrifugation at 500  g for 10 min at 4c , and the resulting supernatants 
 overlaid onto a preformed 3050% sucrose gradient in 20 mm tris - hcl ( ph 8.0 ) . samples were 
 centrifuged at 192,000  g for 1 hr at 4c ( sw55 ti rotor , beckman coulter , 
 brea , ca , u.s.a . ) , and each 400 l fraction was taken from the top for 12 
 fractions . each fraction was subjected to sds - page and immunoblotting with the anti - sv40 vp1 
 antibody overnight at 4c . after washing the 
 membrane with tbst , the membrane was incubated with hrp - conjugated anti - rabbit igg for 1 hr 
 at room temperature ( biosource international ) . ( de3 ) plyss competent cells ( stratagene , la jolla , ca , u.s.a . ) were 
 transformed with the pet15b plasmid ( novagen , madison , wi , u.s.a . ) encoding the full - length 
 jcv vp1 gene , and vlps were purified . expression of vmpyv vp1 in hek293 t cells : the full - length of vmpyv vp1 
 gene was amplified and cloned into the pcmv - flag vector . in addition , we synthesized a 
 c - terminal deletion mutant of vmpyv vp1 ( c vp1 ) . the deleted region was determined based on 
 the amino acid sequences alignment with the reported pyv vp1s ( fig . 1fig . the vp1 sequences of pyvs were obtained from genbank 
 ( abbreviations and accession numbers are indicated in table 1 ) . amino acid identities are shaded as follows : black shading indicates 
 that all amino acid sequences were conserved , whereas grey shading indicates that more 
 than 51% of them were conserved . ) . we synthesized the c vp1 of 1387 amino acid residues of wild - type vmpyv vp1 ( wt 
 vp1 ) . the c - terminal deletion mutant vp1 gene with a stop codon ( taa ) was also amplified and 
 cloned into the pcmv - flag vector . these plasmids were transfected into hek293 t cells 
 individually and incubated for 48 hr . to detect the vp1 protein expression both wt and c vp1s were mainly detected in the nuclei and 
 sometimes in the cytoplasm in transfected cells with encoding wt or c vp1 plasmids ( fig . immunocytochemical and immunoblot 
 analyses ( ic and ib ) of vmpyv vp1 with the anti - sv40 vp1 antibody . the hek293 t cells 
 were transfected with wt vp1 , c vp1 or the corresponding empty vector ( mock ) as a 
 negative control . ( b ) wt and c vp1 signals were 
 detected in cellular lysates from hek293 t cells at the expected molecular weight 
 positions in immunoblotting . we also performed immunoblotting analysis to confirm the expression levels of wt 
 and c vp1s . we detected both wt and c vp1s in the cells using the anti - sv40 vp1 antibody 
 at the expected molecular weights of 56 and 44 kda , respectively ( fig . 2b ) . because c vp1 is devoid of 388503 amino acid residues 
 ( 116 a.a . the expression 
 levels of wt and c vp1s were almost similar relative to the expression levels of internal 
 control protein actin ( fig . these results 
 suggested that the intracellular localization and expression levels of wt and c vp1s were 
 similar in the transfected cells . the vp1 sequences of pyvs were obtained from genbank 
 ( abbreviations and accession numbers are indicated in table 1 ) . amino acid identities are shaded as follows : black shading indicates 
 that all amino acid sequences were conserved , whereas grey shading indicates that more 
 than 51% of them were conserved . immunocytochemical and immunoblot 
 analyses ( ic and ib ) of vmpyv vp1 with the anti - sv40 vp1 antibody . the hek293 t cells 
 were transfected with wt vp1 , c vp1 or the corresponding empty vector ( mock ) as a 
 negative control . ( b ) wt and c vp1 signals were 
 detected in cellular lysates from hek293 t cells at the expected molecular weight 
 positions in immunoblotting . expression of vlps using electron microscopy : because the vmpyv vp1 was 
 detected in transfected hek293 t cells , we confirmed the formation of vlps using transmission 
 electron microscopy ( tem ) . at 48 hr post transfection of wt and c vp1 encoding plasmids , ultra - thin - sections with a 
 thickness of 70 nm were stained with uranyl acetate and lead citrate . tem revealed a large 
 number of vlps with a diameter of approximately 50 nm in the nuclei of wt vp1-expressing 
 cells ( wt vlps ; fig . 3a3cfig . electron micrographs of 
 hek293 t cells expressing wt vp1 ( a  c ) and c vp1 ( d  f ) . ( b , c , e and f ) higher 
 magnification of the regions indicated in panels a , b , d and e , respectively . we were also able to confirm vlps with a diameter of approximately 4550 nm in the 
 nuclei of c vp1-expressing cells ( c vlps ; fig . 3d3f ) ; however , the number of c vp1 vlps was much lower than that of wt vp1 
 vlps . electron micrographs of 
 hek293 t cells expressing wt vp1 ( a  c ) and c vp1 ( d  f ) . ( b , c , e and f ) higher 
 magnification of the regions indicated in panels a , b , d and e , respectively . sucrose gradient sedimentation analysis : to confirm that vlps were formed 
 by vmpyv vp1 in transfected cells , we also performed the sucrose gradient sedimentation 
 analysis , which can distinguish vlps from vp1 pentamers . as a positive control in the analysis the vp1 signal in jcv vlps was mainly detected in fractions 6 to 
 9 ( fig . ( a  c ) immunoblot analyses of vp1 in 
 fractionated samples after sucrose gradient sedimentation of jcv vlps and cellular 
 lysates from hek293 t cells expressing wt vp1 or c vp1 . cellular lysates were 
 separated by 3050% sucrose gradient sedimentation and fractionated into 12 fractions 
 from the tops of the tubes . the 12 fractions were separated by sds - page and subjected 
 to immunoblotting with the anti - sv40 vp1 antibody . ( b ) hek293 t cell lysates transfected with wt vp1 and ( c ) 
 c vp1 . ( d ) electron micrograph of negative staining of fractions 6 to 9 from hek293 t 
 cells transfected with wt vp1 . the vp1 signal in cellular lysates from the wt vp1-expressing cells was also mainly 
 detected in fractions 6 to 9 ( fig . however , 
 the vp1 signal in cellular lysates from c vp1-expressing cells was mainly detected in 
 fractions 1 to 4 and slightly detected in fractions 5 to 8 ( fig . 4c ) . to confirm the formation of wt vlps in fractions 6 to 9 , we 
 collected these fractions and verified them with negative - stained tem . we observed a large 
 number of wt vlps with a diameter of approximately 50 nm ( fig . we also confirmed the presence of jcv vlps in fractions 6 to 9 ( data not 
 shown ) . ( a  c ) immunoblot analyses of vp1 in 
 fractionated samples after sucrose gradient sedimentation of jcv vlps and cellular 
 lysates from hek293 t cells expressing wt vp1 or c vp1 . cellular lysates were 
 separated by 3050% sucrose gradient sedimentation and fractionated into 12 fractions 
 from the tops of the tubes . the 12 fractions were separated by sds - page and subjected 
 to immunoblotting with the anti - sv40 vp1 antibody . ( b ) hek293 t cell lysates transfected with wt vp1 and ( c ) 
 c vp1 . ( d ) electron micrograph of negative staining of fractions 6 to 9 from hek293 t 
 cells transfected with wt vp1 . vmpyv was originally detected in a vm spleen using nested broad - spectrum pcr techniques . it 
 has a longer vp1 orf in the c - terminus region compared with the sequences of other known 
 pyvs vp1s , whereas its functions are still unclear . in general , the pyv capsid contains 360 molecules of vp1 formed with 72 
 pentamers , 5 molecules of vp1 and 1 molecule of vp2/vp3 [ 13 , 22 ] . in the current study , to examine 
 the role of c - terminal of vmpyv vp1 in virion formation , vmpyv vlps consisting of wt vp1 or 
 c vp1 were generated in mammalian hek293 t cells . immunocytochemical analysis revealed that wt and c vp1s were expressed in the transfected 
 hek293 t cells ( fig . both wt and c vp1s were 
 also detected at the expected molecular weights by immunoblot analysis ( fig . furthermore , there was no difference in the expression 
 level between wt and c vp1s ( fig . although 
 vlps were observed in wt and c vp1-expressing cells by using electron microscopy ( fig . 3 ) , the number of wt vlps was higher than that of 
 c vlps . in addition , the number of cells with wt vlps was higher than that of cells with c 
 vlps ( data not shown ) . and c 
 vlps were observed in the nuclei ; however , no vlps were confirmed in the cytoplasm ( data not 
 shown ) . immunoblot analysis revealed that the signal of jcv vlps was mainly 
 detected in fractions 6 to 9 ( fig . the vp1 
 signal in cellular lysates from wt vp1-expressing cells was also mainly detected in 
 fractions 6 to 9 ( fig . we confirmed the 
 presence of jcv vlps and wt vlps in fractions 6 to 9 with negative - stained tem ( fig . however , the vp1 signal in cellular lysates 
 from c vp1-expressing cells was mainly detected in fractions 1 to 4 ( fig . because the protein density in fractions 1 to 4 is lower 
 than that in fractions 6 to 9 , it is supposed that the vp1 signal in fractions 1 to 4 may 
 have represented the pentamers instead of vlps . in addition , the faint vp1 signal in 
 fractions 5 to 8 of cellular lysates from c vp1-expressing cells may have represented vlps . this result is convincing in light of the tem results showing fewer vlps in c 
 vp1-expressing cells ( fig . taken together , 
 the results showed that c vp1 formed vlps ; however , the efficiency of vlp formation was 
 lower than that of wt vp1 . it has been reported that the c - terminal arm of pyvs vp1 can be subdivided into three 
 segments ;  c helix , c insert and  c loop [ 13 , 
 22 ] . the c helix of sv40 ( sfllsdlinrrtq ; 305317 
 a.a . ) mediates contacts between pentamers as described previously [ 13 , 22 ] . as demonstrated in fig . 1 , the c helix of sv40 is predicted to correspond 
 to the 295307 amino acid residues ( sflltdlinrrtp ) of jcv and the 325337 amino acid 
 residues ( tsllgslftglmp ) of vmpyv . in comparison with tripartite , the homology was 85% for 
 sv40-jcv , 23% for sv40-vmpyv and 31% for jcv - vmpyv in the c helix of vp1s . it revealed that 
 the c - helix of jcv may mediate contacts between pentamers , because of the high homology with 
 that of sv40 . however , the c helix of vmpyv has low homology with that of sv40 and jcv . as 
 shown in fig . 3 , we observed that the number of wt 
 vp1 vlps was much higher than that of c vp1 vlps . thus , the deleted c - terminal 116 amino 
 acid residues ( 388503 a.a . ) of vmpyv vp1 affect the efficiency of its vlp formation . chpyv also encodes a unique extended c - terminal vp1 protein of 497 a.a . , and these results suggest that the length of vp1 amino acid 
 residues has no effect on the size ( i.e. , diameter ) of vlps . we observed that 
 vmpyv vlps have a typical shape of pyv virions , and their diameters were approximately 50 nm 
 in size , suggesting that native vmpyv virions also have morphology similar to that of its 
 vlps . in conclusion , we demonstrated that vmpyv vlps were formed in mammalian cells expressing 
 vp1 and found the extra c - terminal region of vp1 does not affect the size and morphology of 
 vlps , whereas the c - terminal of vmpyv vp1 may have some function for efficient vlp 
 formation . the study to investigate function ( s ) of the extra c - terminal region of vmpyv vp1 
 needs to be continued .