cells and viruses : bovine testicle ( bt ) cells were grown in eagle s 
 minimum essential medium ( emem ) ( nissui pharmaceutical , tokyo , japan ) supplemented with 
 0.295% tryptose phosphate broth ( tpb ) ( becton dickinson , san jose , ca , u.s.a . ) and 5% fetal 
 bovine serum ( fbs ) ( mitsubishi chemical , tokyo , japan ) . bovine kidney cell line mdbk - hs 
  and porcine kidney cell line sk - l   were grown in emem supplemented with 0.295% tpb , 10% 
 horse serum ( hs ) ( life technologies , carlsbad , ca , u.s.a . ) and 10 mm n , n - bis 
 ( 2-hydroxyethyl)-2-aminoethanesulfonic acid ( bes ) ( sigma - aldrich , st . bovine fetal muscle ( bfm ) cells were grown in emem supplemented with 0.295% tpb , 5% fbs , 5% 
 hs and 10 mm bes . cells were confirmed to be free from bvdv , and fbs was confirmed to be 
 free from both bvdv and anti - bvdv neutralizing antibodies . the bvdv gbk strain is an adventitious bvdv2 isolated from cells of the bovine kidney cell 
 line gbk . the 
 ndv miyadera strain was propagated in 10-day - old embryonated hens eggs . the new jersey 
 serotype strain of vsv was grown in sk - l cells . cloning of a pair of end and end viruses from viral 
 stocks : a reverse plaque formation technique   was used to obtain a pair of end and end viruses from 
 viral stocks as described previously . sequencing : bvdv gbk_e and gbk_e strains , the 
 full - length cdna clones and the in vitro - rescued viruses were completely 
 sequenced as described previously . in brief , the 
 nucleotide sequences of cdna clones and pcr fragments from viral rna were determined using 
 the bigdye terminator v3.1 cycle sequencing kit ( life technologies ) and a 3130 genetic 
 analyzer or a 3500 genetic analyzer ( life technologies ) according to the manufacturer s 
 protocol . sequencing data were analyzed using genetyx version 10 software ( genetyx , tokyo , 
 japan ) . plasmid constructs : the cdna fragments from the gbk_e strain , 
 obtained by reverse transcription polymerase chain reaction ( rt - pcr ) , were cloned into 
 plasmid pcr - blunt ii - topo ( life technologies ) using the zero 
 blunt topo cloning kit ( life technologies ) . the cdna sequence was 
 flanked by a modified t7 promoter sequence at the 5 end and a pst i 
 restriction site at the 3 end . subclones were assembled into a full - length cdna clone , 
 termed pgbk_e , by replacing the csfv genome of the full - length cdna clone of the 
 csfv alfort187 - 1 strain pa187 - 1   with the genome 
 of the gbk_e strain using appropriate restriction enzymes and the in - fusion hd 
 cloning kit ( clontech , mountain view , ca , u.s.a . ) according to the manufacturer s protocol . the full - length cdna clone 
 pgbk_e was transformed and propagated in competent cell stbl3 cells ( life 
 technologies ) and purified using the qiagen plasmid plus midi kit ( qiagen , hilden , germany ) 
 according to the manufacturer s protocol . g , 
 i2623v , d3148 g and d3502y were constructed in the pgbk_e backbone using the 
 quickchange lightning multi mutagenesis kit ( agilent technologies , santa clara , ca , u.s.a . ) 
 and the in - fusion hd cloning kit . the plasmid pgbk_e / d136 g has a single amino 
 acid substitution at position 136 of gbk_e , whereas the plasmids 
 pgbk_e / i2623v ; d3148 g ; d3502y and pgbk_e / d136 g ; i2623v ; d3148 g ; 
 d3502y have multiple amino acid substitutions at positions 2623 , 3148 and 3502 of 
 gbk_e and at positions 136 , 2623 , 3148 and 3502 of gbk_e , 
 respectively . full - length pcr amplification , in vitro rna transcription , transfection and viral 
 recovery : the cdna - derived viruses were rescued as described previously   with some modifications . the full - length genome 
 amplification strategy   was employed to obtain a 
 full - length pcr amplicon for in vitro rna transcription . the cdna clones 
 were amplified using primers 5gbke_t7 ( 5-taa tac gac tca cta ta gta tac 
 gag att agc taa agt act cg 3 , t7 promoter sequence underlined ) and 3gbke ( 5- 
 ggg gct gtt aga ggc atc ctc tag tc 3 ) with accuprime taq dna polymerase high fidelity 
 ( life technologies ) . viral rna was transcribed in vitro from the purified 
 full - length pcr amplicon using the megascript t7 kit ( life technologies ) , the remaining pcr 
 amplicon was digested using dnase ( life technologies ) , and the viral rna was then purified 
 with a microspin s-400 column ( ge healthcare , buckinghamshire , u.k . ) . mdbk - hs cells ( 3  
 10 cells ) were transfected with 10 g of viral rna in a 0.4 
 cm cuvette using the gene pulser xcell electroporation system ( bio - rad , hercules , ca , 
 u.s.a . ) the cells were incubated at 37c in 5% 
 co2 for 3 days , and the supernatants were then transferred onto naive mdbk - hs 
 cells to obtain infectious viruses . the viruses were named according to the plasmid from which they were rescued , replacing the cdna - derived viruses generated in the present study are 
 shown in fig . 1.schematic representation of the genomic differences between gbk_e virus 
 and gbk_e virus , and mutant viruses derived from cdna clones generated in 
 the present study . ( a ) four amino acid substitutions , d136 g , i2623v , d3148 g and 
 d3502y , were found in the gbk_e virus in comparison with the 
 gbk_e virus . ( b ) three recombinant viruses with combinations of amino 
 acid substitutions were generated in the gbk_e backbone by site - directed 
 mutagenesis and in - fusion techniques . the white and gray boxes indicate the 
 nonstructural and structural proteins , respectively .. schematic representation of the genomic differences between gbk_e virus 
 and gbk_e virus , and mutant viruses derived from cdna clones generated in 
 the present study . ( a ) four amino acid substitutions , d136 g , i2623v , d3148 g and 
 d3502y , were found in the gbk_e virus in comparison with the 
 gbk_e virus . ( b ) three recombinant viruses with combinations of amino 
 acid substitutions were generated in the gbk_e backbone by site - directed 
 mutagenesis and in - fusion techniques . end assay and measurements of type i interferon production : the end assay 
 was conducted using bfm cells as described previously . in brief , bfm cells grown in 96-well plates were infected with bvdv and 
 incubated for 5 days at 37c in 5% co2 . the end 
 phenomenon was regarded as positive , if strong cpe was observed in ndv - infected cells . measurements of type i interferon production in bvdv - infected cells were performed using a 
 previously described plaque reduction method with vsv as a challenge virus , with some modifications . in brief , the supernatants 
 from bvdv - infected cells were inactivated by exposure to uv light ( 254 nm ) in the uv 
 crosslinker ( atto corporation , tokyo , japan ) under the condition of 500 mj / cm . viral inactivation of samples was confirmed using indirect fa techniques with anti - bvdv ns3 
 monoclonal antibody # 46/1   after inoculation of 
 samples onto mdbk - hs cells and incubation for 2 days at 37c in 5% co2 . then , 
 monolayers of sk - l cells in 12-well plates were inoculated with 1 ml of a 
 four - fold dilution of uv - inactivated supernatant and incubated for 24 hr at 37c in 5% 
 co2 . interferon titers were expressed as reciprocals of dilutions that reduced the number of 
 challenge viral plaques by 50% . detection of irf-3 in bvdv - infected cells by sodium dodecyl sulfate - polyacrylamide 
 gel electrophoresis ( sds - page ) and western blotting : sds - page and western 
 blotting were performed as described previously . bfm cells were infected with bvdv ( m.o.i . of 1 ) in six - well plates and incubated for 5 days 
 at 37c in 5% co2 . after 
 transferring the proteins from the gels to the immunobilion - p transfer membrane ( millipore , 
 billerica , ma , u.s.a . ) , the membranes were treated with anti - human irf-3 rabbit polyclonal 
 antibodies ( genetex , hsinchu city , taiwan ) , goat anti - rabbit igg - hrp conjugate ( bio - rad ) and 
 immunobilion western detection reagents ( millipore ) , in that order . the membranes were read 
 using lumi vision pro ( aishin seiki , kariya , japan ) , and specific bands for irf-3 were 
 detected . cloning of a pair of end/ end viruses from the bvdv gbk 
 strain and determining their whole genome sequences : a pair of end 
 and end viruses ( gbk_e and gbk_e , respectively ) was 
 cloned from the bvdv gbk strain by means of reverse plaque formation techniques and limited 
 dilution . the genomes of gbk_e and gbk_e viruses were both 12,284 
 nucleotides in length and encoded 3,897 deduced amino acids . comparison of the complete 
 genome sequences of both viruses revealed only six nucleotide and four amino acid 
 differences ( d136 g , i2623v , d3148 g and d3502y , the numbers refer to the amino acid position 
 in gbk_e ) between the two viruses ( table 
 1table 1.differences of amino acid sequences in the genome of gbk_e and 
 gbk_eviral proteinnns4bns5ans5bamino acid position1362,6233,1483,502gbk_ediddgbk_egvgya ) d : aspartic acid , i : isoleucine , g : glycine , v : valine , y : tyrosine . ) . the complete genome sequences of the gbk_e and gbk_e 
 strains were deposited in the ddbj / embl / genbank databases under accession numbers ab894423 
 and ab894424 , respectively . a ) d : aspartic acid , i : isoleucine , g : glycine , v : valine , y : tyrosine . generation and characterization of in vitro - rescued viruses : the 
 infectious bvdv vgbk_e was successfully rescued by electroporation of mdbk - hs 
 cells with viral rna transcribed in vitro from a full - length pcr amplicon 
 obtained from a cdna clone pgbk_e . the mutant viruses 
 vgbk_e / d136 g , vgbk_e / i2623v ; d3148 g ; d3502y and 
 vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y were also recovered from full - length cdna 
 clones . sequencing of the complete genomes of these three viruses confirmed the mutations at 
 the desired amino acid positions and demonstrated the lack of any other mutations in 
 comparison with the parental gbk_e virus . to investigate the biological properties of the mutant viruses , bfm cells were inoculated 
 with both the parental gbk_e and the in vitro - rescued 
 vgbk_e viruses . the results revealed that both gbk_e and 
 vgbk_e were ncp ( data not shown ) and exhibited the end phenomenon 
 ( end ) ( fig . bfm cells infected with gbk_e , gbk_e 
 or the in vitro - rescued viruses were superinfected with newcastle 
 disease virus ( ndv ) . cells infected with gbk_e , vgbk_e or 
 vgbk_e / i2623v ; d3148 g ; d3502y exhibited distinguishable cpe after 
 superinfection with ndv ( end ) , whereas those infected with 
 gbk_e , vgbk_e / d136 g or vgbk_e / d136 g ; i2623v ; 
 d3148 g ; d3502y did not exhibit cpe ( end ) in addition , titration of both viruses in mdbk - hs cells revealed that 
 vgbk_e exhibited the same growth characteristics as wild - type 
 gbk_e ( data not shown ) . moreover , compared with the wild - type 
 gbk_e strain , other in vitro - rescued viruses grew equally in 
 mdbk - hs cells ( data not shown ) . bfm cells infected with gbk_e , gbk_e 
 or the in vitro - rescued viruses were superinfected with newcastle 
 disease virus ( ndv ) . cells infected with gbk_e , vgbk_e or 
 vgbk_e / i2623v ; d3148 g ; d3502y exhibited distinguishable cpe after 
 superinfection with ndv ( end ) , whereas those infected with 
 gbk_e , vgbk_e / d136 g or vgbk_e / d136 g ; i2623v ; 
 d3148 g ; d3502y did not exhibit cpe ( end ) identification of the amino acid determinants responsible for inhibition of type i 
 interferon production : as expected , the mutant viruses vgbk_e / d136 g , 
 vgbk_e / i2623v ; d3148 g ; d3502y and vgbk_e / d136 g ; i2623v ; d3148 g ; 
 d3502y remained ncp ( data not shown ) . an end assay revealed that the 
 vgbk_e / d136 g and vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y viruses were 
 changed to end ; bfm cells infected with these viruses did not exhibit cpe after 
 ndv superinfection . in contrast , the vgbk_e / i2623v ; d3148 g ; d3502y virus 
 remained end ; bfm cells infected with this virus exhibited clear cpe after 
 superinfection with ndv , as observed in bfm cells infected with gbk_e ( fig . the amount of type i interferon in supernatants from bfm cells infected with the parent or 
 one of the mutant viruses gbk_e , gbk_e , vgbk_e , 
 vgbk_e / d136 g , vgbk_e / i2623v ; d3148 g ; d3502y or 
 vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y was measured in sk - l cells . secretion of 
 type i interferon was inhibited in bfm cells infected with gbk_e , 
 vgbk_e or the virus carrying the mutations without npro 
 ( vgbk_e / i2623v ; d3148 g ; d3502y ) , which is in consistent with the results 
 from the end assay . cells infected with gbk_e or viruses carrying mutations in 
 n(vgbk_e / d136 g and vgbk_e / d136 g ; i2623v ; d3148 g ; 
 d3502y ) did induce type i interferon ( table 
 2table 2.measurements of type i interferon in cells infected with wild - type and 
 in - vitro rescued virusesvirusestype i interferongbk_e<4vgbk_e<4vgbk_e / d136g4vgbk_e / i2623v ; d3148 g ; d3502y<4vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y4gbk_e4 ) . these results clearly indicate that the aspartic acid of n(at position 136 
 of the gbk_e strain ) is the key amino acid residue that determines the capacity 
 to inhibit the production of type i interferon and induce the end phenomenon . detection of irf-3 in cells infected with the parental or mutant viruses : 
 to investigate the expression levels of irf-3 in bvdv - infected cells , irf-3 in the lysates 
 of bfm cells infected with gbk_e , gbk_e , vgbk_e or one 
 of the three mutant viruses was detected by western blotting . irf-3 was not detected in the 
 lysates of cells infected with gbk_e , vgbk_e or 
 vgbk_e / i2623v ; d3148 g ; d3502y , whereas clear bands of irf-3 appeared in the 
 lysates of cells infected with the end gbk_e virus or one of the 
 n mutant viruses ( vgbk_e / d136 g and vgbk_e / d136 g ; 
 i2623v ; d3148 g ; d3502y ) ( fig . 3fig . irf-3 was not 
 detected in western blots of the lysates of cells infected with gbk_e , 
 vgbk_e or vgbk_e / i2623v ; d3148 g ; d3502y , whereas clear bands 
 of irf-3 appeared in western blots of the lysates of cells infected with 
 gbk_e , vgbk_e / d136 g or vgbk_e / d136 g ; i2623v ; 
 d3148 g ; d3502y . ) . irf-3 was not 
 detected in western blots of the lysates of cells infected with gbk_e , 
 vgbk_e or vgbk_e / i2623v ; d3148 g ; d3502y , whereas clear bands 
 of irf-3 appeared in western blots of the lysates of cells infected with 
 gbk_e , vgbk_e / d136 g or vgbk_e / d136 g ; i2623v ; 
 d3148 g ; d3502y . comparison of amino acid sequences of zinc - binding trash motif in 
 n : the amino acid sequences of the zinc - binding trash motifs in the 
 n proteins from various bvdv strains deposited in the ddbj / embl / genbank 
 databases were compared with those of the gbk_e and gbk_e strains . at least one strain per subgenotype [ 1a1o ( except for 1l ) and 2a2c ] 
 [ 11 , 18 ] was 
 chosen in the present study . as a result , bvdv strains , except for the g strain , contain 
 amino acid residues cys112-cys134-asp136-cys138 in the zinc - binding trash motif of 
 n , as observed in gbk_e ( fig . 4.comparison of amino acid sequences of the zinc - binding trash motifs in 
 n. the amino acid sequences of the zinc - binding trash motifs in the 
 n proteins of various bvdv strains deposited in the ddbj / embl / genbank 
 databases were compared with those of gbk_e and gbk_e . at 
 least one strain per subgenotype [ 1a1o ( except for 1l ) and 2a2c ] 
 was chosen . the cysteines ( cs ) at positions 112 , 134 and 138 of the zinc - binding trash 
 motif are highlighted in bold and underlined . the accession numbers of strains from the ddbj / embl / genbank are as 
 follows : nadl ( m31182 ) , sd-1 ( m96751 ) , osloss ( m96687 ) , cp7 ( u63479 ) , bega ( af049221 ) , 
 721 ( af144463 ) , f ( af287284 ) , 3186v6 ( af287282 ) , w ( af287290 ) , a ( af287283 ) , g 
 ( af287285 ) , 23 - 15 ( af287279 ) , ks86 - 1ncp ( ab078950 ) , suwancp ( kc853440 ) , zm-95 
 ( af526381 ) , shitara/02/06 ( ab359930 ) , is25cp/01 ( ab359931 ) , 890 ( u18059 ) , 
 hokudai - lab/09 ( ab567658 ) and nrw 14 - 13_dup (  ) ( hg426485 ) . ) . comparison of amino acid sequences of the zinc - binding trash motifs in 
 n. the amino acid sequences of the zinc - binding trash motifs in the 
 n proteins of various bvdv strains deposited in the ddbj / embl / genbank 
 databases were compared with those of gbk_e and gbk_e . at 
 least one strain per subgenotype [ 1a1o ( except for 1l ) and 2a2c ] 
 was chosen . the cysteines ( cs ) at positions 112 , 134 and 138 of the zinc - binding trash 
 motif are highlighted in bold and underlined . the accession numbers of strains from the ddbj / embl / genbank are as 
 follows : nadl ( m31182 ) , sd-1 ( m96751 ) , osloss ( m96687 ) , cp7 ( u63479 ) , bega ( af049221 ) , 
 721 ( af144463 ) , f ( af287284 ) , 3186v6 ( af287282 ) , w ( af287290 ) , a ( af287283 ) , g 
 ( af287285 ) , 23 - 15 ( af287279 ) , ks86 - 1ncp ( ab078950 ) , suwancp ( kc853440 ) , zm-95 
 ( af526381 ) , shitara/02/06 ( ab359930 ) , is25cp/01 ( ab359931 ) , 890 ( u18059 ) , 
 hokudai - lab/09 ( ab567658 ) and nrw 14 - 13_dup (  ) ( hg426485 ) . both bvdv and csfv inhibit the production of type i interferon in vitro 
 through proteasomal degradation of cellular irf-3 [ 1 , 
 4 , 8 , 28 ] . cells infected with these ( end ) viruses 
 exhibit strong cpe after superinfection with ndv or some orbiviruses [ 15 , 20 ] . one of the two domains of 
 pestivirus n , the c - terminal domain containing a zinc - binding trash motif 
 consisting of cys112-cys134-asp136-cys138 , is required for irf-3 binding and prevention of 
 type i interferon production [ 7 , 32 ] . for csfv , it has been reported that mutations at positions 112 , 134 , 
 136 or 138 in n of end viruses abolish the inhibition of type i 
 interferon production , while mutations at positions 112 or 136 in n of 
 end viruses restore this function [ 24 , 
 32 , 33 ] . for 
 bvdv , previous studies have revealed that amino acid substitutions l8p , e22v or h49l in n abolish its capacity to interfere with type i interferon production [ 2 , 6 ] . however , to 
 the best of our knowledge , there were no studies with bvdv that had approached the basic 
 mechanism of the inhibition of type i interferon production using the amino acid differences 
 between a pair of end and end viruses as well as viruses genetically 
 engineered on the basis of these differences . pestiviruses are quasispecies ; single strains 
 consist of populations with various characteristics , such as end and 
 end . we cloned a pair of 
 end and end viruses ( gbk_e and gbk_e ) from 
 the bvdv gbk strain using reverse plaque formation techniques [ 5 , 19 ] . determination of complete 
 genome sequences of these viruses revealed that there were only four amino acid differences 
 between them ( table 1 ) . to clarify the molecular 
 mechanism of the inhibition of type i interferon production and the end phenomenon , we 
 generated a full - length cdna clone of gbk_e ( pgbk_e ) as well as 
 three other full - length cdna clones with single ( d136 g ) or multiple ( i2623v ; d3148 g ; d3502y 
 and d136 g ; i2623v ; d3148 g ; d3502y ) mutations . the ifn bioassay of vgbk_e and three mutant viruses revealed 
 that vgbk_e and vgbk_e / i2623v ; d3148 g ; d3502y exhibited the end 
 phenomenon and inhibited the production of type i interferon , whereas the n 
 mutant viruses vgbk_e / d136 g and vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y 
 were end and did not inhibit the production of type i interferon ( fig . this result demonstrates that the single aspartic acid residue in the 
 zinc - binding trash motif of n , which occurs at position 136 of the genome of 
 the gbk_e virus , is the key to determine viral capacity to inhibit type i 
 interferon production and display the end phenomenon . because it has been reported that the 
 n proteins of both bvdv and csfv inhibit the production of type i interferon 
 by proteasomal degradation of cellular irf-3 [ 1 , 4 , 8 , 28 ] , we assessed whether n of wild - type 
 gbk_e , wild - type gbk_e , vgbk_e and the three mutant 
 viruses reduced the amount of cellular irf-3 in infected cells by western blotting . an 
 apparent reduction in cellular irf-3 was observed in cells infected with wild - type 
 gbk_e , vgbk_e or vgbk_e / i2623v ; d3148 g ; d3502y , 
 whereas wild - type gbk_e and mutant viruses with mutations in n 
 showed no reduction in cellular irf-3 ( fig . the results indicate that the inhibition of type i interferon production and the 
 end phenomenon displayed by gbk_e occurs by the degradation of cellular irf-3 
 caused by combining with the zinc - binding trash motif . comparison of 
 vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y ( the same amino acid sequence as 
 gbk_e ) with vgbk_e / i2623v ; d3148 g ; d3502y revealed that the 
 function of n was restored by a single g136d mutation in n of 
 the gbk_e virus . in the present study , a comparison of the amino acid sequences of gbk_e , 
 gbk_e and viruses from ddbj / embl / genbank databases revealed that amino acid 
 residues cys112-cys134-asp136-cys138 in the zinc - binding trash motif of n were 
 well conserved among field bvdv isolates ( fig . it is reported that these four residues are required for the inhibition of type i interferon 
 production [ 7 , 32 ] . taken together , the above - mentioned results suggest that these field isolates 
 inhibit the type i interferon production in infected cells , although there remains a 
 possibility that amino acid residues other than those of trash motif contribute to this 
 phenomenon . the g strain contains the amino acid residue glu136 in the trash motif of 
 n ( fig . 4 ) , and this may affect 
 the structure of n and result in the production of type i interferon 
 in vitro . it was reported that 35 out of 45 ( 77.8% ) field isolates of 
 bvdv in japan contained end virus as the predominant virus population compared 
 with end virus and seven isolates ( 15.6% ) contained similar titers of 
 end and end viruses , whereas two isolates ( 4.4% ) contained only 
 end virus . therefore , further 
 studies are needed to investigate how quasispecies of bvdv ( end and 
 end ) contribute in vivo . in conclusion , our results indicate that a single mutation in the trash motif at position 
 136 of bvdv n abolishes its interaction with bovine irf-3 and halts the 
 degradation of irf-3 . moreover , the mutation of the amino acid residue at position 136 of 
 gbk_e restores its function as an interferon antagonist , as shown for csfv . however , how n interacts with irf-3 and the nature of the cascade after 
 interaction with n and irf-3 are hardly understood . in addition , it is unknown 
 how the inhibition of type i interferon production contributes to the viral infection 
 strategy when cells are infected with bvdv in vivo . therefore , an 
 additional study is also required to reveal the fundamental mechanism by which pestiviruses 
 inhibit the production of type i interferon and how this mechanism functions in 
 vivo .
the exaltation of newcastle disease virus ( end ) phenomenon is induced by the 
 inhibition of type i interferon in pestivirus - infected cells in vitro , 
 via proteasomal degradation of cellular interferon regulatory factor ( irf)-3 with the 
 property of the viral autoprotease protein npro . reportedly , the amino acid 
 residues in the zinc - binding trash motif of npro determine the difference in 
 characteristics between end - phenomenon - positive ( end+ ) and 
 end - phenomenon - negative ( end ) classical swine fever viruses ( csfvs ) . 
 however , 
 the basic mechanism underlying this function in bovine viral diarrhea virus ( bvdv ) has not 
 been elucidated from the genomic differences between end+ and end 
 viruses using reverse genetics till date . in the present study , comparison of complete 
 genome sequences of a pair of end+ and end viruses isolated from 
 the same virus stock revealed that there were only four amino acid substitutions 
 ( d136 g , 
 i2623v , d3148 g and d3502y ) between two viruses . based on these differences , viruses with 
 and without mutations at these positions were generated using reverse genetics . 
 the end 
 assay , measurements of induced type i interferon and irf-3 detection in cells infected 
 with these viruses revealed that the aspartic acid at position 136 in the zinc - binding 
 trash motif of npro was required to inhibit the production of type i interferon 
 via the degradation of cellular irf-3 , consistently with csfv .