Patent Application: US-48898704-A

Abstract:
the present invention relates to mutant mva vaccinia viruses , which are used for the generation of recombinant mva viruses , as well as host cells , which have been infected with these mutant mva viruses . the present invention further relates to dna - vector constructs , and a method for the generation of recombinant mva by using the mutant mva viruses and the dna - vector constructs . the mutant mva vaccinia viruses of the present invention are characterized in that the mva orf 050l gene or a functional part thereof has been inactivated in the viral genome .

Description:
the mva virus is a greatly attenuated vaccinia virus produced by serial passages of the original cva strain on chicken embryo fibroblast ( cef ) cultures . for a general rewiew of the history of the production , the properties and the use of the mva strain of vaccinia , reference may be made to the summary published by mayr et al . in infection 3 , 6 – 14 [ 1975 ]. owing to the adaptation to cef , growth of the mva virus on other cell systems is greatly restricted . exceptionally , baby hamster kidney cells ( bhk - 21 ), a well characterized , easily maintained cell line , supports mva growth and as proficient expression of recombinant genes as the highly efficient cef and has been recommended for standardized mva propagation during the development of expression vectors and live recombinant vaccines ( drexler et al . 1998 , j . gen . virol ., 79 , 347 – 352 ). the mva virus was normally grown on cef cells , the host cell for which it had been adapted . to prepare the cef cells , 11 - days old embryos were isolated from incubated chicken eggs , the extremities were removed , and the embryos were cut into small pieces and slowly dissociated in a solution composed of 25 % trypsin at room temperature for 2 hours . the resulting cell suspension was diluted with one volume of medium i ( mem eagle , for example obtainable from gibco , basle , switzerland ; order no . 072 - 1500 ) containing 5 % fetal calf serum ( fcs ), penicillin ( 100 units / ml ), streptomycin ( 100 mg / ml ) and 2 mm glutamine and filtered through a cell screen ( for example obtainable from technomara ag , zurich , switzerland , order no . bellco 1985 , 150 mesh ), and the cells were sedimented by centrifugation at 2000 rpm in a bench centrifuge ( hermle kg , d - 7209 gosheim , frg ) at room temperature for 5 minutes . the cell sediment was taken up in ¼ of the original volume of medium i , and the cef cells obtained in this way were spread on cell culture dishes . they were left to grow in medium i in a co 2 incubator at 37 ° c . for 1 – 2 days , depending on the desired cell density , and were used for infection either directly or after 1 – 2 further cell passages . a clear description of the preparation of primary cultures can be found in the book by r . i . freshney , “ culture of animal cell ”, alan r . liss verlag , new york [ 1983 ] chapter 11 , page 99 et seq . the mva - δ e3l is routinely propagated in baby hamster kidney bhk - 21 ( american type culture collection atcc ccl - 10 ) cells which were grown in minimal essential medium ( mem ) supplemented with 10 % fetal calf serum ( fcs ). bhk - 21 cells were maintained in a humidified air − 5 % co 2 atmosphere at 37 ° c . the viruses were used for infection as follows . cells were cultured in 175 cm 2 cell culture bottles . at 80 – 90 % confluence , the medium was removed and the cells were incubated for one hour with an mva virus suspension ( 0 . 01 infectious particles (= pfu ) per cell , 0 . 01 ml / cm 2 ) in phosphate - buffered saline ( pbs / dulbecco , for example animed ag , muttenz , switzerland , order no . 23 . 100 . 10 ). then medium was added ( 0 . 2 ml / cm 2 ) and the bottles were incubated at 37 ° c . for 2 – 3 days until about 80 % of the cells had rounded . the virus lysates were stored with the cells and medium , without treatment , in the cell culture bottles at − 30 ° c . before further processing ( purification etc .) the purification steps undertaken to obtain a virus preparation which was as pure as possible and free from components specific to the host cell were identical for the mva and wr viruses ( joklik , virology 18 , 9 – 18 [ 1962 ], zwartouw et al ., j . gen . microbiol . 29 , 523 – 529 [ 1962 ]). the cell cultures which had been infected and then stored at − 30 ° c . were thawed , the residual cells were shaken off or scraped off the plastic substrate , and cells and virus were removed from the medium by centrifugation ( sorvall centrigue , gsa rotor , 1 hour at 5000 rpm and 10 ° c .). the sediment , composed of viral and cell particles , was suspended once in pbs ( 10 – 20 times the volume of the sediment ), and the suspension was centrifuged as above . the new sediment was suspended in 10 times the volume of rsb buffer ( 10 mm tris - hcl ph 8 . 0 , 10 mm kcl , 1 mm mgcl2 ), and the suspension was briefly treated with ultrasound ( labsonic 1510 equipped with a 4 mm diameter tip , obtainable from bender and hobein , zurich , switzerland , 2 × 10 seconds at 60 watts and room temperature ) in order to disintegrate remaining still intact cells and to liberate the virus particles from the cell membranes . the cell nuclei and the larger cell debris were removed in the subsequent brief centrifugation of the suspension ( sorvall gsa rotor obtainable from dupont co ., d - 6353 bad nauheim , frg ; 3 minutes at 3000 rpm and 10 ° c .). the sediment was once again suspended in rsb buffer , treated with ultrasound and centrifuged , as described above . the collected supernatants containing the free virus particles were combined and layered over a pad composed of 10 ml of 35 % sucrose in 10 mm tris - hcl , ph 8 . 0 , and centrifuged in a kontron tst 28 . 38 / 17 rotor ( kontron instrumente , zurich , switzerland ; corresponds to a beckman sw 27 rotor ) for 90 minutes with 14 , 000 rpm at 10 ° c .). the supernatant was decanted , and the sediment containing the virus particles was taken up in 10 ml of 10 mm tris - hcl , ph8 . 0 , homogenized by brief treatment with ultrasound ( 2 × 10 seconds at room temperature , apparatus as described above ), and applied to a stepped gradient for further purification . the steps of the gradient were each composed of 5 ml of sucrose in 10 mm tris - hcl , ph 8 . 0 ( sucrose concentration steps : 20 %, 25 %, 30 %, 35 % and 40 %). the gradient was centrifuged in a kontron tst 28 . 38 / 17 rotor at 14 , 000 rpm 10 ° c . for 35 minutes . after this centrifugation , several discrete zones containing virus particles were visible in the region of the gradient between 30 % and 40 % sucrose . this region was siphoned off from the gradient ( 10 ml ), the sucrose solution was diluted with pbs ( 20 ml ) and the virus particles were sedimented therefrom by centrifugation ( kontron tst 28 . 38 / 17 rotor , 90 minutes at 14 , 000 rpm , 10 ° c .). the sediment , which now consisted mostly of pure virus particles , was taken up in pbs in such a way that the virus concentrations corresponded on average to 1 – 5 × 10 9 pfu / ml . the purified virus stock solution was used either directly or diluted with pbs for the subsequent experiments . mva genomic dna sequences flanking the e3l gene were amplified by polymerase chain reaction using dna of mva ( cloned isolate f6 , 582 nd passage on cef ) as a template . the primers of the upstream flanking region of e3l were 5 ′- atatgatt gggccc actagcggcaccgaaaaagaattcc - 3 ′ ( seq id no : 1 ) ( apai site underlined ) and 5 ′- gtcaata gcggccgc agacatttttagagagaactaac - 3 ′ ( seq id no : 2 ) ( noti site underlined ). the primer for the downstream region were 5 ′- tacatgaa acgcgt tctattgatgatagtgactatttc - 3 ′ ( seq id no : 3 ) ( mlui site underlined ) and 5 ′- gttacgtc ggatcc agattctgattctagttatc - 3 ′ ( seq id no : 4 ) ( bamhi site underlined ). the constucts were cut with apai / noti or mlui / bamhi and inserted into the corresponding sites of the plasmid p δ k1l ( staib et al . 2000 , biotechniques , 28 : 1137 – 1148 ) to obtain the e3l deletion plasmid p δ e3l - k1l mutant virus mva - δ e3l was generated using previously described methodology ( staib et al . 2000 , biotechniques , 28 : 1137 – 1148 ). briefly , monolayers of 1 × 10 6 confluent bhk - 21 cells grown in 6 - well tissue - culture plates ( costar , corning n . y ., usa ) were infected with mva at a multiplicity of infection ( moi ) of 0 . 01 iu per cell . ninety min after infection cells were transfected with 10 μ g of plasmid p δ e3l - k1l dna per well using calcium phosphate ( ce / lphect transfection kit , amersham pharmacia biotech , freiburg , germany ) as recommended by the manufacturer . forty - eight hours after infection the cells were harvested , freeze - thawed three times , and homogenized in a cup sonicator ( sonopuls hd 200 , bandelin , germany ). ten - fold serial dilutions ( 10 − 1 to 10 − 4 ) of the harvested material in medium were used to infect subconfluent monolayers of rk - 13 cells grown in 6 - well tissue - culture plates . after three days incubation at 37 ° c . foci of rk - 13 cells infected with mutant mva - δ e3l were picked in a 20 μ l volume by aspiration with an air - displacement pipette , transferred to microcentrifuge tubes containing 500 μ l medium , and processed by freeze - thawing and sonication for another infection of rk - 13 cell monolayers . after elimination of all parental mva during passage on rk - 13 cells , ten - fold serial dilutions ( 10 − 1 to 10 − 6 ) of the recombinant viruses were used for infection of subconfluent bhk - 21 cells grown in 6 - well tissue - culture plates ( costar , corning n . y ., usa ). well separated foci of infected bhk - 21 cells were harvested to isolate k1l - negative mutant mva - δ e3l . viral dna from cloned mva isolates was routinely analyzed by pcr as described previously ( staib et al 2000 ). to monitor the e3l gene locus in genomes of mutant mva , we used oligonucleotides from gene sequences adjacent to orf e3l : 5 ′- ata tga ttg ggc cca cta gcg gca ccg aaa aag aat tcc - 3 ′ ( seq id no : 1 ) and 5 ′- tac atg aaa cgc gtt cta ttg atg ata gtg act att tc - 3 ′ ( seq id no : 3 ). southern blot analysis . total dna isolated from virus - infected bhk cells was digested with eco ri , separated by gel electrophoresis in 0 . 8 % agarose , transferred to a hybond n + membrane , and hybridized to a dna probe consisting of a pcr fragment spanning the downstream flanking region of the e3l gene labeled with [ α - 32 p ] ctp . prehybridization and hybridization was performed according to the quickhyb protocol ( stratagene gmbh , heidelberg , germany ). membranes were washed twice with 2 × sodium chloride / sodium citrate ph 7 . 0 solution ( ssc )/ 0 . 1 % sds at 65 ° c . for 30 min and twice at room temperature . the blots were exposed to a kodak x - omat film . western blot analysis . confluent bhk - 21 cell monolayers grown in 6 - well tissue culture plates were inoculated with 10 iu virus / cell for one hour . infected cells were briefly washed with medium and incubated with fresh medium with and without addition of cytosine arabinoside ( arac , 40 μ g / ml ). after 24 h cell lysates were prepared and separated by sds - 12 % polyacrylamide gel electrophoresis ( page ). proteins were then electroblotted onto nitrocellulose membranes ( biorad ) for 2 h in a buffer containing 25 mm tris , 19 , 2 mm glycine and 20 % methanol ( ph 8 . 3 ). after blocking overnight in blocking buffer containing 2 % ( w / v ) bsa , 0 . 05 % ( v / v ) tween , 50 mm tris , 150 mm nacl , ( ph 7 . 5 ), the blot was probed with 10 - fold diluted supernatants from hybridoma tw2 . 3 producing an anti - e3l mouse monoclonal antibody ( yuwen et al . virology 195 , 732 – 744 , 1993 ) in blocking buffer for 1 h . after being washed , the blot was incubated for 1 h with peroxidase - conjugated polyclonal goat anti - mouse antibody ( igg ( h + l )), cat . no . 111 - 035 - 114 ; dianova , hamburg , germany , dilution 1 : 10000 ) diluted 5000 - fold in blocking buffer , washed again , and developed by visualization with enhanced chemiluminescence procedure ( ecl , manufacturer ) using kodak x - omat film . to determine low - multiplicity - growth profiles , virus multiplication was monitored after infecting cef or bhk - 21 monolayers with 0 . 05 infectious units ( iu ) uva , mva - δ e3l or mva - e3rev per cell . one - step - growth of mva , mva - δ e3l and mva - e3rev was analyzed infecting cells at a multiplicity of infection ( moi ) of 5 iu . for all infection experiments confluent monolayers grown in 6 - well tissue culture plates were used . after virus adsorption for 60 min at 37 ° c ., the inoculum was removed . the infected cells were washed twice with rpmi 1640 and incubated with fresh rpmi 1640 medium containing 10 % fcs at 37 ° c . in a 5 % co 2 atmosphere . at multiple time points post infection ( p . i .) infected cells were harvested and virus was released by freeze - thawing and brief sonication . serial dilutions of the resulting lysates were plated on confluent bhk - 21 monolayers grown in 6 - well plates as replicates of two . for vaccinia virus - specific immunostaining of virus infected cells , media were removed 48 hours p . i ., cells were briefly fixed in acetone : methanol ( 1 : 1 , 1 ml / well ). after washing , and blocking in pbs - 2 % fcs cells were incubated for 60 min with polyclonal rabbit anti - vaccinia antibody ( igg fraction , biogenesis ltd , poole , england , cat . no . 9503 - 2057 , diluted 1 : 10000 in pbs - 3 % fcs ). after washing cells with pbs , peroxidase - conjugated polyclonal goat anti - rabbit antibody ( e . g . 2nd antibody horseradish peroxidase - conjugated ( igg ( h + l )), cat . no . 111 - 035 - 114 ; dianova , hamburg , germany , dilution 1 : 1000 in pbs - 3 % fcs ) was added , and incubated for 45 min . after washing with pbs , antibody - labeled cells were developed with dianisidine ( sigma no . 09143 ) substrate solution , foci of stained cells were counted , and virus titers were calculated ( iu / ml ) as with vaccinia virus plaque assay . analysis of viral dna . genomic viral dna was isolated from infected cells as described previously ( earl p & amp ; moss b , current protocols in molecular biology ). to assess viral dna replication total dna was transferred with a slot blot apparatus to hybond n + membranes and hybridized to a 32 p - labeled mva dna probe . radioactivity was quantitated with a phosporimager analyser ( biorad ). analysis of [ 35 s ] methionine - labeled polypeptides . bhk and cef cell monolayers , in 12 - well plates , were either mock infected or infected with uva , mva - δ e3l or mva - e3rev at an moi of 20 . following 45 min adsorption at 4 ° c ., virus inocula were replaced by pre - warmed tissue culture medium and incubated at 37 ° c . in a 5 % co 2 atmosphere . at indicated time points post - infection , cells were washed with methionine - free medium at 37 ° c . for 10 min , and 50 μ ci of [ 35 s ] methionine were added to each well and incubated for 30 min at 37 ° c . cytoplasmic extracts of infected cell monolayers were prepared by adding 0 . 2 ml 0 . 5 % nonidet p - 40 lysis buffer ( 20 mm tris - hcl , 10 mm nacl ( ph 8 . 0 )) for 10 min . polypeptides from cell extracts were separated by 10 % sodium dodecyl sulfate ( sds ) polyacrylamide gel electrophoresis ( page ) and analyzed by autoradiography . analysis of dna fragmentation . cef were mock - infected or infected with mva , mva - δ e3l and mva - e3rev at an moi of 20 . cells were harvested at 16 h and 24 h p . i . and total dna was extracted as described . precipitated dna was resuspended in 100 μ l h 2 o , treated with rnase ( final concentration : 1 mg / ml ) for 15 min at 37 ° c . and resolved in an 1 % agarose gel . dna fragments were visualized by staining with ethidium bromide . hoechst staining . cef cells grown to confluency in a 12 - well plates containing ø 12 mm glass coverslips were either non - infected or infected with mva , mva - δ e3l or mva - e3rev at mois of 5 or 20 . cells were stained at 16 or 24 h p . i . with hoechst 3343 for 30 min at room temperature and photographed under a fluorescence microscope . the ratio of apoptotic cells to non - apoptotic cells was determined by counting and presented as the mean and three times the standard error of the mean ( mean / 3 × the sem ). assay for caspase activity . monolayers of 5 × 10 5 confluent cef grown in 12 - well tissue - culture plates were either mock - infected or infected with mva , mva - δ e3l and mva - e3rev at an moi of 20 . ninety minutes after infection at 4 ° c ., cells were washed twice with mem containing 10 % lactalbumin and 5 % bms and incubated for 16 h at 37 ° c . cells were harvested , collected by centrifugation , washed once in pbs , and lysed by incubating 2 × 10 7 cells / ml in lysis buffer ( 1 % nonidet p - 40 , 50 mm tris - hcl , 150 mm nacl , ( ph 8 . 0 )) for 10 min on ice followed by vigorous vortexing . extracts were cleared by centrifugation for 5 min at 10 . 000 × g at 4 ° c . and transferred to fresh vials . to determine devd - cleaving activity , extracts were diluted 1 : 10 in reaction buffer ( mitotic dilution buffer ( lazebnik , y . a ., cole , s ., cooke , c . a ., nelson , w . g . and eamshaw , w . c . ( 1993 ). nuclear events of apoptosis in vitro cell free mitotic extracts : a model system for analysis of the active phase of apoptosis . j . cell . biol . 123 : 7 – 22 ) containing 10 mm hepes - koh , 40 mm β - glycerophosphate , 50 mm nacl , 2 mm mgcl ; 5 mm egta , and 1 mm dithiothreitol [ dtt ], ( ph 7 . 0 ) supplemented with 0 . 1 % chaps { 3 ′-[( 3 ′- cholamidopropyl )- dimethylammonio ]- 1 - propanesulfonate }, 100 μ g of bovine serum albumin and acetyl - devd - 7 - amino - 4 - methylcoumarib ( devd - amc ) ( final concentration : 10 μ m ). reactions were performed in triplicate in flat - bottomed 96 - well pates at 37 ° c . for 1 h . free amc was then measured by determining the fluorescence at 390 nm ( excitation ) and at 460 nm ( emission ) in a millipore cytofluor 96 reader . values were calculated by subtracting the background fluorescence ( buffer and substrate alone ) and are presented as the mean and three times standard error of the mean ( mean / 3 × the sem ). measurement of apoptosis by elisa . confluent cef monolayer with 1 × 10 5 cells were mock infected or infected with mva , mva - δ e3l and mva - e3rev at a moi of 5 , 10 and 20 , respectively . 16 h p . i . the extent of apoptosis was analyzed using the cell death detection elisa kit ( roche diagnostics , mannheim ) according to the manufacture &# 39 ; s instructions . briefly , 16 h p . i . medium was removed and cells were incubated in lysis buffer . after lysis , intact nuclei were pelleted by centrifugation and aliquots of supernatant were transferred to streptavidin - coated wells of microtiter plates . the amount of apoptoic nuclesomes present in the sample was determined using mouse monoclonal antibodies directed against dna and histones , and spectrophotometrical analysis . propidium iodide staining . to determine nuclear fragmentation , monolayers of 1 × 10 6 confluent cef grown in 6 - well tissue - culture plates were infected with mva , mva - δ e3l and mva - e3rev at an moi of 5iu and 20iu . ninety minutes after infection at 4 ° c . cells were washed twice with mem containing 10 % lactalbumin and 5 % bms . at various time points post infection cells were trypsinized , collected and stored in 70 % ethanol at 4 ° c . on the following day , cells were washed twice in pbs and resuspended in pbs containing propidium iodide with a final concentration of 50 μ g / ml . samples were stored for 1 h at 4 ° c . in the dark and analyzed with a becton dickinson facscalibur apparatus as described previously ( nicoletti , i . ; migliorati , g . ; pagliacci , m . c . ; grignani , f . and riccardi , c . ( 1991 ). a rapid and simple method for measuring thymocyte apoptosis by propidium staining and flow cytrometry . j . immunol . methods 139 : 271 – 279 ). to allow the generation of recombinant mva using e3l based selection novel vector plasmids were constructed on the basis of piii plasmid vectors which contain mva flanking sequences ( flank iii - 1 and flank iii - 2 ) to target insertion of recombinant dna precisely to the site of the naturally occuring deletion iii within the mva genome ( sutter & amp ; moss 1992 , staib et al . 2000 ). to generate the plasmid vector piii - e3 - p sh5 a 627 bp dna fragment containing the complete coding sequence of the mva e3l gene under transcriptional control of its authentic promoter sequence was prepared by pcr , treated with klenow and inserted between the flank iii - 1 and flank iii - 2 dna sequences in plasmid plw9 via the restriction site bamhi ( wyatt , l . et al . 1996 , vaccine 14 : 1451 – 1458 ). a 723 - bp dna fragment containing the gfp open reading frame was excised from pegfp - n1 ( clonetech laboratories gmbh , heidelberg , germany ) using the restriction endonucleases bamhi and noti , treated with klenow polymerase , and inserted into the restriction site smai of piii - e3 - p sh5 to obtain piii - e3 - p sh5 - gfp . monolayers of 1 × 10 6 confluent bhk - 21 cells grown in 6 - well tissue - culture plates ( costar , corning n . y ., usa ) were infected with mva - δ e3l at a multiplicity of infection ( moi ) of 0 . 01 iu per cell . ninety min after infection cells were transfected with 10 μ g of piii - e3 - p sh5 - gfp dna per well using calcium phosphate ( cellphect transfection kit , amersham pharmacia biotech , freiburg , germany ) as recommended by the manufacturer . forty - eight h after infection the cells were harvested , freeze - thawed three times , and homogenized in a cup sonicator ( sonopuls hd 200 , bandelin , germany ). ten - fold serial dilutions ( 10 − 1 to 10 − 4 ) of the harvested material in medium were used to infect subconfluent monolayers of cef cells grown in 6 - well tissue - culture plates . after three days incubation at 37 ° c . foci of cef cells infected with recombinant uva were monitored for gfp synthesis and picked in a 20 μ l volume by aspiration with an air - displacement pipette , transferred to microcentrifuge tubes containing 500 μ l medium , and processed by freeze - thawing and sonication for another infection of cef cell monolayers . after three consecutive rounds of plaque purification in cef cells the recombinant mva - psh 5 - gfp viruses were amplified by infection of cef monolayers , and the dna was analyzed by pcr to confirm genetic homogenity of the virus stock . to evaluate the relevance of the key interferon - resistance gene e3l in the mva life cycle , we used homologous recombination to replace the complete promoter and coding sequences of the e3l open reading frame in the mva genome with a vaccinia virus k1l gene expression cassette ( fig1 a ). this marker allows for stringent growth selection of mutated viruses upon infection of rabbit kidney rk - 13 cells , in a second step using nonselective growth conditions it is simply removed again by further homologous recombination between flanking repetitive dna sequences ( staib et al ., 2000 ). recombinant mva with deleted e3l gene sequences were isolated during several rounds of plaque purification on rk - 13 and bhk - 21 cell monolayers . during plaque cloning the expected genome alterations were monitored by pcr ( fig1 b ). primary virus stocks were amplified on bhk - 21 cells , re - assessed by southern blot analysis of viral dna ( fig1 c ), and designated mva - δ e3l . western blotting of cell lysates which were prepared in the presence or absence of arabinoside c confirmed the synthesis of e3l protein in cells infected with wild - type mva , whereas no e3l polypeptides could be detected after infection with mva - δ e3l ( fig1 d ). in the attempt to generate secondary virus stocks of mva - δ e3l we infected confluent cef monolayers at low multiplicity of infection ( moi ), but to our surprise the virus failed to amplify . suspecting a host range phenotype , we wished to comparatively analyze virus growth after infection of bhk - 21 and cef cells . a growth deficiency could result from a defect in virus replication or virus spread . first , we determined virus yields in multiple - step growth experiments infecting cells with mva or mva - δ e3l at 0 . 1 infectious units ( iu ) per cell ( fig2 a , 2 b ). at this multiplicity , replication and spread of mva and mva - δ e3l in bhk - 21 cells were close to identical with very similar peaks of infectivity titers reached between 20 and 48 h after infection ( fig2 a ). as expected mva replicated efficiently in cef . in contrast , titers of mva - δ e3l in cef steadily decreased in comparison to input infectivity suggesting a virtual absence of productive virus growth ( fig2 b ). in addition , we analyzed one - step virus growth using a moi of 10 for infection , and again we found equal capabilties of mva and mva - δ e3l to multiply in bhk - 21 cells . paralleling the data from multiple - step - growth analysis , infectivity titers of mva - δ e3l detected in cef after 8 , 22 , or 48 h of infection never reached the level of infectivity found after virus adsorption ( fig2 c , fig2 d ). we concluded from these experiments that mva - δ e3l has a specific replication defect in cef . to verify that this host range phenotype is solely due to the deletion of the e3l gene sequences we reinserted the e3l gene under transcriptional control of its authentic promoter sequence into the genome of mva - δ e3l to generate the revertant virus mva - e3rev . briefly , we used the plasmid vector pe3lrev containing a dna fragment comprising the complete e3l gene expression cassette together with its genomic flanking sequences for transfection of mva - δ e3l - infected bhk cells . revertant virus mva - e3rev could be readily isolated by plaque purification in cef monolayers . stable reinsertion of e3l was confirmed by pcr with genomic mva - e3rev dna , and western blot analysis of cell lysates revealed synthesis of e3l protein at levels equal to wild - type mva ( data not shown ). already the fact that mva - e3rev could be isolated without need for additional screening or selection suggested a successful reversion of the growth defect of mva - δ e3l in cef . correspondingly , upon multiple - step and one - step growth analysis of mva - e3rev in both bhk - 21 and cef cells we found levels of virus replication similar to wild - type mva ( fig2 a - d ). from this data we concluded that e3l function is necessary to maintain mva replication in cef , the cell culture system in which the virus had been developed upon longterm passage . reduced viral protein and dna synthesis in cef infected with mva - δ e3l to better characterize the non - permissive infection of cef with mva - δ e3l , we sought to determine wether the failure to produce infectious viral progeny resulted from reduced viral protein synthesis . cef and bhk cells were metabolically labeled with [ 35 s ] methionine at various times after infection with mva , mva - δ e3l , or mva - e3rev . after each labeling period , lysates were prepared and analyzed by sds - page and autoradiography . in bhk cells infected with mva , late viral protein synthesis occurred at 5 h after infection and became prominent with profound shut - off of cell protein synthesis at 10 h after infection ( fig3 a ). similar patterns of viral proteins were found in bhk cells infected with mva - δ e3l or mva - e3rev ( fig3 a ). in cef infected with mva or mva - e3rev abundant late viral protein synthesis was found at times 5 h and 10 h after infection ( fig3 b ). by contrast , in cef cells infected with mva - δ e3l we could hardly detect polypeptide bands specific for viral protein production ( fig3 b ). some weak bands of polypeptides co - migrating with typical late viral proteins became visible with the prominent shut - off of host cell protein synthesis at times after 5 h of infection . because synthesis of the abundant viral late proteins is dependent on the replication of viral dna , we further examined this step in the life cycle of mva - δ e3l comparing infection of bhk - 21 and cef cells . viral dna synthesis was monitored by isolating total dna from infected cells at different times during a one - step infection and transferring this dna onto a membrane which was hybridized with a radioactively labeled mva - dna probe ( fig3 c , d ). as shown by fig3 c , in bhk - 21 cells infected with mva - δ e3l or mva the accumulation of viral dna occured with very similar kinetics and quantities . also in cef cells we found viral dna being increasingly made after infection with both viruses ( fig3 d ). in the case of the non - permissve cef infection with mva - δ e3l , however , we detected , compared to permissive conditions , lesser amounts of dna at time points 6 h and 12 h after infection . thus the drastically diminished protein synthesis found in mva - δ e3l - infected cef was not correlated with a gross block of virus - specifc dna replication , while production of viral dna appeared arrested at later times during non - permissive infection . this infection phenotype being characterized by prominent shutdown of viral protein synthesis together with maintained capacity for viral dna replication was reminiscent of the non - permissive vaccinia virus infection of chinese hamster ovary ( cho ) cells ( spehner et al ., 1988 ; ramsey - ewing and moss , 1995 ). the abortive vaccinia virus - cho infection is associated with induction of apoptosis that can be overcome by co - expression of the cowpox virus gene cho hr ( ink et al ., 1995 , ramsey - ewing and moss , 1998 ). furthermore , also the vaccinia e3l gene product was described to inhibit induction of apoptosis in vaccinia virus - infected hela cells ( lee and esteban , 1994 ; kibler et al ., 1997 ). because we had observed an unusual cell shrinkage when monitoring cef cultures by light microscopy within 24 h of infection with mva - δ e3l , it appeared important to probe if the growth restriction of mva - δ e3l in cef cells could be possibly linked to apoptosis . in a first standard assay for apoptosis we monitored for the characteristic cleavage of dna into 180 bp - multimers corresponding to a nucleosomal “ dna ladder ” ( wyllie et al ., 1980 ). total cellular dna was isolated from cef cultures infected either with mva - δ e3l , mva or mva - e3rev , separated by agarose gel electrophoresis , and visualized upon ethidium bromide staining . we observed the typical fragmentations of cellular dna indicative for apoptosis in samples from cef cells infected with mva - δ e3l for 16 h and 24 h . in contrast , no such dna laddering was detectable in samples from cef cells infected with mva or revertant virus mva - e3rev ( fig4 a ). another hallmark for apoptosis is the appearance of extranuclear nucleosomes . therefore , we stained cef cultures with hoechst 3343 and screened for apoptotic bodies in individual cells . at 16 h after infection with mva - δ e3l we could easily detect cytoplasmic vacuoles that stained extensively with hoechst 3343 ( fig4 b ). in addition , this assay allowed to quantify the number of apoptotic cells by determining the percentage of cells with extranuclear staining in randomly selected areas with 300 to 500 cells ( in triplicate , fig4 c ). after infection with mva - δ e3l , we found clear signs of apoptosis in about 20 % of all cells when using a multiplicity of 5 iu / cell . in experiments done at a multiplicity of 20 iu / cell the percentage of apoptotic cells increased to about 30 %. in contrast , at the most 4 % of cells infected with wild - type mva or revertant virus mva - δ e3rev , and only 1 % of mock - infected cells were counted positive for extranuclear staining . to demonstrate that this apoptotic process does not only occur on a dna level , we also investigated intracellular proteolysis by caspases using the specific peptide substrate asp - glu - val - asp ( devd ) ( salvesen and dixit , 1997 ). using an established fluorescence assay devd - cleaving activity was measured in cell extracts prepared from cef at 16 h after infection ( linsinger et al . 1999 ). again after infection with mva - δ e3l we found strongly enhanced devd - cleavage activities , while extracts from mva or mva - e3rev infected cells contained little if any detectable activity ( fig4 d ). these results clearly demonstrated that the growth defect of mva - δ e3l in cef was associated with an induction of apoptosis . furthermore , we wished to assess wether the onset of apoptosis in mva - δ e3l - infected cef could be more obviously linked to the host range restriction phenotype . although our previous experiments were done a multiplicity of 20 iu / cell , which should guarantee an infection of all the cells , only approximately 30 % of the cells were scored apoptotic upon hoechst staining . therefore , first , we were interested to more carefully titrate mva - δ e3l with regard to the extent of apoptosis . we infected cef cells with 1 , 5 , 10 or 20 iu mva - δ e3l , mva , or mva - e3rev , and at 16 h after infection we quantified apoptosis using an elisa based on the specific detection of apoptotic nucleosomes with mouse monoclonal antibodies ( cell death detection elisa kit , roche diagnostics , mannheim ) ( fig5 a ). we found clear evidence for apoptosis in cef inoculated with the lowest dose of 1 iu mva - δ e3l per cell . levels of apoptosis steadily augmented with increasing amounts of mva - δ e3l used for infection , while even higher dose infections with mva or mva - e3rev resulted in amounts of cell death that barely exceeded background levels . the accuracy of an enumeration of apoptotic cells following hoechst staining could have been limited by the fact that a fraction of apoptotic cells is likely to be lost for analysis due to detachment during handling of infected cell monolayers . to use an alternative protocol for analyzing the percentage of dead cells present after infection with an moi of 20 , we stained apoptotic nuclei with propidium iodide and counted them by flow cytrometry at various time points ( nicoletti et al ., 1991 ). as early as 6 h after infection with mva - δ e3l we detected an increased number of apoptotic nuclei in comparison to cef infected with mva or mva - e3rev ( fig5 b ). at following time points the percentage of dead cells in mva - δ e3l - infected cef raised continuously accounting for about 70 % of all the cells at 24 h post infection . this data suggested that extent and kinetics of apoptosis found in mva - δ e3l - infected cef correlated with the growth restriction phenotype being characterized by reduced protein and dna synthesis . induction of ifn type i in cef infected with mva - δ e3l induction of apoptosis in hela cells by vaccinia virus wr with e3l deleted has been linked to the function of e3l as potent dsrna - binding protein which interferes with the activation of the principal ifn - regulated antiviral enzymes pkr and rnase l ( lee and esteban , 1994 ; kibler et al . 1997 ). another characteristic feature of this vaccinia virus mutant is its increased sensitivity to ifn treatment ( beattie et al . 1991 ; beattie et al ., 1995 ). interestingly , deletion of e3l function upon vaccinia virus copenhagen infection also results in the induction of ifn - β synthesis through activation of interferon regulatory factor 3 ( irf - 3 ). therefore , we wished to comparatively monitor for the presence of ifn after infection of cef with mva - δ e3l . we inoculated cef monolayers grown in 6 - well tissue culture plates with 10 iu / cell of mva - δ e3l , mva , mva - e3rev , wild - type vaccinia viruses cva , or wr . cell - free supernatants were collected at 24 h after infection and tested for activities of chicken ifn type i in comparison to recombinant chicken ifn ( recifn , 250 u / ml ) using an established assay ( fig6 ). supernatants from cells infected with wild - type vaccinia virus cva contained no detectable ifn activity ( fig6 a ). the same result was obtained after infection with vaccinia virus wr ( data not shown ). in contrast , ifn activity was clearly present in medium from mva infected cef ( fig6 b ). surprisingly , highest levels of ifn were found after infection with mva - δ e3l reaching activities similar to those obtained with recifn used as control ( fig6 c ). also after infection with mva - e3rev we detected ifn activity albeit lower levels and very comparable to the activity found after infection with non - mutated mva ( fig6 d ). this data demonstrated that already the permissive infection of cef with mva induced an accumulation of biologically active type i ifn , while the non - permissive mva - δ e3l infection produced even more ifn . upon infection of mammalian cells vaccinia virus replication has been found to be relatively resistant to ifn activity . interestingly , this appears to be different in cef cultures in which pretreatment with chicken interferon can inhibit vaccinia virus growth . to determine the effect of ifn on mva infection , we pre - treated cef monolayers with increasing amounts of recombinant chicken ifn type i for 24 h , before infecting the cultures at low moi with mva , mva - δ e3l , or mva - e3rev , or for comparison with vaccinia viruses cva or wr . at 48 h after infection cell monolayers were fixed and foci of virus infected cells were visualized using vaccinia virus - specific immunostaining ( fig7 ). after infection with vaccinia viruses wr or cva , we revealed multiple virus plaques that had formed in monolayers being mock treated or treated with low amounts of ifn ( 1 or 10 u ifn / ml medium ). yet , the presence of 10 u ifn / ml medium resulted in less virus plaques of usually smaller size . no more plaque formation was detectable in monolayers preincubated with 100 u ifn / ml medium or 1000 u ifn / ml medium ( data not shown ). immunostaining of mva infected cef revealed foci of virus - positive cells which remained associated with the cell monolayer demonstrating the typical lower cytopathogenicity of mva infection . despite of this difference in plaque phenotype , formation of mva - infected cell foci upon ifn treatment was very comparable to plaque formation seen with vaccinia virus wr or cva . we found mva - infected cells in monolayers treated with up to 10 u ifn / ml medium . this latter amount clearly affected number and size of the foci , while higher ifn concentrations resulted in complete growth inhibition . in sharp contrast , there were no detectable virus - infected cells in monolayers that were inoculated with mva - δ e3l irrespective of ifn treatment confirming the incapability of this mutant to productively replicate in cef . whereas upon infection with mva - e3rev we found again a foci formation that was identical to the pattern established with wild - type mva . this data confirmed earlier work that suggested a relative ifn sensitivity of vaccinia virus infection in cef , and additionally demonstrated that the ifn - mediated inhibition of cef - adapted mva is very comparable to the ifn effect found upon infection with vaccinia virus strains wr and cva . single gene dependent host range phenotypes in vaccinia virus infection can be elegantly used for efficient selection of recombinant viruses through reinsertion of the host range gene into the mutant virus genome . to verify if the growth restriction of mva - δ e3l in cef would allow for such host range selection we constructed an mva insertion plasmid to target reintroduction of the e3l coding sequences under transcriptional control of its authentic promoter into the site of deletion iii in the mva genome . the addition of an expression cassette of the a . victoriae gfp gene that served as a model recombinant gene being expressed with the vaccinia virus - specific promoter pmh5 resulted in the mva vector plasmid piii - e3l - p h5 - gfp ( fig7 a ). after transfection of this plasmid into mva - δ e3l infected bhk - 21 cells we observed a few gfp gene expressing cell clusters among the many cells showing virus - specific cytopathic effects which suggested that recombinant mva had formed . to test for a selective restoration of virus growth on cef we inoculated ten - fold dilutions of the virus material obtained following transfection on cef monolayers grown in 6 - well tissue culture plates . after three days we observed the formation of virus plaques which virtually all contained gfp producing cells . from ten well separated plaques being processed for further amplification on cef , we recovered eight different isolates of recombinant mva - p h5 - gfp after one additional passage . pcr analysis of viral dna demonstrated that all virus isolates represented bona fide recombinants carrying stable insertions at the site of deletion iii within their genomes ( fig7 b ). the ease with which these virus isolates were obtained reflects the essential requirement of e3l function for mva growth on cef , and suggests that the e3l - 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