stable transfectants , the   lac repressor expression vector , p3ss ( stratagene , la jolla , ca ) ,   was transfected into nih3t3 cells by calcium phosphate coprecipitation .
stable transfectants were selected in 250 g / ml hygromycin and clonal cell lines were isolated .
lac repressor expression   was measured on western blots reacted to anti - lac repressor antibody ( stratagene ) .
cell lines expressing high levels of lac repressor were transfected with the isopropyl -d - thiogalactopyranoside ( iptg ) inducible pop13 ( stratagene ) plasmid containing the   human rnase l cdna in the sense orientation , cloned into the   not 1 site .
stable transfectants were selected in 500 g / ml g418   and clonal cell lines were isolated for analysis . for rnase l activity measurements , after indicated treatments ,   cells were washed with pbs and then incubated in serum - containing medium for an additional 3.5 h before harvesting .
total   cellular rna was isolated using trizol ( gibco brl , gaithersburg , md ) and 5 g was electrophoresed on 1.4% agarose ,   stained with ethidium bromide , and visualized under uv fluorescence .
alternatively , rna ( 10 g ) was separated by glyoxal - agarose   gel electrophoresis and then transferred to nytran membranes   ( schleicher and schuell , inc . , keene , nh ) before hybridization to   p - labeled 18s rrna cdna ( 12 ) .
the cdna probe was radiolabeled with -p - deoxycytidine 5triphosphate using prime - a - gene system ( promega corp . ,
cells were cultured overnight at 5  10 cells / ml in eagle 's   minimum essential medium ( emem ) , containing 10% fcs , penicillin / streptomycin , gentamycin , and for stably transfected cells   500 g / ml g418 ( gibco brl ) and 250 g / ml hygromycin .   for protein synthesis inhibition assays , cells , in 96-well flat - bottomed plates
, were incubated in leucine - free rpmi media containing 0.5 ci [ c]leucine for 1.25 h. cells were harvested   onto glass fiber filters with a cell harvester and radioactivity was   counted . for in situ
dna fragmentation end - labeling , cells were cultured to 5  10 cells / well on glass coverslips precoated with   poly - l - lysine ( 0.1 mg / ml in distilled water ) .
after 24 h of treatment , cells were fixed for 10 min in 4% formaldehyde , washed   twice , incubated for 15 min in methanol , and permeabilized in   1% bsa , 0.1% saponin , and 1.5 mg / ml glycine .
cells were   stained for dna fragmentation using either t7 dna polymerase   ( 13 ) or tdt ( 14 ) ( trevigen , gaithersburg , md ) , then counterstained with methyl green .
for transient cotransfections with gfp , hela cells and l929   cells were plated at 3  10 cells / well and incubated overnight .   the gfp plasmid ( 1 g ) , cloned into pcdna3 ( invitrogen   corp . ,
carlsbad , ca ) , was mixed with 4 g of either empty vector pcdna3 , rnase lzb1 in pcdna1 , or bcl - xl in pcdna3 and   10 l of lipofectamine ( gibco brl ) in a volume of 1 ml/ transfection for 5 h. for viral studies ,
poliovirus type i at 10   pfu / well was added to hela cells 36 h after transfection and was   allowed to adsorb for 30 min .
after incubation , cells were washed   twice and incubated in serum - containing media .
cells expressing   gfp were counted at each of the indicated time points using fluorescent microscopy and calculated as a percentage of untreated   gfp - positive cells ( 15 ) .
to investigate the potential role of rnase l in apoptosis ,   we generated nih3t3 cell lines stably transfected with the   human rnase l gene under the control of an iptg - inducible lac promoter .
one representative clone of seven independent clones characterized , 3t3/rnasels , expressed   the human rnase l protein constitutively ( fig .
1 a , lane   2 ) and 24 h after induction with iptg , expression levels   increased by severalfold ( fig .
1 a , lane 3 ) . to examine   rnase l activity in the 3t3/rnasels cells ,
the rnase l   activator , trimeric 25a [ ppp5(a2p5)2a ] was introduced into 3t3/rnasels cells .
specific 18s and 28s   rrna degradation products , characteristic of 25a - dependent rnase l cleavage ( 16 ) , were detected in uninduced   3t3/rnasels cells ( fig .
1 b and c , lane 2 ) and this ribonuclease activity increased after iptg induction ( fig . 1 b   and c , lane 3 ) .
degradation of 28s rrna , a   characteristic of human rnase l ( 16 ) , was not observed in   3t3/neo control cells expressing only the endogenous murine rnase l ( data not shown ) or in 3t3/rnasels cells   induced by iptg in the absence of 25a .
active human   rnase l is expressed and inducible in 3t3/rnasels cells .   in the absence of iptg
, 3t3/rnasels cells grew more   slowly than control 3t3/neo cells ( table 1 ) .
the rate of   [ h]thymidine incorporation was identical in the two cell   lines ( table 1 ) , suggesting that the 3t3/rnasels cells ,   chronically expressing human rnase l , had a higher cell   death rate rather than a slower cell division rate .
l in 3t3/rnasels cells by exposure to iptg for 72 h decreased the protein synthesis   rate to 20% of uninduced cells ( fig .
2 ) and decreased cell   viability measured by trypan blue dye exclusion to 38% .  
similar results were found in two of two independent   clones examined .    using in situ dna fragmentation detection ( 13 , 14 )   ( fig .
3 ) , we quantitated the number of cells that died with   the characteristic dna cleavage of apoptosis .
the 3t3/ rnasels fibroblasts spontaneously underwent apoptosis at   a low rate ( 02.2% ) and induction of higher levels of   rnase l expression for 48 h increased the number of cells   staining for dna fragmentation to 8.813.5% of the total   cells in four separate experiments ( p < 0.003 ) .
vector control cell lines displayed either considerably less or undetectable apoptosis either in the absence ( 0 cells of 1,116   counted ) or presence ( 0 cells of 1,105 counted ) of iptg   ( fig .
3 , a and c ) . interestingly , almost all of the 3t3/ rnasels cells had pyknotic nuclei when stained with methyl green after induction of high rnase l levels with   iptg whereas vector control cells did not show the pyknotic morphology upon iptg induction ( fig .
3 , c and   d ) . thus , increasing rnase l expression increases the rate   of nih3t3 cell apoptosis .  
the only   known biological activity of 25a is to bind rnase l.   rnase l binds 25a with high specificity , resulting in a   large increase in enzyme activity .
trimeric 25a   ( ppp5a2p5a2p5a ) introduced into l929 cells directly   triggered apoptosis whereas mock transfection of cells did   not ( table 2 ) .
moreover , apoptosis was not triggered by   the structurally related analogue - inhibitor , ppp5a2p5   a2p5u ( 17 ) ( table 2 ) , which can bind to rnase l but is   10-fold less effective as an activator due to the missing   n1/n6 domain of the third adenine ring of parent 25a   ( 18 ) .
these results correlate an increase in rnase l enzymatic activity with an induction in apoptosis .  
poly ( i)poly ( c ) has been shown to activate oas , to increase levels of endogenous 25a , and to cause death of interferon - treated fibroblasts ( 6 ) .
neither interferon nor dsrna alone significantly reduced l929 cell   viability or induced apoptosis ( fig .
however , the combination of interferon and dsrna   caused an 8891% decrease in cell viability and caused a   large increase in the number of apoptotic cells ( fig .
4 , c   and f ) . to determine whether rnase l was required for   this apoptosis pathway , as opposed to the interferon - inducible protein kinase ( 19 ) , we transiently transfected l929   cells with a dominant negative inhibitor of rnase l. the   inhibitor is a truncated version of rnase l , designated   rnase lzb1 , which lacks 89 cooh - terminal amino acids   and lacks ribonuclease activity ( 12 , 20 )
. this truncated protein can function as a potent inhibitor of the catalytic activity of wild - type rnase l both in cell - free systems and in   intact cells ( 12 ) .
cells expressing the rnase l inhibitor remained viable significantly longer than the vector control   cells after interferon and poly ( i)poly ( c ) treatment ( fig .
thus , dsrna can directly induce apoptosis in interferon - treated cells and inhibition of rnase l inhibits this   cell death .
interestingly , transfection of cells with the bcl - xl   gene did not protect cells from poly ( i)poly ( c)induced   apoptosis ( fig . 5 a ) .
either poly ( i)poly ( c ) induces an apoptosis pathway which is not blocked by bcl - xl or it activates   apoptosis downstream of the bcl - xl regulatory point .   
these results suggested that rnase l activity and the 25a   pathway may mediate viral - induced apoptosis .
we therefore examined poliovirus , a single - stranded rna virus   classified in the picornavirus family , which has been shown   recently to induce apoptosis in hela cells ( 21 ) .
hela cells   were transiently transfected with rnase lzb1 , the dominant   negative mutant , and then infected with poliovirus .
after   45 h , only 22% of the vector control transfected hela cells   remained viable , whereas 80% of the rnase lzb1 transfected cells remained viable ( fig .
interestingly , and in contrast to poly ( i)poly ( c)induced apoptosis , the poliovirus - induced cell death was blocked by   transfection with the bcl - xl gene ( fig .
thus , rnase l   activity was required for the poliovirus induced , bcl - xl sensitive pathway of apoptosis .  
rnase l was initially discovered as a mediator of the 25a   dependent rna breakdown stimulated by interferon and   viral infections ( 4 , 6 ) .
however , the precise mechanism for   the antiviral effect of the 25a system in vivo is unknown .  
we show here that overexpression of rnase l triggers apoptosis , that allosteric activation of rnase
l with 25a triggers apoptosis , and that a dominant negative inhibitor of   rnase l blocks apoptosis induced by both dsrna and   poliovirus infection .
this suggests that activation of rnase   l by viral infection could serve to eliminate infected cells   by apoptosis , preventing viral spread through the cell population .
this is a particularly intriguing hypothesis because   several studies indicate that apoptosis is a cellular mechanism of viral defense ; for example , virus infection itself can   trigger apoptosis ( 22 , 23 ) , and many viruses express virulence factors which block apoptosis ( 24 ) .
the results presented here suggest that rnase l activates apoptosis of   virus - infected cells to mediate the established antiviral activity of the 25a system .
to investigate the potential role of rnase l in apoptosis ,   we generated nih3t3 cell lines stably transfected with the   human rnase l gene under the control of an iptg - inducible lac promoter .
one representative clone of seven independent clones characterized , 3t3/rnasels , expressed   the human rnase l protein constitutively ( fig .
1 a , lane   2 ) and 24 h after induction with iptg , expression levels   increased by severalfold ( fig .
1 a , lane 3 ) . to examine   rnase l activity in the 3t3/rnasels cells ,
the rnase l   activator , trimeric 25a [ ppp5(a2p5)2a ] was introduced into 3t3/rnasels cells .
specific 18s and 28s   rrna degradation products , characteristic of 25a - dependent rnase l cleavage ( 16 ) , were detected in uninduced   3t3/rnasels cells ( fig .
1 b and c , lane 2 ) and this ribonuclease activity increased after iptg induction ( fig . 1 b   and c , lane 3 ) .
degradation of 28s rrna , a   characteristic of human rnase l ( 16 ) , was not observed in   3t3/neo control cells expressing only the endogenous murine rnase l ( data not shown ) or in 3t3/rnasels cells   induced by iptg in the absence of 25a .
active human   rnase l is expressed and inducible in 3t3/rnasels cells .   in the absence of iptg
, 3t3/rnasels cells grew more   slowly than control 3t3/neo cells ( table 1 ) .
the rate of   [ h]thymidine incorporation was identical in the two cell   lines ( table 1 ) , suggesting that the 3t3/rnasels cells ,   chronically expressing human rnase l , had a higher cell   death rate rather than a slower cell division rate .
l in 3t3/rnasels cells by exposure to iptg for 72 h decreased the protein synthesis   rate to 20% of uninduced cells ( fig .
2 ) and decreased cell   viability measured by trypan blue dye exclusion to 38% .  
similar results were found in two of two independent   clones examined .    using in situ dna fragmentation detection ( 13 , 14 )   ( fig .
3 ) , we quantitated the number of cells that died with   the characteristic dna cleavage of apoptosis .
the 3t3/ rnasels fibroblasts spontaneously underwent apoptosis at   a low rate ( 02.2% ) and induction of higher levels of   rnase l expression for 48 h increased the number of cells   staining for dna fragmentation to 8.813.5% of the total   cells in four separate experiments ( p < 0.003 ) .
vector control cell lines displayed either considerably less or undetectable apoptosis either in the absence ( 0 cells of 1,116   counted ) or presence ( 0 cells of 1,105 counted ) of iptg   ( fig .
3 , a and c ) . interestingly , almost all of the 3t3/ rnasels cells had pyknotic nuclei when stained with methyl green after induction of high rnase l levels with   iptg whereas vector control cells did not show the pyknotic morphology upon iptg induction ( fig .
3 , c and   d ) . thus , increasing rnase l expression increases the rate   of nih3t3 cell apoptosis .
the only   known biological activity of 25a is to bind rnase l.   rnase l binds 25a with high specificity , resulting in a   large increase in enzyme activity .
trimeric 25a   ( ppp5a2p5a2p5a ) introduced into l929 cells directly   triggered apoptosis whereas mock transfection of cells did   not ( table 2 ) .
moreover , apoptosis was not triggered by   the structurally related analogue - inhibitor , ppp5a2p5   a2p5u ( 17 ) ( table 2 ) , which can bind to rnase l but is   10-fold less effective as an activator due to the missing   n1/n6 domain of the third adenine ring of parent 25a   ( 18 ) .
these results correlate an increase in rnase l enzymatic activity with an induction in apoptosis .  
poly ( i)poly ( c ) has been shown to activate oas , to increase levels of endogenous 25a , and to cause death of interferon - treated fibroblasts ( 6 ) .
neither interferon nor dsrna alone significantly reduced l929 cell   viability or induced apoptosis ( fig .
however , the combination of interferon and dsrna   caused an 8891% decrease in cell viability and caused a   large increase in the number of apoptotic cells ( fig .
whether rnase l was required for   this apoptosis pathway , as opposed to the interferon - inducible protein kinase ( 19 ) , we transiently transfected l929   cells with a dominant negative inhibitor of rnase l. the   inhibitor is a truncated version of rnase l , designated   rnase lzb1 , which lacks 89 cooh - terminal amino acids   and lacks ribonuclease activity ( 12 , 20 ) .
this truncated protein can function as a potent inhibitor of the catalytic activity of wild - type rnase l both in cell - free systems and in   intact cells ( 12 ) .
cells expressing the rnase l inhibitor remained viable significantly longer than the vector control   cells after interferon and poly ( i)poly ( c ) treatment ( fig .
thus , dsrna can directly induce apoptosis in interferon - treated cells and inhibition of rnase l inhibits this   cell death .
interestingly , transfection of cells with the bcl - xl   gene did not protect cells from poly ( i)poly ( c)induced   apoptosis ( fig . 5 a ) .
either poly ( i)poly ( c ) induces an apoptosis pathway which is not blocked by bcl - xl or it activates   apoptosis downstream of the bcl - xl regulatory point .  
these results suggested that rnase l activity and the 25a   pathway may mediate viral - induced apoptosis .
we therefore examined poliovirus , a single - stranded rna virus   classified in the picornavirus family , which has been shown   recently to induce apoptosis in hela cells ( 21 ) .
hela cells   were transiently transfected with rnase lzb1 , the dominant   negative mutant , and then infected with poliovirus .
after   45 h , only 22% of the vector control transfected hela cells   remained viable , whereas 80% of the rnase lzb1 transfected cells remained viable ( fig .
thus , inhibition of   rnase l activity blocked apoptosis due to poliovirus . interestingly , and in contrast to poly ( i)poly ( c)induced apoptosis , the poliovirus - induced cell death was blocked by   transfection with the bcl - xl gene ( fig .
thus , rnase l   activity was required for the poliovirus induced , bcl - xl sensitive pathway of apoptosis .  
rnase l was initially discovered as a mediator of the 25a   dependent rna breakdown stimulated by interferon and   viral infections ( 4 , 6 ) .
however , the precise mechanism for   the antiviral effect of the 25a system in vivo is unknown .  
we show here that overexpression of rnase l triggers apoptosis , that allosteric activation of rnase l with 25a triggers apoptosis , and that a dominant negative inhibitor of   rnase l blocks apoptosis induced by both dsrna and   poliovirus infection .
this suggests that activation of rnase   l by viral infection could serve to eliminate infected cells   by apoptosis , preventing viral spread through the cell population .
this is a particularly intriguing hypothesis because   several studies indicate that apoptosis is a cellular mechanism of viral defense ; for example , virus infection itself can   trigger apoptosis ( 22 , 23 ) , and many viruses express virulence factors which block apoptosis ( 24 ) .
the results presented here suggest that rnase l activates apoptosis of   virus - infected cells to mediate the established antiviral activity of the 25a system .
a )   western blot analysis was performed for   the detection of human rnase l expression in vector control 3t3/neo cells   ( lane 1 ) and in nih3t3 cells transfected   with a lac inducible vector containing   the human rnase l gene ( 3t3/rnasels ) incubated 24 h in the absence   ( lane 2 ) or in the presence ( lane 3 ) of 3   mm iptg .
western blot analysis was   performed as previously described ( 25 )   using a 1:2,500 dilution of a monoclonal   antibody specific for the human rnase   l enzyme which did not detect the endogenous murine rnase l ( lane 1 ) .  
proteins were detected using ecl reagents ( amersham , arlington heights ,   il ) .
rna degradation after   induction of rnase l. ( b ) 3t3/rnasels cells were incubated in the absence ( lanes 1 and 2 ) and presence ( lane 3 ) of 3 mm iptg for 24 h then transfected with 1 m of ppp5(a2p5)2a   ( lanes 2 and 3 ) by calcium phosphate coprecipitation as previously described ( 12 ) .
( c ) northern blot analysis using an 18s rrna probe is shown after incubation of cells in the absence ( lane 2 ) and presence ( lanes 1 and 3 ) of 3 mm iptg for 24 h followed by transfection with 1 m of ppp5(a2p5)2a   ( lanes 2 and 3 ) .
growth rates of 3t3/neo and 3t3/rnasels cell lines  cells were plated at 10 cells / well and immediately after adherence   they were washed twice and incubated in leucine - free rpmi media   containing 0.5 ci [ h]thymidine for 1.25 h. cells were harvested   onto glass fiber filters and radioactivity was determined by scintillation   counting .  
cells were plated at 1.5  10 cells / well and after 24 h of   incubation , trypsinized , resuspended in trypan blue dye , and cells excluding dye were counted .     
after iptg treatment , cellular protein synthesis was determined in 3t3/neo cells ( circles ) and 3t3/rnasels cells ( squares ) and   plotted as a percentage of untreated cells .
3t3/neo ( a and c ) and 3t3/rnasels ( b and d ) cells were incubated for 24 h in the absence ( a and b ) and presence ( c and d ) of 3 mm iptg .
apoptotic cells were detected in situ by using t7 dna polymerase   with methyl green counterstaining .
induction of apoptosis by 25a  l929 cells were transfected by calcium phosphate coprecipitation for   75 min with a buffer control ( mock ) , with 1 m ppp2a5p2a5pu   as a negative control or with 1 m ppp2a5p2a5pa(25a ) .
cells   were washed , incubated for 24 h , and then processed for apoptosis .   
apoptosis was measured by counting cells in random fields positive for   dna fragmentation detected by tdt in situ labeling .     
apoptosis of interferon - treated fibroblasts after activation with dsrna . in situ dna end - labeling ( a  c ) and hoechst dye no .
33342 staining ( d  f ) of l929 cells . ( a and d ) after 48 h incubation in interferon-/ ( 1,000 u / ml ) ( sigma chemical co. , st .
( b and e ) 24 h incubation in synthetic dsrna poly ( i)(c ) ( 25g / ml ) .
( c and f ) , 24 h preincubation in interferon-/ followed by an additional 24 h in poly ( i)poly ( c ) .  
apoptotic cells were detected in situ by using terminal deoxynucleotidyl transferase with methyl green counterstaining .
cells were stained with hoechst   dye ( 0.1 mg / ml in pbs for 15 min ) and photographed using fluorescence on an axiovert microscope ( carl zeiss , inc . , thornwood
( g ) l929 cells were transiently cotransfected with the gfp ( green fluorescent   protein ) gene and either control vector ( open circles ) or the rnase lzb1 gene ( closed squares ) then incubated in interferon-/ ( 1,000 u / ml ) for 24  h. cells   were then treated with poly ( i)poly ( c ) ( 25 g / ml ) and green fluorescent cells were quantified as a measure of cell viability at each of the indicated time   points .
after transient cotransfection of the gfp gene   with control vector , ( gray bars ) , rnase lzb1 ( black bars ) , or bcl - xl ( hatched   bars ) genes , l929 cells were treated with ( a ) interferon-/ ( 1,000 u / ml )   and poly ( i)poly ( c ) ( 25 g / ml ) for 24 h and hela cells were treated   with ( b ) poliovirus at 10 pfu for 45 h. cell viability was assessed by   quantitation of green fluorescent cells and presented as a percentage of   untreated transfected cells .
hela cells were transiently cotransfected with gfp gene and either control vector ( gray bars ) or the rnase lzb1 gene ( black bars ) .
after incubation   in 10 pfu type i poliovirus , gfp - positive cells were quantitated at each   of the indicated time points .
cell numbers were calculated as a percentage   of uninfected cells transfected with each of the vectors .
 the 25a system contributes to the antiviral effect of interferons through the synthesis of 25a   and its activation of the ribonuclease , rnase l. rnase l degrades viral and cellular rna after   activation by unique , 25 phosphodiester - linked , oligoadenylates [ 25a , ( pp)p5 a2(p5a2)]n ,   n 2 . because both the 25a system and apoptosis can serve as viral defense mechanisms and   rna degradation occurs during both processes , we investigated the potential role of rnase l   in apoptosis . 
 overexpression of human rnase l by an inducible promoter in nih3t3 fibroblasts decreased cell viability and triggered apoptosis . 
 activation of endogenous rnase l , specifically with 25a or with dsrna , induced apoptosis . 
 inhibition of rnase l with a dominant negative mutant suppressed poly ( i)poly ( c)induced apoptosis in interferon - primed   fibroblasts . 
 moreover , inhibition of rnase l suppressed apoptosis induced by poliovirus . 
 thus ,   increased rnase l levels induced apoptosis and inhibition of rnase l activity blocked viral - induced apoptosis . 
 apoptosis may be one of the antiviral mechanisms regulated by the 25a   system . 
Materials and Methods
Results and Discussion
Increased RNase L Expression Induces Apoptosis in Fibroblasts.
Allosteric Activation of RNase L Induces Apoptosis.
Inhibition of RNase L Blocks Poliovirus-induced Apoptosis.
Figures and Tables