junn virus ( junv ) is a south american arenavirus , the etiological agent of a severe endemo - epidemic disease called argentine hemorrhagic fever ( ahf ) .  

arenaviridae is a family composed of a growing number of enveloped viruses with a bipartite single stranded rna genome .
members of the arenaviridae family were subdivided into two groups based on the geographical site of isolation , serological cross - reactivity and genetic data .
the prototype of the family , lymphocytic choriomeningitis virus ( lcmv ) is a member of the old world arenavirus group , which also includes ippy ( ippv ) , lassa ( lasv ) , mobala ( mobv ) , and mopeia ( mopv ) viruses .
junv is a member of the new world arenavirus group , that also includes allpahuayo ( allv ) , amapari ( amav ) , bear canyon ( bcnv ) , flexal ( flxv ) , guanarito ( gtov ) , latino ( latv ) , machupo ( macv ) , oliveros ( olvv ) , paran ( parv ) , pichind ( picv ) , pirital ( pirv ) , sabi ( sabv ) , tacaribe ( tcrv ) , tamiami ( tamv ) , and whitewater arroyo ( wwavs ) viruses .
in addition , there are several recently described species that have not yet been classified by the ictv ( http://www.ictvonline.org/virustaxonomy.asp?version=2008 ) .  
the two rna segments of the junn virus genome are designated l and s and have approximate sizes of 7.2 and 3.5  kb , respectively .
each rna segment directs the synthesis of two proteins ; their open reading frames are arranged in opposite orientations ( ambisense coding strategy )   and are separated by a noncoding intergenic region that folds into a stable stem - loop structure .
the s rna codes for the major structural proteins of the virion : the precursor of the envelope glycoproteins ( gpc ) and the viral nucleocapsid protein ( n ) .
posttranslational cleavage of gpc renders a signal peptide and the two viral glycoproteins ( gp1 and gp2 ) .
the l rna segment codes for the viral rna - dependent rna polymerase ( l ) and the small protein ( z ) .
z is a 94 residue long polypeptide , and its central portion is predicted to fold into a ring finger domain .
moreover , z has been implicated in several aspects of arenavirus biology [ 46 ] .
based on nucleotide sequence data , a better comprehension of the taxonomy and evolution of the arenaviridae has been achieved .
it has been suggested   that new world arenaviruses should be classified into four different lineages , named a , b , c , and rec .
lineage a would contain allv , flxv , parv , picv and pirv ; lineage b contains amav , gtov , junv , macv , sabv , and tcrv , lineage c contains latv and olvv , and lineage rec , with the wwav , tamv , and bcnv .
currently ( ictv website ) the last three viruses are classified with clade a.   the role of z in the virus life cycle is not completely elucidated , and homologues of z are not found in other ambisense or negative - stranded rna viruses .
z is a structural component of the virion , and by means of in vivo and in vitro experiments , the interaction of z with several cellular factors has been reported , including the promyelocytic leukemia protein and the eukaryotic translation initiation factor 4e [ 9 , 10 ] . because of this latter interaction , it was proposed that z inhibits cap - mediated translation [ 11 , 12 ] .
other researchers suggested that z could be a transcriptional regulator of the viral cycle   or even an inhibitor of viral replication .
furthermore , prez and coworkers   proposed , for lcmv and lasv , that z is the functional counterpart of the matrix proteins found in other negative - stranded enveloped rna viruses .
late domains ( lds ) , found in matrix proteins from negative - stranded rna viruses and in gag protein from retroviruses , have an essential role in the viral budding process .
three types of motifs have been defined within viral lds : p[ts]ap , ppxy , and yxxl , where   x  is any amino acid .
later , martn serrano and coworkers   redefined the last as : ypxl / lxxlf .
lds are highly conserved and have been shown to mediate interaction with host cell proteins , in particular with members of the vacuolar protein - sorting pathway [ 19 , 20 ] .
for instance , the ptap motif from ebola virus vp40 matrix protein and from hiv gag protein interacts with tsg101 , a member of the vacuolar protein - sorting pathway . in this work
, we show the strategies employed for the expression , purification , and specific antibody generation against z protein from candid#1 strain of junn virus . here
we report the optimized expression from a synthetic gene of z protein tagged with different peptides , using three expression systems ( two bacterial and a baculoviral one ) , in order to obtain recombinant z protein suitable for functional characterization studies .
the parental junn virus xj strain was isolated in junn city ( buenos aires , argentina ) from a human ahf patient .
the bhk21 cells were cultured in growth medium ( dulbecco 's minimal essential medium  dmem  supplemented with 10% fetal bovine serum and 2  mm l - glutamine ) to 50% confluence and infected at a multiplicity of infection ( moi ) of 1 plaque forming unit ( pfu ) per cell . after virus adsorption for 1  h at room temperature ,
infected cells were washed with phosphate - buffered saline ( pbs ) and maintained at 37c in mem containing 2% fetal bovine serum .
virions were recovered and purified from the supernatant media ; viral and total infected cell rnas were isolated according to procedures described previously .
virus titers were determined by plaque assay on vero e6 ( atcc , ccl 1586 , c1008 ) cell monolayers as described elsewhere .
the cell monolayers were harvested 72  h post infection by scraping and recovered by centrifugation .
rna was isolated from pelleted virions or infected cells , using the qiamp viral rna mini kit or rneasy mini kit ( qiagen , valencia , ca ) .
the z open reading frame ( orf ) from junv candid#1 strain , was amplified by rt - pcr ( superscript iii reverse transcriptase for rt , and platinum taq dna polymerase high fidelity for pcr , invitrogen ) , from junn virus rna using the forward primer jzv   5-atgggcaactgcaacggggcatc-3 ( z orf translation initiation sequence is underlined ) , and the reverse primers jzvc  5- tggtggtggtgctgttggctccac -3 , and jzvc - stop :  5-ctatggtggtggtgctgttggctccac -3 ( sequence complementary to the z stop codon is underlined ) . the amplicon was cloned into pzero and the recombinant plasmid ( zpz ) was amplified in escherichia coli strain top10 ( invitrogen ) .
the recombinant plasmids were confirmed by nucleotide sequencing . for cloning z orf into pet102/d - topo ( invitrogen ) ,
a variant from jzv primer was used , named jzvclamp : 5-ccac  atgggcaactgcaacggggcatc-3 ( the four nucleotides , added to facilitate cloning , upstream of the atg initiation codon , are indicated in italics ) .
the recombinant clone was named pet - z and used for expression experiments in bacterial cell culture ( e. coli bl21 ) .
the produced protein was fused to different tags : thioredoxine by the n - terminus , and his tag and v5 epitopes by the c - terminus .
this fusion protein was called tio - z - v5-his . for the subsequent cloning procedures the plasmid zpz was digested with xhoi and hindiii .
this digestion rendered a product of 366  bp , comprising z orf and a portion of the alpha fragment of lacz ( from pzero mcs ) , hereafter designated as z. this fragment was cloned into different expression vectors : 
 pgz ( pgex - b , novagen ) .
the z fragment was ligated into pqe-30 ( qiagen , valencia , ca ) previously digested with hindiii and sali .
the obtained plasmid , pqz was digested with smai and hindiii and the 374  pb fragment was ligated into pgex - b , previously digested with the same enzymes . the recombinant plasmid ( pgz )
the recombinant protein , called gst - z , contains glutathione - s - transferase ( gst ) fused to the n - terminus of z. 
 pbac - z ( pfastbac - hta , invitrogen ) .
the z fragment was ligated into the vector pfastbac - hta , previously digested with hindiii and sali .
z orf was fused to a his - tag to the amino end of z. this his - tagged version of z was named z - his .
the recombinant plasmid ( pbac - z ) was amplified in e. coli top10 and the insert was confirmed by nucleotide sequencing .
the bac - to - bac system ( invitrogen ) was used to generate recombinant baculoviral genomes ( acmnpv ) harboring z - his gene ( bacmid_ac - z ) .
briefly , z orf in pbac - z was transposed to an acmnpv genome using e. coli strain dh10 bac , which carries the target baculoviral genome and the machinery required for transposition .
the recombinant acmnpv genomes ( bacmid_ac - z ) were purified by minipreparation from bacterial culture and then used to generate recombinant baculoviruses ( v_ac - z ) by transfecting different insect cell lines ( cellfectin , invitrogen ) .
the recombinant baculovirus ( v_ac - z ) was amplified by infection of insect cells .
viruses were recovered from the supernatant media of infected cell monolayers and purified by ultracentrifugation .
optimization procedures of the expression levels consisted in testing different multiplicities of infection and insect cell lines .
two cell lines were used , sf9 from spodoptera frugiperda   and high five ( bti - tn-5b1 - 4 ) from trichoplusia ni . these cells were cultured on 25  cm polystyrene flasks with grace medium ( invitrogen ) , supplemented with 10% fetal bovine serum and 0.1% gentamicin , to 70% confluence and infected with the recombinant baculovirus ( > 10  pfu / ml ) at different multiplicities of infection ( moi ) . after virus adsorption for 1  h at room temperature , infected cells were washed with phosphate - buffered saline ( pbs ) and maintained at 25c in grace containing 10% fetal bovine serum .
the protein expression was analyzed by sds - page and western blotting using z protein - specific antibodies and his - tag antibodies .
a variety of expression strains were tested : rosetta plys and origami b ( de3 ) from novagen ; bl21 ( de3 ) , bl21 ( plyss ) , bl21-codonplus ( de3 ) ripl and xl1blue from stratagene ; bl21 si from invitrogen ; overexpress c41 ( de3 ) and c43 ( de3 ) from lucigen .
cells were grown on luria broth ( lb ) growth medium   and supplemented with the proper antibiotic when required ( 34  g / ml kanamycin , 100  g / ml ampicillin , 50  g / ml chloramphenicol , 12.5  g / ml tetracycline ) . 
							
tio - z - v5-his ( pt - z ) . for tio - z - v5-his fusion protein ,
transformed cells were cultured in 500  ml of lb medium containing 100  g / ml ampicillin , at 37c .
when the culture absorbance at 600  nm reached a value of 1.5 , cells were induced with 600  m isopropyl--d - thiogalactopyranoside ( iptg ) for 4  h at 37c .
bacteria were then harvested by centrifugation at 4000    g for 15  min , and resuspended in 25  ml of 50  mm nah2po4 ph 8.2 ; 300  mm nacl ; 20  mm imidazole ; 1% triton x-100 and complete protease inhibitor cocktail tablets 1x ( roche ) .
cells were disrupted by french press and , after centrifugation at 20,000    g for 30  min , the soluble fraction was collected and analyzed by sds - page ( 4x loading sample buffer containing 200 mm tris - hcl , 8% sds , 40% glycerol , and 0.4% bromophenol blue and 400 mm dtt , ph 6.8 ) .
sds - page analyses were performed according to laemmli , using 1215% polyacrylamide gels stained with r250 coomassie blue .
very similar expression levels were obtained from the different host cells studied for the gst - z fusion protein , as detected by tris - glycine 15% sds - page .
the selected strain and culture conditions were the same as employed by volpon and coworkers , with buffers containing zn .
bacteria were harvested by centrifugation at 4,000    g for 15  min , and the pellet was resuspended in 25  ml of pbs , 200  mm nacl , ph 8 ;
0.1% triton x-100 , 100  m dtt , and complete protease inhibitor cocktail tablets 1x ( roche ) .
cells were disrupted by french press and , after centrifugation at 20,000    g for 30  min , the soluble fraction was collected and analyzed by sds - page .
analyses were performed according to laemmli , using 1215% polyacrylamide gels stained with r250 coomassie blue .
tio - z - v5-his 
						 the soluble fraction ( 25  ml ) obtained after the induction of pet - z was clarified by centrifugation at 12,000    g for 30  min and loaded on a his - trap 5  ml column ( amersham ) , using a running buffer 50  mm nah2po4  ph 8.2 , 300  mm nacl .
after column wash , a step gradient of 500  mm imidazole was applied at 1  ml / min flow for 20  min using an akta fplc ( amersham ) .
each fraction collected was analyzed by sds - page and those containing tio - z - v5-his were pooled , and   concentrated down to 1  ml on a spin concentrator ( vivaspin 15 , 10,000 mwco , sartorius ) .
this was then run on a gel filtration column superdex 200 10/300  gl ( amersham ) using running buffer 50  mm nah2po4 ph 8.2 , 300  mm nacl at 0.5  ml / min flow .
again , every fraction was analyzed by sds - page and those containing tio - z - v5-his were pooled and dialyzed overnight against tris - hcl 20  mm ph 8.0 at 4c . 
 
the soluble fraction ( 25  ml ) obtained after the induction of pet - z was clarified by centrifugation at 12,000    g for 30  min and loaded on a his - trap 5  ml column ( amersham ) , using a running buffer 50  mm nah2po4  ph 8.2 , 300  mm nacl .
after column wash , a step gradient of 500  mm imidazole was applied at 1  ml / min flow for 20  min using an akta fplc ( amersham ) .
each fraction collected was analyzed by sds - page and those containing tio - z - v5-his were pooled , and   concentrated down to 1  ml on a spin concentrator ( vivaspin 15 , 10,000 mwco , sartorius ) .
this was then run on a gel filtration column superdex 200 10/300  gl ( amersham ) using running buffer 50  mm nah2po4 ph 8.2 , 300  mm nacl at 0.5  ml / min flow .
again , every fraction was analyzed by sds - page and those containing tio - z - v5-his were pooled and dialyzed overnight against tris - hcl 20  mm ph 8.0 at 4c . 

cleavage and purification of tio - z - v5-hispurified tio - z - v5-his was dialyzed against   enterokinase cleavage buffer ( 50  mm tris - hcl  ph 8 ; 1  mm cacl2 ; 0.1% tween-20 ) and then concentrated with a spin concentrator ( vivaspin 6 10,000 mwco , sartorius ) down to 1  ml at a final concentration of 0.3  mg / ml . a fraction of 500  l of this sample was subjected to proteolysis with enterokinase on a final volume of 600  l overnight at 4c . after this
, the sample was loaded on a his - trap 5  ml column ( amersham ) .
the running buffer was 50  mm nah2po4 ph 8.2 ; 300  mm nacl .
finally , a 500  mm imidazole step gradient was applied for 20  min .
purified tio - z - v5-his was dialyzed against   enterokinase cleavage buffer ( 50  mm tris - hcl  ph 8 ; 1  mm cacl2 ; 0.1% tween-20 ) and then concentrated with a spin concentrator ( vivaspin 6 10,000 mwco , sartorius ) down to 1  ml at a final concentration of 0.3  mg / ml . a fraction of 500  l of this sample was subjected to proteolysis with enterokinase on a final volume of 600  l overnight at 4c . after this
, the sample was loaded on a his - trap 5  ml column ( amersham ) .
the running buffer was 50  mm nah2po4 ph 8.2 ; 300  mm nacl .
finally , a 500  mm imidazole step gradient was applied for 20  min .
gst - zthe soluble fraction ( 25  ml ) , obtained after the induction of pgz and cell disruption by cell press , was clarified by centrifugation at 12,000    g for 30  min , loaded into 1  ml of glutathione sepharose 4b ( ge healthcare ) , and incubated at 4c overnight . after five washes with 50  ml of pbs ,
the sample was eluted with 2  ml of 25  mm reduced glutathione ( sigma ) in 50  mm tris - hcl ph  9.5 .
the eluted fraction was dialyzed against phosphate buffer 20  mm na2hpo4  ph  7.2 at 4c overnight , concentrated down to 1  ml ( vivaspin 2 , 10,000 mwco , sartorious ) , and loaded into a superdex 200 column ( amersham ) using the same buffer conditions at 0.5  ml / min flow . 
 
the soluble fraction ( 25  ml ) , obtained after the induction of pgz and cell disruption by cell press , was clarified by centrifugation at 12,000    g for 30  min , loaded into 1  ml of glutathione sepharose 4b ( ge healthcare ) , and incubated at 4c overnight .
after five washes with 50  ml of pbs , the sample was eluted with 2  ml of 25  mm reduced glutathione ( sigma ) in 50  mm tris - hcl ph  9.5 .
the eluted fraction was dialyzed against phosphate buffer 20  mm na2hpo4  ph  7.2 at 4c overnight , concentrated down to 1  ml ( vivaspin 2 , 10,000 mwco , sartorious ) , and loaded into a superdex 200 column ( amersham ) using the same buffer conditions at 0.5  ml / min flow . 

cleavage and purification of gst - zgst - z was dialyzed against factor xa cleavage buffer ( 50  mm tris - hcl ph  7.5 ; 150  mm nacl ; 1  mm cacl2 ) and 600  l of this sample were subjected to proteolysis with factor xa protease at room temperature overnight .
the reaction product was incubated with glutathione sepharose for 1  h at room temperature to retain the cleaved gst fusion peptide , while z protein was recovered in the supernatant .
finally the bound gst protein was eluted with 25  mm reduced glutathione in 50  mm tris - hcl ph  9.5 .
the reaction products were analyzed by sds - page and western blotting with a polyclonal antiserum specific for the z protein . 
 
gst - z was dialyzed against factor xa cleavage buffer ( 50  mm tris - hcl ph  7.5 ; 150  mm nacl ; 1  mm cacl2 ) and 600  l of this sample were subjected to proteolysis with factor xa protease at room temperature overnight .
the reaction product was incubated with glutathione sepharose for 1  h at room temperature to retain the cleaved gst fusion peptide , while z protein was recovered in the supernatant .
finally the bound gst protein was eluted with 25  mm reduced glutathione in 50  mm tris - hcl ph  9.5 .
the reaction products were analyzed by sds - page and western blotting with a polyclonal antiserum specific for the z protein .
columns packed with 24  ml bead volumes of superdex 200  gl and superdex 75  gl were used to obtain the target protein in its monomeric form .
columns were loaded with 1  ml of sample and run at 0.5  ml / min flow using an akta fplc ( amersham ) . for tio - z - v5-his recombinant protein , the running buffer was 50  mm nah2po4 , 300  mm nacl ph 8 . for the gst - z recombinant protein it was 20  mm na2hpo4  ph 7.2 . before each protein separation , a molecular weight marker ( bio - rad ) was run for calibration purposes using the corresponding running buffer . a serial dilution 1/10 to 1/10,000 from a stock of 0.1  mg / ml of chymotrypsin ( roche ) and trypsin ( roche )
were incubated with a 0.5  g/l of gst - z recombinant protein , for 2  h at room temperature .
the reaction was stopped by adding sds - page sample buffer or by incubating the reaction at 20c .
the samples subjected to limited proteolysis with chymotrypsin were loaded on a 15% sds - page   and blotted to a pvc membrane ( amersham ) .
the n - terminal sequence of relevant peptides was then obtained using an applied biosystems sequencer abi494 .
briefly , the purified tio - z - v5-his fusion protein was used to inoculate two female new zealand white rabbits with an average weight of 2.5  kg . before each inoculation , the animals were bled from the marginal ear vein for evaluation of antibody titers .
for the first injection , 50  g of purified recombinant protein / kg of body weight were emulsified in an equal volume of complete freund 's adjuvant ( sigma chemicals co ) and administered subcutaneously .
after 25 days , the animals were bled as described above and inoculated with 50  g of purified recombinant protein / kg of body weight emulsified in an equal volume of incomplete freund 's adjuvant ( sigma chemicals co ) .
this procedure was repeated twice with a time interval of 15 days and a last bleeding was carried out by cardiac puncture .
the antibody titer was estimated by inhouse eia as described in argelles et al . .
tio - z - v5-his fusion protein was used as antigen in different assays , with the exception of the cutoff determination , where gst - z was used as antigen against the heterologous sera .
purification of g type immunoglobulin ( igg ) fractions from antiserum was carried out by affinity chromatography on protein g - sepharose fast flow ( pharmacia ) according to the manufacturer 's recommendations .
igg yield , determined by absorbance at 280  nm , was 3.2  mg / ml serum . for inhouse eia ,
microtiter plates ( nunc , roskilde , denmark ) were coated overnight at 4c with tio - z - v5-his fusion protein diluted to 10  g / ml in 0.1  m bicarbonate buffer ( ph  9.6 ) as antigen .
after this , and after each of the following steps , the plates were washed three times with washing solution , that is , phosphate - buffered saline ( pbs ) , 0.5  m nacl , and 0.2% ( v / v )
fifty microliters of each serum sample diluted 1/300 in sample buffer ( i.e. , 1% wt / vol bovine serum albumin in washing solution ) were added to the antigen - coated wells , and the plates were incubated at 37c for 1  h. peroxidase - conjugated antirabbit igg ( santa cruz biotechnology ) diluted 1/16,000 in sample buffer was added at 50  l / well and incubated further for 1  h at 37c .
bound antibodies and conjugates were then developed with orthophenylenediamine ( sigma chemical co ) , 30% h2o2,and citrate buffer ph  5.0 at a ratio of 1  mg/l / ml according to standard procedures .
the optical density ( od ) was measured at a wavelength of 490  nm ( od490 ) ( max line tm enzyme - linked immunosorbent assay reader ; molecular devices , sunnyvale , ca ) .
each serum was simultaneously tested with corresponding control antigens , that is , preimmune serum .
the results were expressed as the value of the od of the control antigen subtracted from the od of the fusion protein antigen for each serum sample .
the enzyme - linked immunosorbent assay cutoff value was estimated as the mean od obtained with 20 certified negative specimens plus 3 standard deviations .
these negative specimens were heterologous sera , diluted 1/300 , and the antigen used was the gst - z fusion protein .
the extracts of bacterial cells and insect cell lines infected with recombinant baculovirus were separated by sds - page ( 1215% polyacrylamide gel ) and blotted onto nitrocellulose membranes ( hybond p , amersham pharmacia ) in tris - glycine buffer containing 20% ( v / v ) methanol . to avoid nonspecific binding of the antibodies ,
the membranes were blocked by incubation with 5% wt / vol skimmed powder milk in pbs for a minimum of 2  h at 37c . for the primary antibody incubation the blocked membrane was probed with a 1/1,000 dilution of the polyclonal antiserum specific for the z protein on pbs 2% casein 0.1% tween-20 at 37c for 1  h , followed by an incubation with horseradish - peroxidase conjugated goat anti - rabbit igg ( santa cruz biotechnology ) , diluted 1/10,000 on pbs 0.1% tween-20 at 37c for 1  h. after each step the membrane was washed three times with pbs 0.1% tween-20 , 5  minutes each time .
supernatants were then ultracentrifuged in order to pellet down small particles , and these fractions were treated with proteinase k ( 0.1  mg / ml , 37c , 30  min ) , or a combination of proteinase k plus 1% triton x-100 in the same conditions .
proteolysis was stopped by adding 100  mm pmsf and boiling the samples for 10  min .
sequencesamino acid sequences of the following arenavirus were obtained from the genbank database ( accession numbers are indicated between brackets ) : amav - bean70563 ( aby59841.1 ) , cpxv - bean119303 ( aby59842.1 ) , gtov - cvh-961104 ( aat77691.1 ) , gtov - vhf-3990 ( aat77689.1 ) , junv - mc2 ( aby59838.1 ) , junv - candid#1 ( aau34182.1 ) , macv - maru-222688 ( aay27821.1 ) , macv-9530537 ( aay27823.1 ) , tacv ( np_694847.1 ) , latv - maru-10924 ( aay27824.1 ) , olvv-3229 ( aby59840.1 ) , pirv - vav488 ( aby59836.1 ) , pirv-1743 ( aat77682.1 ) , allv - clhp2472 ( aby59833.1 ) , picv - an3739 ( yp_138535.1 ) , bcnv - a0060209 ( aby59834.1 ) , bcnv - ava0070039 ( aax99343.1 ) , wwav - av9310135 ( aax99351.1 ) , tamv - w10777 ( aax99348.1 ) , lcmv - clone#13 ( abc96003.1 ) , lcmv - mx ( caa10342.1 ) , lasv - csf ( aao59514.1 ) , lasv - av ( aao59508.1 ) , ippy - dakanb188 ( yp_516232.1 ) , mobv - acar3080 ( yp_516228.1 ) , mopv - an20410 ( yp_170707.1 ) , mopv - mozambique ( abc71136.1 ) , lujo ( yp_002929492 ) , and morogoro ( acj24975.1 ) . 
 
amino acid sequences of the following arenavirus were obtained from the genbank database ( accession numbers are indicated between brackets ) : amav - bean70563 ( aby59841.1 ) , cpxv - bean119303 ( aby59842.1 ) , gtov - cvh-961104 ( aat77691.1 ) , gtov - vhf-3990 ( aat77689.1 ) , junv - mc2 ( aby59838.1 ) , junv - candid#1 ( aau34182.1 ) , macv - maru-222688 ( aay27821.1 ) , macv-9530537 ( aay27823.1 ) , tacv ( np_694847.1 ) , latv - maru-10924 ( aay27824.1 ) , olvv-3229 ( aby59840.1 ) , pirv - vav488 ( aby59836.1 ) , pirv-1743 ( aat77682.1 ) , allv - clhp2472 ( aby59833.1 ) , picv - an3739 ( yp_138535.1 ) , bcnv - a0060209 ( aby59834.1 ) , bcnv - ava0070039 ( aax99343.1 ) , wwav - av9310135 ( aax99351.1 ) , tamv - w10777 ( aax99348.1 ) , lcmv - clone#13 ( abc96003.1 ) , lcmv - mx ( caa10342.1 ) , lasv - csf ( aao59514.1 ) , lasv - av ( aao59508.1 ) , ippy - dakanb188 ( yp_516232.1 ) , mobv - acar3080 ( yp_516228.1 ) , mopv - an20410 ( yp_170707.1 ) , mopv - mozambique ( abc71136.1 ) , lujo ( yp_002929492 ) , and morogoro ( acj24975.1 ) . 

alignmentssequence alignments were done using the clustal x program [ 31 , 32 ] . 
 
sequence alignments were done using the clustal x program [ 31 , 32 ] . 

synthetically , the sequence logo is a graphic representation of a multiple alignment of sequences .
the stacking height indicates the local information content in this position , while the height of each symbol inside the stacking is the fraction of information content representing the frequency of each residue in that position .
because of the characteristics of the software , and to avoid bias towards particular species that are found over - represented in the sequence data bank , at most two strains for each viral species were selected . moreover , to get a more accurate representation , gap positions within the sequences in the alignment were considered as another character .
the usual behavior of the software does not take into account the gaps , calculating the values relative to the number of sequences with an amino acid in that position .
all the motif descriptions were annotated according to the syntax rules of prosite , and represent more than 60% of all z protein sequences . 
 
synthetically , the sequence logo is a graphic representation of a multiple alignment of sequences .
the stacking height indicates the local information content in this position , while the height of each symbol inside the stacking is the fraction of information content representing the frequency of each residue in that position .
because of the characteristics of the software , and to avoid bias towards particular species that are found over - represented in the sequence data bank , at most two strains for each viral species were selected . moreover , to get a more accurate representation , gap positions within the sequences in the alignment were considered as another character .
the usual behavior of the software does not take into account the gaps , calculating the values relative to the number of sequences with an amino acid in that position .
all the motif descriptions were annotated according to the syntax rules of prosite , and represent more than 60% of all z protein sequences . 

biochemical properties of different recombinant proteins the different biochemical parameters were calculated from the amino acid sequence data using the protparam tool found at the expasy proteomics server ( www.expasy.ch , proteomics tools , primary structure analysis ; ) . 
  
the different biochemical parameters were calculated from the amino acid sequence data using the protparam tool found at the expasy proteomics server ( www.expasy.ch , proteomics tools , primary structure analysis ; ) .
in figure 1 , three z protein alignments ( old world , new world , and all arenaviruses ) are shown separately . in the top of the figure a diagram of the z protein
we observe a myristoylation recognition motif in the amino end region , a ring finger domain , characterized by the seven cysteines ( at positions 43 , 46 , 56 , 62 , 65 , 76 , and 79 in the alignment ) and one histidine at the 59 position in the central core region , and different late domains ( found in other viral matrix proteins ) at the carboxyl end region . in the amino end region
the most conserved island contains 7 amino acids comprising the first two amino acids ( mg ) that include the glycine modified by the myristic acid aggregate ( gly2 , completely conserved ) ( gray shadowed in figure 1 ) . the other amino acids in this region are basic residues ( k or r ) , or amino acid residues with amide ( q or n ) , sulfhydryl ( c ) , or hydroxyl ( s or y ) groups , probably constituting additional factors for membrane attachment .
the rest of the fragment has poor conservation degree , with the exception of another island for the old world arenavirus group ( rx4pd , where x is any amino acid , underlined in figure 1 ) .   in the z protein core region ,
particularly , the 7 cysteines and the histidine residues required for zn binding and folding of the predicted ring finger domain ( gray shadowed in figure 1 ) .
these residues are totally conserved in the viral family , indicating strong selection pressure for keeping the predicted ring finger domain .
other positions within this region also show high conservation degree between both groups ( new and old world arenavirus ) .
for example residues k44 , w47 , and l53 are completely conserved among all arenaviruses , suggesting they might play a critical role in protein folding , or maybe protein - protein interactions required for function , such as eif4e interaction . furthermore , the amino end of this domain , shows a sequence motif characteristic for each group ( l[yh]gr[yf]n , for the new world arenavirus , and gp[elq][fns ] , for the old world arenavirus ) . until the moment , the role of this region is unknown , but it is interesting to highlight that the degree of conservation is unique within the z protein , although differing among groups . it is therefore possible to hypothesize that this region could be associated with the arenavirus host range .
in addition , at the carboxyl end of the ring finger domain , another conserved site within groups is detected , described as kx(0,1)plptx[il ] ( where x can be any amino acid ) .
this putative site is underlined in figure 1 , where it can be seen that the conservation is stronger in the old world arenavirus group .
furthemore , by modifying the first conserved leucine in this site ( l83 ) , it was demonstrated that this residue is involved in both the rescue of nucleocapsids and the incorporation of glycoproteins into infectious virus - like particles .
finally , a cluster of conserved sites , in an island configuration , can also be found at the carboxyl end region .
this island includes the characteristic motifs found in viral matrix proteins of retroviruses ( late domains ) : p[ts]ap and pppy ( gray shadowed in figure 1 ) .
these two motifs are present in the old world arenavirus group , while in the new world arenavirus group only the first one is found .
another late domain found but situated in the core region was ylcl ( yxxl [ 18 , 19 , 39 , 40 ] ) ( underlined in figure 1 ) .
the ylcl motif is also present in the tacaribe virus z protein and was observed not to be involved with budding of virus like - particles .
interestingly , tacaribe z protein   does not posses the p[ts]ap motif present in the majority of arenaviruses including junin z. another late domain frequently identified in matrix proteins of negative - stranded rna viruses is pxv ( where  indicates a hydrophobic residue and x any aminoacid , ) , however the same was not found in the sequence of arenaviruses z protein . for the different fusion proteins , several parameters were calculated and are shown in table 1 .
the z protein indicates the product of translation from the 366  bp fragment ( z ) . near 90 days after first inoculation with the tio - z - v5-his protein
, the inoculated rabbits were heart bled , obtaining 30  ml of blood . after serum separation , the same was used to determine the cutoff value , yielding a number of 0.165 .
afterwards , the polyclonal serum titer was determined , defined as the bigger serum dilution that renders a positive reaction ( more than the cutoff value ) . for this determination , the assay conditions were the same as those in the assay cutoff determination , obtaining an antiserum titer of 128,000 .
then , the iggs were purified by affinity chromatography to later obtain more specific z detection .
the different fractions were analyzed by sds - page ( data not shown ) and quantified by bradford method using a goat igg as concentration reference in a calibration curve .
the expression profile of the recombinant tio - z - v5-his protein is shown in figure 2 . in figure 2(a ) a diagram of the expressed recombinant orf is shown . in figure 2(b ) the lanes corresponding to t0 indicate the moment when the iptg was added to the culture , while the tf lanes indicate the final culture time .
after induction , a protein of expected size was expressed ( figure 2(b ) ) . following cell disruption ,
sds - page analysis confirmed it was possible to obtain a considerable amount of tio - z - v5-his in the soluble fraction ( figure 2(c ) ) .
the soluble fraction of tio - z - v5-his was loaded into a his - trap column and monitored at uv280 and uv254 .
the peak fractions were collected and analyzed by 12% sds - page , containing a main band corresponding to the molecular weight of tio - z - v5-his ( ca .
27 kda ) ( figure 2(d ) ) . final yield of pure tio - z - v5-his protein was between 10 and 12  mg per liter of culture . in order to further purify tio - z - v5-his , selected fractions
were pooled , concentrated and loaded into a superdex 200 gel filtration column ( figure 2(e ) ) .
the obtained tio - z - v5-his was dialyzed , concentrated and subjected to proteolysis with enterokinase .
the cleaved products , thioredoxine ( 12.8 kda ) and z - v5-his ( 14.5 kda ) were visible after 2  h at 37c incubation ( figure 2(f ) ) .
the products of this reaction were loaded into a his - trap column , as previously described and two major peaks were observed after elution .
the first peak corresponded to thioredoxine , which eluted at 100  mm imidazole concentration , and the second peak corresponded to z - v5-his protein , which eluted at 250  mm imidazole concentration ( data not shown ) .
the fractions of the second peak were pooled , concentrated ( vivaspin 2 5,000 mwco , sartorius ) and analyzed by sds - page and western blotting with anti - z igg ( figure 2(g ) and 2(h ) ) . in both cases a band of ca .
the expression profile of the recombinant gst - z protein is shown in figure 3 .
a diagram of the expressed recombinant orf can be seen in figure 3(a ) . in figure 3(b ) , the overexpression of a protein with a size of about 37 kda is shown , where the lane t0 indicates the moment when the iptg was added to the culture , while the   tflane indicates the final culture time .
the soluble fraction , after cell lysis , was mixed with glutathione sepharose resin for batch purification of the gst - z protein . a protein of about 37 kda was present in the first wash fraction ; nevertheless , it was possible to obtain significant quantities of a ca .
37 kda protein retained in the glutathione sepharose ( figure 3(c ) , lanes e1 and e2 ) .  
these two peptides , retained together with the bigger protein , were probably degradation products of the ca .
. final yield of pure gst - z protein was between 5 and 8  mg per liter of culture .
the eluted fractions were pooled , concentrated and loaded into a superdex 200 gel filtration column .
the molecular weight of the peaks was estimated from the chromatogram profile ( figure 3(d ) ) .
37 kda band and the other two , of approximately 29 kda and 25 kda , were present in different proportions .
most of the 37 kda polypeptide was present at the high molecular weight peak ( ca .
700 kda ) , indicating that most of the overexpressed gst - z protein was aggregated ( figure 3(d ) ) , lanes 6 to 10 ) . in the collected fractions of the second peak , the 37 kda band was also detected , indicating that a portion of the overexpressed protein was soluble and monomeric ( figure 3(d ) , lanes 11 to 16 ) . to obtain the z protein without the gst fusion peptide , the purified gst - z obtained after the glutathione sepharose purification was dialyzed against cleavage buffer and subjected to proteolysis with factor xa .
the expected cleavage products , gst fusion peptide and z protein were observed . to confirm the identity of the last product the sample was analyzed by western blot with polyclonal antiserum specific for the z protein ( -tio - z - v5-his ) and antibodies specific for fusion protein ( -gst ) .
finally , as shown in figure 3(f ) , it was possible to detect a ca .
11.5 kda protein corresponding to z. however , it was evident that the cleavage reaction was incomplete , because the full - length ca .
29 kda peptide was also recognized by the z polyclonal antiserum , strengthening the hypothesis that it was a degradation product of gst - z . to confirm this hypothesis , the purified gst - z ( obtained after gel filtration in the 40 kda peak )
the peptides thus obtained were subjected to n - terminal sequencing ( figure 3(g ) ) .
the bands indicated with i match with the first six amino acids of the gst protein sequence , and the bands pointed out with ii and iii matched the c - terminal of gst and the middle region of z protein , respectively .
expression results obtained for the recombinant his - z protein are shown in figure 4 .
a diagram of the tagged protein obtained using the bac - to - bac expression system is shown in figure 4(a ) .
the v_ac - z construct ( recombinant bacmid ) was transfected into high five or sf9 insect cells , and the resulting viral stock was amplified by infection in the same type of cells .
the infection assays , optimized to obtain high virus titers , rendered a stock with a titer of 1.2  10  pfu / ml .
in addition to infecting different cell lines , different multiplicities of infection ( moi ) were tested , as shown in figure 4(b ) .
sf9 insect cells were infected and , after 36  h , the monolayer fraction was collected and analyzed by 16% sds - page . in this case
z - his protein was detected in the cellular fraction ( lane 1 and 2 , figure 4(c ) ) when the purified -tio - z - v5-his igg was used in a western blot . in this experiment
, two z corresponding bands appeared , same as observed previously by jcamo and coworkers when tacaribe virus recombinant z protein was expressed .
nevertheless , by using anti - his polyclonal serum for western blotting it was not possible to visualize his - z expression ( figure 4(d ) ) .
this suggests that possibly in insect cell lines his - z could be subjected to a post - translational process that modifies the amino end of his - z . in order to ascertain the location of this protein in the supernatant fraction , the same was analyzed by western blot and a proteinase k protection assay ( figure 5 )
the general aim of any heterologous expression system is to obtain a purified protein in its native conformation .
11 kda ) viral protein normally expressed in the late phases of the arenavirus infection in mammalian cells .
however , there are some applications for which it is not necessary to obtain the protein in the native conformation , for example for antibodies production .
also , it has been reported that post - translational modifications are few in many proteins ; therefore in these cases the bacterial expression systems are really advantageous because they have higher expression levels and lower costs when compared to eukaryotic expression systems .
thus , the system will be selected depending on the application of the recombinant product . using the sequence logo ( figure 1 ) the conservation of amino acids for the arenaviral z proteins
z proteins could be divided into three characteristic regions named amino , core and carboxyl regions .
each region comprises previously described z protein domains . the myristoylation domain into the amino region , the ring finger domain into the core , and the late domains into the carboxyl region .
although the role of these last domains is today unknown , they could be a likely target for studies associated with the arenavirus host range .  
simple methodologies that allowed z protein expression and facilitated its subsequent purification were employed . during the analysis of the z protein expression in bacteria it was not possible to obtain the product without a fusion protein or tags . probably , the reason for low z protein yield during purification is its hydrophobicity , causing it to form relatively insoluble intracellular inclusion bodies that must be denatured . the borden laboratory   discovered that including zn in the iptg - induction media or in the lysis buffers greatly improved yields of recombinant z protein .
although we included zn in the purification process , we have not yet tested a purification protocol that uses denaturing buffers .
several expression strategies were tested , including cell hosts with different biological properties ; it was only possible to achieve the goal when z 's amino terminus was fused to a bacterial protein .
this fusion stabilized the recombinant protein and improved the expression levels . in this work the over - expression of three different recombinant variants of z protein : ( 1 ) tio - z - v5-his , ( 2 ) gst - z , and ( 3 ) his - z , was achieved .
this last fusion protein was obtained from a baculoviral system in an insect cell line , while the other two were obtained from bacterial systems .
the three variants of z protein obtained can be cleaved from their n - fusion protein ; tio - z - v5-his after proteolysis keeps the v5 epitope and the his tag .
any of the expressed recombinant z proteins could be used to obtain polyclonal or monoclonal antibodies that would allow immunological experiments to answer questions about the molecular biology of junn virus .
for example , studies of biological activity in different cell lines , cellular localization and protein interactions
. moreover , this serum could be useful for affinity chromatography designed to allow simple purification of z protein without tags , or z protein obtained after proteolysis of its fusion partner .
tio - z - v5-his recombinant protein was selected for antibody production , mainly because it showed the best expression level and solubility .
purified igg fraction for subsequent western blotting and eia , was also obtained . on the other side ,
the purified tio - z - v5-his was used as substrate for the enk reaction , in order to obtain z - v5-his , employed as positive control in different assays .
tio - z - v5-his and his - z fusion proteins can be purified by imac , since both peptides have his - tags . however
, this purification method could be counterproductive for the purification of native z , since it is not known how co or ni ions could modify z structure by binding and affecting its ring finger domain .
it was observed that the native structure of z protein contains two zn atoms , and other transition metals could interfere with these sites . on the other hand ,
the gst - z fusion protein can be purified by affinity chromatography with glutathione resins , without using immobilized metals .
its expression and purification was optimized with the aim to obtain the viral protein without tags on its native conformation , ready for crystallographic studies .
the proteolysis of gst - z protein with factor xa , produced only two peptides : gst and z. the proteolysis reaction was successful because : ( 1 ) in sds - page analysis , at different times of the proteolysis reaction with factor xa , decreasing of gst - z and increasing of individual gst and z was observed ; and ( 2 ) by western blot analysis , the z protein identity after the proteolysis reaction was asserted ( figures 3(e ) and 3(f ) ) .
consequently , high concentrations of z protein ( necessary for crystallographic studies ) could be obtained .
in addition , after gst - z purification , the presence of two proteins that copurified with gst - z was observed ( indicated with i in the figure 3(g ) ) . a similar phenomenon has been previously reported .
the western blot , with igg purified from polyclonal anti - z serum recognized the gst - z protein and other copurified peptide ( ca .
29 kda ) , whiles the control , gst alone , did not generate signal . nevertheless , apparently this peptide is not a substrate of factor xa . in summary ,
the copurified peptides had high affinity for glutathione resin , so they probably contained gst derived amino acids , and one of them was recognized by the anti - z serum , so it had z derived amino acids . to confirm these facts
the n - terminus of the copurified peptides , and some peptides obtained by limited proteolysis with chymotrypsin were sequenced ( figure 3(g ) ) .
this assay allowed the identification of structured domains , which were less sensitive to proteolysis , and could be better candidates for crystallographic studies .
we found that one of the copurified peptides , the one of approximately 25 kda , was stable even at high chymotrypsin concentrations .
the n - terminus of this peptide shared the sequence with the first six amino acids of gst , indicating that degradation occurred by the carboxyl terminus of the recombinant protein .
the same results were obtained after analyzing the n - terminus of the approximately 29 kda copurified protein , which was also stable after the chymotrypsin treatment .
thus , it was possible that these low molecular weight peptides contained fragments of z protein at its c - terminus .
two of them were sequenced at the n - terminus because , according to the molecular weight estimated by the electrophoretical migration , the z protein sequence would be included ( indicated with ii and iii in the figure 3(g ) ) .
we could not establish the c - terminal sequence of the two copurified peptides , so we still do not know the specific site of cleavage of gst - z in order to determine a stable domain .
however , the approximate stable polypeptide corresponds to most of gst , which is not of our interest for crystallographic studies .
a subsequent approach to obtain z protein in a native conformation could take advantage of its interactions with host proteins , such as the promyelocytic leukemia protein ( pml ) and others ; it might be possible to overexpress z and its interaction partner fused to a suitable tag for affinity purification .
this would allow the copurification of the complex by affinity chromatography to later obtain native z. for lassa arenavirus it was demonstrated that expression of z in mammalian cell lines was sufficient for budding of  pseudo - virions  or z - containing membranous particles .
apparently , glycine myristoylation at position 2 of z sequence is critical for this phenomenon [ 4749 ] .
this glycine residue is completely conserved in z protein from all members of arenaviridae ( figure 1 ) .
this suggests that all z homologues within the arenaviridae family are myristoylated at this position .
currently , all reports of pseudo - virion budding employed mammalian cell lines . at the present it
is not known whether insect cell lines are capable of recognizing this post - translational modification signal present in z , so during this work we started testing this possibility . when insect cell lines were used for z expression , high levels of expression were not achieved ( figure 4(b ) ) . for this system ,
z was his - tagged so it could be purified by imac and then untagged by tev protease cleavage .
interestingly , z was detected in the cellular fraction by means of western blotting with -tio - z - v5-his iggs , but it was not possible to detect it using an anti -his tag antibody ( figures 4(c ) and 4(d ) , resp . ) .
the absence of his - tag signal could indicate the removal of the his - tag from the protein , by a yet unidentified cellular process , or possible protein degradation at this particular site .
besides protein western blot , a purification of the protein by imac was attempted , but no retention of protein from a cellular protein extract was observed ( data not shown ) .
interestingly , when the cell culture supernatant fraction was analyzed using a proteinase k protection assay , the results indicated that z was included within small lipid vesicles , although other confirmatory assays such as immuno electron microscopy will be required in order to unambiguously confirm this .
the next step will be to further investigate the z - membrane vesicles obtained from these cell lines .
if n - myristoylation of z in insect cells is proven to be effective , it will be necessary to modify the constructions for purification purposes .
the employment of insect cell lines would represent a much safer methodology to obtain virus like particles , which have potential use as vaccine against arenaviruses for which successful treatment has not yet been established .
 arenaviridae comprises 23 recognized virus species with a bipartite ssrna genome and an ambisense coding strategy . 
 the virions are enveloped and include nonequimolar amounts of each genomic rna species , designated l and s , coding for four orfs ( n , gpc , l , and z ) . 
 the arenavirus junn ( junv ) is the etiological agent of argentine hemorrhagic fever , an acute disease with high mortality rate . 
 it has been proposed that z is the functional counterpart of the matrix proteins found in other negative - stranded enveloped rna viruses . here 
 we report the optimized expression of a synthetic gene of z protein , using three expression systems ( two bacterial and a baculoviral one ) . 
 one of these recombinant proteins was used to generate antibodies . 
 a bioinformatic analysis was made where z was subdivided into three domains . 
 the data presented contributes methodologies for z recombinant production and provides the basis for the development of new experiments to test its function . 
1. Introduction
2. Materials and Methods
3. Results
4. Conclusions