arenaviridae , bunyaviridae , and orthomyxoviridae , the principal families of segmented negative single - stranded rna viruses ( snsv ) , each include serious human pathogens such as lassa fever , crimean - congo haemorrhagic fever , and influenza viruses , respectively . orthomyxoviruses have six to eight genome segments , whereas bunyaviridae ( reviewed in elliott , 2014 ) have  three , and arenaviridae two . for each segment , transcription , generating capped viral mrnas , and replication , generating full - length genome or antigenome copies ( vrna and crna , respectively ) , are performed by the same virally encoded rna - dependent rna polymerase ( rdrp ) . for arena- and bunyaviruses , which replicate in the cytoplasm , the rdrp is the single - chain l protein , whereas for orthomyxoviruses , which replicate in the nucleus , it is a hetero - trimeric complex , formed by the pa , pb1 , and pb2 subunits ( fodor , 2013 ; guu et  al . , 2012 ; vrna genome segments are always packaged by multiple copies of the viral nucleoprotein ( np ) together with one copy of the rdrp into filamentous ribonucleoprotein particles ( rnps ) , which are the functional replication and transcription units ( reguera et  al . , 2014 ) . mechanism , whereby short 5 capped rna fragments are cleaved from host cell mrna by an endonuclease intrinsic to the rdrp and then used to prime synthesis of viral mrnas ( morin et  al . , 2010 ; , 1981 ; reguera et  al . , 2010 ; reich et  al . , second , they recognize each genome segment via their highly conserved , quasi - complementary 3 and 5 extremities ( over a length of 1319 nucleotides ) , known as the promoter ( barr and wertz , 2004 ) . correlated with this , snsv rnps are  generally circularized , which is thought to occur by base pairing between the genome ends ( forming a double - stranded  panhandle  ) and/or the simultaneous binding of both ends to the polymerase ( reguera et  al . , 2014 ) . vrna promoter binding to influenza polymerase was visualized recently for the first time in a co - crystal structure . this revealed that each vrna extremity binds sequence specifically as a single strand to distinct sites on the polymerase but then come  together to form a short duplex of about four base pairs  ( pflug et  al . furthermore , this mode of promoter binding is required for activation of diverse influenza polymerase functions ( fodor , 2013 ) . for bunyaviruses , exact self - complementarity of the genome ends extends for 1519 nts ( except for only  one g - u mismatch in the case of orthobunyaviruses [ barr  and wertz , 2004 ; kohl et  al . , 2004 ] ) , potentially allowing formation of a much more stable panhandle than for influenza vrna . however , the exact nature of the vrna - vrna and vrna - l interactions that circularize bunyavirus rnps are not known . cross linking suggests that the vrna ends within bunyavirus rnps are base paired  at least partially ( raju and kolakofsky , 1989 ) and a distal duplex region is essential for rna synthesis by bunyavirus ( barr  and wertz , 2004 ; kohl et  al . , 2004 ) and arenavirus ( kranzusch et  al . , 2010 ) polymerases . the absence of significant sequence similarity , outside of the cap - snatching endonuclease ( reguera et  al . , 2010 ) and the conserved rdrp motifs ( mller et  al . , 1994 ) , between arenaviridae and bunyaviridae l proteins and orthomyxoviridae heteotrimeric polymerases also poses the question as to whether all snsv have a structurally and evolutionary conserved architecture to match their functional similarity . to answer this question and those related to promoter binding , we determined the crystal structure of 77% of the l protein from la crosse orthobunyavirus ( lacv ) in complex with just the 3 or both 3 and 5 conserved genomic rna ends and the cryo - em structure of the apo - form . lacv is a potentially serious but rare , mosquito - transmitted human pathogen that causes 50100 cases of encephalitis per year in the usa ( http://www.cdc.gov/lac/ ) ( elliott , 2014 ; haddow and odoi , 2009 ) . the structure reveals high similarity , but also interesting differences , to the equivalent influenza complex ( pflug et  al . , 2014 ; reich et  al . , in particular , it shows the structural basis of the specific recognition of the vrna 3 end , the allosteric regulation mediated by vrna 5 end binding , and the likely path of the template into the polymerase active site cavity and out again . these findings , combined with those gained from the influenza polymerase structures , provide new insight into the common mechanism of action , the conserved features , and the diversity among snsv polymerases . l1750 , a construct comprising residues 11750 ( out of 2263 ) of  the lacv l protein , was expressed in insect cells and purified to homogeneity in milligram amounts ( figure  s1a , figures s2a and s2b ) . in  vitro rna - protein interaction experiments show that the separated single stranded lacv genomic extremities each bind with high affinity and specificity to the polymerase with dissociation constants of 13.8  2.6  nm and 9.3  1.6  nm for the 5 and the 3 ends , respectively , whereas the polymerase only has low affinity ( 1.5  m ) for the pre - annealed double stranded panhandle ( figure  s3a ) . however , as shown by mobility shift assays , the polymerase still binds with high affinity to partially double stranded 3 vrna provided the first eight nucleotides from the 3 end are single stranded ( figure  s3b ) . similar conclusions were previously found for machupo arenavirus polymerase ( kranzusch et  al . , 2010 ) . using these results , co - crystals of l1750 diffracting up to 2.6   resolution were obtained with nucleotides 116 of the genomic 3 vrna ( 3oh - ucaucacaugaugguu ) , to which was annealed a complementary 8-mer ( 5oh - gcuaccaa ) , corresponding to nucleotides 916 from the 5 vrna extremity . the structure was solved by multiple isomorphous replacement with anomalous scattering ( figures s1b  s1d ) . soaking the first 10 nucleotides of the 5 vrna ( 5p - aguagugugc ) into the crystals gave a new structure at 3.0   resolution that revealed the 5 end in a distinct binding site . crystallographic statistics are given in table s1 and a sequence alignment of representative orthobunyavirus polymerases , annotated with the secondary structure , is shown in data s1 . the l1750 model contains 1652 residues ( 94.4% complete ) with several connecting loops missing , some of which become ordered upon 5 vrna binding ( figure  1 , data s1 ) . the structure of apo - l1750 was determined from cryo - em images by single particle 3d reconstruction at 8.3   resolution and allows visualization of secondary structure elements in most of the protein ( figure  2 , figure  s2 ) . the rna - bound and apo - lacv l1750 structures display the same overall shape with a large globular central core and a flexible protrusion ( figures 1 and 2 ) . the overall structural organization is strikingly similar to that of the influenza polymerase ( pflug et  al . , 2014 ) despite the complete lack of extended sequence homology ( figures 1a and 1c , figure  s4 ) . in fact  l1750 corresponds precisely to pa , pb1 , and pb2-n ( residues 1250 of pb2 ) , confirming the linear , head - to - tail mapping of the influenza heterotrimeric polymerase onto the orthobunyavirus l protein , as previously proposed ( reguera et  al . , 2010 ) . the central pb1-like rdrp region of l1750 ( residues 7581433 ) contains the canonical fingers , fingertips , palm , and thumb domains with the conserved polymerase motifs exposed into the internal rna synthesis chamber . it is buttressed on one side by the pa - c like region , which also has distinct pockets for the 3 and 5 vrna extremities , and on the other by the pb2-n like region . the previously described n - terminal endonuclease domain ( residues 1184 ) ( reguera et  al . , 2010 ) is solvent exposed and differently orientated compared to influenza polymerase ( figure  1c , figure  s4a ) . however , it is clearly flexibly linked to the central polymerase core , as revealed by its lack of density in one 3d class of the em map ( figure  2b ) . an extended linker ( residues 185270 ) , analogous to the influenza pa - linker , that packs on and stabilizes the fingers and palm domains of the rdrp ( figure  1b , figures s4a and s4b ) , connects the endonuclease to the pa - c like domain ( residues 271759 ) , which is divided into two lobes ( figures 1a and 1b ) . the larger  core - lobe  is -helical and buttresses the thumb and palm domains of the rdrp . the second lobe is mainly involved in vrna promoter interactions and is therefore called the vrna binding lobe ( vrbl ) . it has a central  sheet with , on one side , a structure denoted the  clamp  ( residues 355400 ) that binds the 3 of the vrna ( see below ) and which has no equivalent in influenza pa . on the opposite side is a long loop ( residues 420440 ) , analogous to the influenza pa - arch , which binds the vrna 5 end ( figure  1b , figure  s4 ) . 3d classification of the apo l1750 cryo - em data allows generation of em maps corresponding to two states of the vrbl . in the first , the same conformation is observed as in the crystal structure , but the arch is invisible , whereas in the second state , the most of the vrbl is invisible , suggesting its enhanced flexibility in the absence of bound vrna ( figures 2c and 2d ) . -ribbon  ( residues 847905 ) , structurally equivalent to the influenza pb1 -ribbon ( but emerging from a different fingers domain loop , figure  s4b ) and the  fingernode  ( residues 11051135 ) , functionally equivalent to the pb1 -hairpin . the fingernode folds into two  helices ( 4344 ) linked by a flexible loop , and together with the vrbl arch , it plays a central role in sequence specific binding of the vrna 5 end ( see below ) . the palm domain appears unusually elongated , partly due to the insertion of a solvent exposed helical hairpin of unknown function that is specific for the california orthobunyavirus serogroup including lacv ( figure  1b , data s1 ) . the thumb domain is surrounded by the pa - c like domain core lobe and a set of helices ( 5862 and 67 ) and strands ( 3134 ) , denoted the thumb ring which is structurally homologous to the influenza pb2 n1 and n2 domains . a loop ( residues 1402 to 1422 ) at the c - terminal end of the thumb is likely deployed into the polymerase cavity but lacks electron density map , indicating mobility . the thumb domain is followed by a helical bundle called the bridge ( residues 14331503 ) , which replaces the helical pb1-pb2 interface and closes the circular architecture of the polymerase around its internal cavity . the highly conserved connection between the bridge and the thumb ring ( residues 14981506 ) partly defines the template entry channel ( see below ) . inserted in the thumb ring is the lid ( residues 16141703 ) which as in influenza pb2 , borders the exit channel . nucleotides 18 from the vrna 3 end are bound in an extended , single - stranded configuration in a narrow cleft over which the clamp closes ( figure  3a ) . diverse regions of the polymerase contribute 3 end rna binding loops , including both lobes of the pa - c like domain , the thumb ( residues 13071315 ) and the  thumb ring domain ( residues 15131517 ) . the protein - rna interface buries a total of 3460   of surface area and includes > 30 protein - rna hydrogen bonds , indicating a high degree of sequence specificity ( figure  s5a , table s2 ) . nucleotides u1 , c2 , and a3 are orientated into individual pockets by an extensive hydrogen bond network with residues from helices 14 and 21 and two loops from the vrbl ( residues 312316 and 535539 ) and another loop from the core lobe ( residues 469473 ) ( figure  3b ) . the very 3 end is completely sequestered by the stacking of his312 onto the u1 ribose and hydrogen bonds from the 2 and 3 oh to pro314 and asn538 ( figure  3b ) . nucleotides u4 , c5 , and a6 are stacked on each other with their bases facing the protein and their phosphates interacting with his1515 from the thumb ring and arg372 from the clamp ( figures 3b and 3c ) . nucleotides c7 and a8 are again orientated into separate pockets mainly formed by clamp residues , with c7 stacking between conserved trp395 and tyr524 as well as making base specific hydrogen bonds to residues gln398 and arg531 . a8 stacks on ile 378 and its n6 makes a base - specific hydrogen bond with the backbone of lys381 ( figure  3c ) . these structural observations are consistent with rna binding experiments with all possible single substitutions in 3 end nucleotides 111 , which show that nucleotides 68 are the most critical for sequence specific binding ( figure  s3c ) . 3 end nucleotides 9-ugaugguu-16 form a duplex with the co - crystallized complementary oligonucleotide 5oh - gcuaccaa ( figure  3a ) and the 3 end backbone is neutralised by basic residues lys859 , lys862 , arg869 , and lys870 from helix 33 of the -ribbon ( figure  s5a ) . the 10 first nucleotides of the 5 vrna , soaked into the crystal , binds as a stem - loop to the polymerase in a similar configuration and location to the ten nucleotide 5 hook of influenza vrna promoter ( pflug et  al . , 2014 ) ( figure  4 , figure  s6a ) . however , lacv 5 hook is less compact with only two base pairs between g2-c10 and u3-g9 , compared to four in influenza . bases g5 and a4 are consecutively stacked on u3 , whereas u6 , g7 , and u8 are splayed out in the loop region , compared with only one in influenza . the 5 vrna interaction with the polymerase buries a total of 3030  of surface area and includes 37 protein - rna  hydrogen bonds ( figure  s5b , table s2 ) . upon rna binding both the arch and the fingernode are structurally reconfigured to promote protein - rna interactions ( figures s6b and s6c ) . an array of conserved , mainly positively charged , residues stabilizes the 5 vrna backbone phosphates ( a1-lys423 , g2-lys302/arg592 , u3-his306 , a4-arg600/thr642 , g5-lys643/tyr677 , u6-arg292 , g7-lys768 , u8-his760/his761 ) ( figure  s5b ) . nucleotide a1 stacks onto the planar backbone of cys419 and gly420 and consecutive base stacking of nucleotides 15 is interrupted by conserved arch residue pro440 , which stacks on base g5 forcing base u6 to flip out ( figures 4a and 4b ) . highly specific , induced fit interactions are made from residues 11161123 of the fingernode loop to flipped out bases g7 and u8 . the loop structurally reconfigures to allow g7 to stack on tyr1120 and make base specific interactions with gln1116 , asp1123 , and lys768 ( 30 ) . u8 stacks on gln1116 and makes three base specific main - chain interactions with the peptide 11181120 ( figure  4c , figure  s6c ) . adjacent , conserved his760 and his761 on 30 , further stabilize the rna loop conformation by binding the u8 phosphate and by stacking onto the g9 and g5 ribose moieties , respectively ( figures 4b and 4c ) . biochemical evidence for the involvement of the clamp and arch in , respectively , 3 and 5 end binding comes from proteolysis experiments that show clamp residue lys368 and arch residue lys430 are protected from trypsination by 3 and 5 end binding , respectively ( figures 3a and 4a , figure  s3d ) . however , 5 vrna binding not only induces conformational changes in rna binding loops but also structures elements of the polymerase active site ( figure  4d ) . most significant is the complete ordering of the fingertips loop ( residues 950958 ) , which contains motif f. the interactions of his760 and his761 pull helix 30 toward the 5 vrna , and the consequent displacements of val762 and leu766 make room for the fingertips to order into a structured active form through multiple contacts with residues from the pa - c like domain 29 , fingers domain 30 and the linker joining them ( figure  4d ) . conserved his757 plays a key role in stabilizing the fingertips -turn through multiple hydrogen bonds to backbone carbonyl groups ( figure  4d ) . fingertips ordering has a knock - on effect on stabilization of motif b notably through the interaction of conserved motif b gln1145 with arg958 and glu959 ( figure  4d ) . this is the first observation of functionally important allosteric effects associated with 5 vrna binding and is only observed when 1011 5 end nucleotides are bound , but not eight ( data not shown ) . interestingly , soaking in 5 crna nucleotides 110 ( which differ from vrna only in the substitution g9 to a ) shows that the wobble base pair becomes canonical u3-a9 with no discernible difference on the induced polymerase rearrangements compared to vrna ( data not shown ) . this is consistent with only the identity of 3 position 9 leading to a significant difference in propensity for transcription between vrna and crna ( barr and wertz , 2005 ) . in the 5 vrna end 111 soaked structure , nucleotide u11 is only partially ordered and can not base pair with a1 without displacement of conserved arg595 which hydrogen bonds to n1 of a1 ( figure  s6d ) . furthermore soaking 5 rnas longer than 11 nucleotides resulted in no binding in the crystal , suggesting that further rearrangements of the polymerase ( notably the -ribbon ) and probably a sharp turn in the rna ( as observed in influenza 5 end between nucleotides 1011 ) may be required to allow binding of the complete 5 end , but which are incompatible with the crystal packing ( figure  s6d ) . the l protein internal active site chamber , where nucleotide addition occurs , is formed by the conserved polymerase motifs a - f and configured like other rna polymerases , notably influenza polymerase(pflug et  al . , 2014 ; te velthuis , 2014 ) motif f forms part of the fingertips , the flexible loop between fingers strands 20 and 21 that is only fully ordered when the 5 vrna is bound ( see above ) . the other conserved polymerase motifs are all in the palm domain : motif a ( 1060-dmskws ) between palm 22 and 41 with divalent cation binding d1060 ; motif b ( 1145-qgnfnytssy ) between 24 and the long 45 with conserved n1149 ; the catalytic motif c ( 1186-sdd ) in the turn between strands 25 and 26 ; motif d ( 1223-qanmkkty ) just before 27 and motif e ( 1236-kefvslfn ) forming the tight loop between 28 and 29 . interestingly , structural alignment with influenza polymerase allows identification of two new active site motifs , denoted g and h , which appear to be conserved specifically in snsv polymerases ( figure  5a , figures s7a and s7b ) . motif g ( 653-rymi in lacv , 658-rkll in influenza pa ) is in helix 24 in the core - lobe of the pa - c like domain ( figure  s7a ) , the conserved arginine being positioned to interact with the priming ntp ( figure  5a ) . motif h ( 1101-kelil in lacv and 347-kvarl in influenza pb1 ) forms a  strand ( 23 in lacv ) with the conserved lysine stabilizing the motif b backbone conformation by hydrogen bonding to multiple carbonyl - oxygens ( figure  s7b ) . four positively charged solvent accessible tunnels , visualized using mole 2.0 ( sehnal et  al . , 2013 ) converge into a central inner cavity where the rdrp motifs mediate template directed rna synthesis ( figure  5b , figure  s7c ) . the tunnels are delimited by residues conserved among orthobunyavirus polymerases ( figure  s7d ) . the configuration of the template entry channel , the ntp entry channel and the nascent strand exit channel is similar to that described for influenza polymerase ( figure  5c ) ( pflug et  al . , 2014 ) . the template channel entrance is defined by the vrbl , fingers and bridge and is partially obscured by the -ribbon ( figure  5d ) , which together with several loops of the vrbl  sheet that are deployed toward the entrance but disordered in the structure , may modulate access . the ntp entry channel is lined by conserved basic residues r287 , k673 ( pa - c like ) , k956 , r958 ( fingertips ) , k1063 ( motif a ) , k1227 , and k1228 ( motif d ) , some of which are only positioned correctly upon 5 vrna binding ( figure  s7e ) . the product strand exit tunnel is surrounded by the lid domain and the thumb ring mainly by the extended joining linkers and by fingers and palm opposite side of the ntp entry channel ( figure  s7f ) . in a lacv l there is a more obvious extra channel that we postulate is for the template to exit . in influenza polymerase the equivalent channel is present , but narrowed by the presence of the putative priming loop ( figure  5c ) , which is 15 residues longer than in lacv l. the putative template exit channel is defined by the thumb , thumb ring , bridge , and lid domains and lined by conserved basic residues r1430 , k1492 , r1493 , k1686 , and r1690 ( figure  5e ) . as discussed below , the arrangement of the tunnels in lacv l protein suggests an elegant strategy for rna synthesis whereby the polymerase forces separation of the template and product strands and directs each down distinct exit channels on opposite sides of the molecule . in the l1750-vrna complex , the 3 end of the template is tightly and specifically bound but not accessing the entry tunnel , corresponding perhaps to the highly stable state that occurs in inactive rnps , for example within virions . for active rna synthesis , the 3 end clearly has to be relocated into the polymerase active site ( figures 6a and 6b ) . furthermore it has been shown that complementarity and presumably base pairing between at least 3 and 5 nucleotides 12 to 16 are required for rna synthesis by orthobunyavirus polymerases ( barr and wertz , 2004 ; kohl et  al . , 2004 ) . a pre - initiation configuration , with the 3 end specifically bound on the outside but not entering the polymerase active site , was also visualized for influenza polymerase ( pflug et  al . , 2014 ; reich et  al . , however , whereas in the influenza structures , both 3 and 5 extremities of the promoter simultaneously bind their separate single - stranded binding sites and form a distal duplex region ( with 1114 of 5 base pairing with 1013 of the 3 end ) , for lacv polymerase in the  crystallized conformation , this appears to be impossible . first , nucleotides 11 of the 3 and 5 ends are around 25   apart , too far to see how 3 and 5 nucleotides 12 to 16 could base  pair ( figure  6a ) . second , in the all l1750 structures , bases 916 of the  3 end are already base paired with the co - crystallized crna , corresponding in sequence to 5 nucleotides 916 . thus when  5 nucleotides 111 are soaked into the crystal , nucleotides 911 are present in two distinct locations greater than 20   apart , which obviously can not happen physiologically ( figure  6a ) . given the similar mode of 5 end binding to both lacv and influenza polymerases ( and that the 5 end remains bound as observed during initiation ; reich et  al . , 2014 ) and the similar requirement of a short distal 35 duplex , we propose that an alternative configuration of the bound promoter likely exists more analogous to the influenza pre - initiation conformation ( figure  6b ) . release and repositioning of the 3 end could occur  by swinging of the clamp , without necessarily letting go  of the 3 end rna , with a concomitant reorientation of the -ribbon into a position analogous to the -ribbon in influenza polymerase ( reich et  al . , 2014 ) , to stabilize duplex formation ( figure  6b , figure  s6d ) . the flexibility of the vrbl and -ribbon as seen by 3d classification of the em images , together with the lower local resolution of these regions in the cryo - em map , shows that such movements are plausible ( figure  2 , figure  s2f ) . in the case of influenza virus , extrapolation of the template from the observed duplex region , based on the poliovirus polymerase elongation complex model ( gong and peersen , 2010 ) , would result in the 3 end overshooting the polymerase active site by three nucleotides as previously discussed ( reich et  al . , 2014 ) . assuming that for lacv the duplex is from nucleotides 1215 of both strands ( barr and wertz , 2004 ; kohl et  al . , 2004 ) , based on the influenza / polio models , the template would overshoot the polymerase active site by 5 or 4 nucleotides , depending on whether the lacv 35 1212 base pair corresponds to influenza 3-5 1011 or 1112 base pair , respectively ( figure  6b ) . in the case of cap - dependent transcription , this overhang could favor base pairing with the incoming capped primer . in the case of replication , this situation could be explained by ( 1 ) a different , less direct path of the single stranded template , so that the 3 nucleotide 1 was placed directly in the polymerase active site ( i.e. , the modeling is misleading ) ; ( 2 ) a mechanism of internal initiation followed by realignment , dependent on the triplet repeat at the beginning of the template ( 3-ucauca ) , as has been described for some bunya- and arena - viruses ( guu et  al . , 2012 ) ( i.e. , internal initiation at position 4 followed by realignment of the agu triplet ) ; or ( 3 ) initiation starting at nucleotide 1 but duplex formation between 3 and 5 nucleotides 12 to 16 only occurring after 45 nucleotides have been synthesized and the template has translocated further into the active site cavity . this latter possibility would be consistent with the observed position of lacv 3 end nucleotide 8 being close to that of the nucleotide 8 counting from the active site along the polio template ( figure  6b ) . whatever mode of initiation , during elongation , a duplex formed by template and nascent strands starts to grow in the active site cavity as visualized in the structure of the poliovirus polymerase elongation complex ( gong and peersen , 2010 ) ( pdb : 3ol7 ) ( figure  6c ) . however , after a complete double - helical turn , the nascent and emerging template strands would appear to clash with the thumb and c - terminal lid domains , respectively , as described for the influenza case ( reich et  al . , 2014 ) . to resolve this situation , we propose that the strands are forced to separate and each is extruded along a separate exit tunnel ( figure  6c ) . the nascent strand would exit to the rear of the polymerase and , by analogy to the influenza case , the cap - snatched transcription primer would probably enter by the same route ( reich et  al . , the template would exit to the front of polymerase through the template exit channel on the same side and not far from the template entrance ( figure  6c ) . the proximity of the entry and exit channels would allow reintegration of the vrna genome template into the rnp with minimal disruption ( figure  7 ) . modeling shows that the template path through the entry channel , cavity , and the exit channel would accommodate around 20 nucleotides . given that 11 nucleotides of the 5 and 9 of the 3 ends directly bind the polymerase in the pre - initiation state , and also a single lacv np can accommodate 11 nucleotides ( reguera et  al . , 2013 ) , no free rna needs to be exposed , nor nps removed or added , during template reading . the mobile clamp together with the -ribbon and proximal nps our structures of the lacv l protein in the apo - state and with one or both vrna ends bound suggest that assembly of a functional initiation complex is a multistep process . the structures clearly show that the polymerase has highly specific and distinct sites for the single - stranded 3 and 5 vrna ends , preventing them from forming an extended panhandle . the mode of 5 end hook binding is similar to that observed for influenza polymerase , but here we directly observe the associated allosteric effects that are essential for structuring critical active site loops . on the other hand the 3 end appears to be preferentially and tightly bound in a groove closed by a clamp on the side  of the polymerase . elucidation of the exact purpose of this binding site and the mechanism for 3 end relocation into the template tunnel for the initiation of rna synthesis are clear  questions for future studies . furthermore , the extended complementarity of the lacv 3 and 5 ends appears to be a major obstacle to reconstitute the lacv initiation complex in  vitro , since incubation , even sequentially , of the complete 3  and 5 ends leads to the preferred formation of a long , stable duplex , which has low affinity for the polymerase . probably for the same reason , we have not yet been able to demonstrate robust template directed rna synthesis activity for either l1750 or full - length polymerase ( see supplemental experimental procedures ) . it is therefore likely that in the case of bunya- , and probably , arenavirus polymerases ( kranzusch et  al . , 2010 ) , to avoid stable base pairing of the highly complementary vrna promoter , the free ends are prevented from ever meeting each other by the sequential mode of assembly of nascent rnps starting at one end of a growing replicate ( figure  7 ) . thus it is plausible that , unlike the situation for recombinant influenza polymerase , which is fully active when reconstituted only with the vrna promoter ( reich et  al . , 2014 ) , np - l and np - rna interactions may be required for bunyavirus polymerase activity . the striking structural similarities between the single - chain lacv and heterotrimeric influenza polymerases strongly support the idea of an evolutionary common ancestor . indeed , it now seems plausible that all snsv polymerases ( i.e. , from arena- , bunya- , and orthomyxovirus families ) have a similar architecture , despite very low overall sequence homology , and this is supported by structure - based identification of new common motifs ( figures s7a and s7b ) . however , this does not mean that these polymerases will not have idiosyncratic family and sub - family differences . for example , whereas the arch and fingernode that bind the 5 hook have structural and functional homologs in influenza , the lacv 3 end binding site and the clamp structure that pins it in place has no such equivalent . similarly , it is reasonable to suppose that each polymerase is adapted to its cognate nucleoprotein , whose size , structure , mode of rna binding , and number of nucleotides bound ( e.g. , 11 for orthobunyaviruses , 7 for phleboviruses ) , are very different for each snsv family ( reguera et  al . , 2014 ) . in this context , it is intriguing that lacv and influenza contain , respectively , an - or -ribbon , equivalently located extended and flexibly hinged structures that could both play a role in both rna and np interactions and could be adapted to the respective np structures ( figure  s6d ) ( reich et  al . , 2014 ) . finally , it is highly significant that the l1750 construct ends precisely at the same position as separates pb2-n and pb2-c in influenza polymerase ( figure  1 ) . pb2-c , which includes the cap - binding domain and the c - terminal nuclear localization motif ( not relevant for cytoplasmic lacv ) , has already been shown to be loosely associated with the rest of influenza polymerase ( reich et  al . , 2014 ) . l , missing in l1750 , are structurally homologous to pb2-c . in particular it is still unknown whether there is a cap - binding domain , for which there is no direct evidence yet , but , in the case of lassa arenavirus a specific requirement for c - terminal residues for mrna transcription has been established ( lehmann et  al . , 2014 ) . based on the l1750 structure , we propose a model for rna replication in which there are clearly separated exit tunnels for the single - stranded template and product ( figure  6c , figure  7 ) . the proximity of the template entrance and exit , on one side of the polymerase , is compatible with processive template reading with minimal disruption of the rnp . template rna would progressively dissociate from proximal np as it translocates through the entrance tunnel , into the polymerase internal chamber and out again , to be reincorporated into the rna free np that concomitantly translocates round the outside of the polymerase between the entrance and exit tunnels , held together by flexible np - np interactions ( reguera et  al . , 2013 ) meanwhile , the products exit to the other side of the polymerase , thus allowing spatial separation of template translocation and product processing . in the case of ( anti)-genome replicates , product processing involves assembly into progeny rnps , possibly first with an incoming apo - polymerase binding specifically the emerging , nascent 5 end and subsequently progressive packaging by incoming nps ( figure  7 ) . we propose that robust polymerase dimerization is necessary to ensure efficient and correct circularisation of progeny rnp ( figure  7 ) , and this is consistent with some observations concerning replication by influenza polymerase ( jorba et  al . , 2008 ; york et  al . , 2013 ) , but other influenza replication models involve more complicated higher order polymerization ( chang et  al . , 2015 ; jorba et  al . , 2009 ) . in the case of transcription , as suggested by the structure of influenza polymerase , the cap - snatched primer would enter the internal cavity via the product exit channel and then , upon elongation , extrude out in the same direction , where it likely interacts with host translation factors ( in the case of bunyaviruses , transcription is closely coupled to translation ; barr , 2007 ) . as suggested for influenza polymerase ( reich et  al . , 2014 ) , the main role of the pb2-c like region may be in these product processing mechanisms . we think these concepts are likely to be applicable to all snsvs polymerase and possibly those of nsnsv as well , which also operate in an rnp context . residues 11750 ( l1750 ) of the polymerase ( l protein ) sequence of la crosse virus ( lacv ) were expressed in insect cells from a synthetic gene inserted in a pfastbac vector . purified protein at 5  mg / ml was crystallized with an equimolar mixture of nucleotides 116 from the 3 and nucleotides 916 from the 5 vrna ends ( dharmacon ) . diffraction data were collected on beamlines id23 - 1 or id29 at the european synchrotron radiation facility and integrated with xds ( kabsch , 2010 ) . the structure was solved by the multiple isomorphous replacement with anomalous signal method using selenium , platinum , and tantalum cluster derivatives with sharp ( bricogne et  al . , 2003 ) . lacv endonuclease ( pdb : 2xi5 ) and influenza polymerase pa and pb1 subunits ( pdb : 4wsb ) were used as a guide to model building . selenomethionine positions from the anomalous difference map helped align the sequence and autobuilding with buccanneer ( cowtan , 2006 ) was useful to extend the model , which was refined with refmac ( murshudov et  al . , 1997 ) and phenix ( afonine et  al . , 2012 ) . 5 end nucleotides 110 or 111 were soaked into pre - grown crystals to reveal the 5 end binding site . cryo - em grids were prepared by applying 4  l of l1750 at 0.2  mg / ml to a quantifoil grid , blotting excess solution and then freezing in liquid ethane . cryo - em images of apo - l1750 were collected on a krios microscope at 80 kv with a falcon ii direct detector ( fei ) at magnification 138,129 times . fei epu automatisation software was used to collect 6,129 micrographs with a defocus between 0.5 and 2  m , an exposure time of 0.5  s and a dose of 14e /  . after contrast transfer function correction , 10,102 manually picked particles were used to derive initial class averages with imagic ( van heel et  al . , 1996 ) . these were then used to select 180,000 particles for input to 3d reconstruction and  refinement with relion 1.3 ( scheres , 2012 ) , using as initial model the l1750-vrna crystal structure filtered at 30   resolution , leading to a map at 8.3   resolution . the dataset was subsequently partitioned by 3d classification resulting into three structures which revealed the less well - defined , flexible regions . for electrophoretic mobility shift assays , radioactively labeled rnas were produced by in  vitro transcription with t7 polymerase . for binding assays , 10  m of l1750 in 10 l buffer was mixed with radiolabelled rna and 1  l of non - specific poly(u ) rna ( sigma ) . mixtures were incubated at room temperature for several hours and resolved on native tg gels . radioactive signal from shifted bands was recorded with a typhoon and quantified with imagequant . for fluorescence anisotropy measurements , 5  nm 25-nucleotide long rna oligos corresponding to 3 or 5 vrna ( iba ) , labeled with fluorescein on the appropriate non - interacting end , were titrated with l1750 in order to obtain 1015 protein concentration points ranging from 3  nm to 1  m . fluorescence and fluorescence anisotropy measurements used 495  nm excitation and 515  nm emission wavelengths . kaleidagraph ( synergy software ) was used to evaluate the data and derive dissociation constants . for proteolysis protection experiments , l1750 was incubated at 1  mg / ml with 1:1 molar ratios of 25-nucleotide long 3 or 5 genomic ends and then digested for 1  hr at room temperature with trypsin ( 1:1,000 w / w ) . products of digestion were analyzed by various techniques including : sds - page , western - blot , esi - tof - ms , maldi - tof - ms , maldi - tof - ms with n - terminal acetylation , and n - terminal sequencing of protein fragments by edman degradation . for more details did protein production , biochemistry , rna binding studies , and x - ray data collection and structural analysis . co - directed the project and wrote the paper with input from p.g . and h.m .
summarysegmented negative - strand rna virus ( snsv ) polymerases transcribe and replicate the viral rna ( vrna ) within a ribonucleoprotein particle ( rnp ) . 
 we present cryo - em and x - ray structures of , respectively , apo- and vrna bound la crosse orthobunyavirus ( lacv ) polymerase that give atomic - resolution insight into how such rnps perform rna synthesis . 
 the complementary 3 and 5 vrna extremities are sequence specifically bound in separate sites on the polymerase . 
 the 5 end binds as a stem - loop , allosterically structuring functionally important polymerase active site loops . 
 identification of distinct template and product exit tunnels allows proposal of a detailed model for template - directed replication with minimal disruption to the circularised rnp . 
 the  similar overall architecture and vrna binding of monomeric lacv to heterotrimeric influenza polymerase , despite high sequence divergence , suggests that all snsv polymerases have a common evolutionary origin and mechanism of rna synthesis . 
 these results will aid development of replication inhibitors of diverse , serious human pathogenic viruses .