Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus
[['Double-stranded ( ds ) RNA viruses are a diverse group of viruses that vary widely in host range ( humans , animals , plants , fungi , and bacteria ) , genome segment number ( one to twelve ) , and in the number of capsid layers , with many of them considered important pathogens of either agriculture or human health .', 'A common feature of dsRNA viruses is that their capsid associated polymerase performs both of its functions , namely replicating as well as transcribing the viral genome , from within the confines of the virus capsid .', 'This sequestration of the polymerase and the dsRNA genome prevents the activation of the host’s RNA induced antiviral response [1] .', 'During the viral replication cycle , dsRNA viruses have been shown to encapsidate up to twelve RNA-dependent RNA polymerase ( RdRP ) molecules in each virus particle [2–5] To date , several different mechanisms of incorporating the RdRP molecules into the capsid have been identified .', 'Those that possess multi-layered capsids , such as the bacteriophage ϕ6 , rotavirus , and reovirus , as well as the single-layered capsids of cypoviruses have been shown to attach their polymerase molecules to the inner surface of the capsid through direct protein-protein interactions [6–10] , suggesting that non-covalent protein-protein interaction plays an important role in RdRP incorporation .', 'A number of single-shelled dsRNA viruses , such as the yeast L-A virus , express their polymerase as a capsid protein ( CP ) -RdRP ( gag-pol ) fusion protein , which is then incorporated into viral particles as a minor structural component during capsid assembly [11 , 12] .', 'With few exceptions , most notably the polymerase of bacteriophage ϕ6 [13] , polymerases from dsRNA viruses are not fully active when their respective capsid proteins are not present [14–16] .', 'It has been proposed that the dependence of polymerase activity on the presence of capsid proteins may help to ensure that dsRNA products are preferentially produced only within a capsid enclosure [17] .', 'The crystal structures of the RdRPs from several dsRNA viruses ( i . e . ϕ6 , reovirus , rotavirus , and birnavirus ) have been determined , and all have been found to contain a core polymerase domain with a right-hand shape [18–21] .', 'In reovirus and rotavirus polymerases , which catalyze conservative RNA transcription , possess elaborate N- and C-terminal domains that interact with the core polymerase domain , thus creating a cage-like structure with four channels leading in and out of the active site at the center of the molecule [22] .', 'These polymerases also possess an mRNA cap binding site that may facilitate the initiation of viral RNA transcription [19 , 21] .', 'In contrast , the ϕ6 and birnavirus polymerases , which produce RNA transcripts in a semi-conservative manner , are relatively smaller in size with a structure containing only three active site channels .', 'Distinct structural features have been identified in the RdRPs of ϕ6 , reovirus and rotavirus that function as a structural platform for the binding of a single priming nucleotide to allow for de novo initiation of RNA synthesis [18 , 19 , 21 , 23 , 24] .', 'Picobirnaviruses ( PBV ) are small , non-enveloped dsRNA viruses infecting a wide range of mammalian and avian species [25–29] .', 'Human PBV ( hPBV ) has been identified on almost every continent , and has been associated with acute gastroenteritis primarily in children and people that are immunocompromised [30 , 31] .', 'It has a bi-segmented genome with the genome segment two ( PBV2 ) encoding the viral RdRP and the genome segment one ( PBV1 ) encoding the CP and a protein of unknown function [32] .', 'The crystal structure of a rabbit PBV virus-like particle ( VLP ) shows that the capsid possesses T = 1 icosahedral symmetry in which the asymmetric unit is a dimer .', 'Such an organization is sometimes referred to as “T = 2” structure and is unique for dsRNA viruses [33] .', 'However , the overall capsid organization of PBV appears to be somewhat different from the organization of the larger dsRNA viruses like reoviruses [34] .', 'Instead of having side-by-side CP dimers clustering around the 5-fold symmetry axes ( i . e . CP decamers ) , the PBV capsid is made of diamond-shaped dimers of dimers ( i . e . CP tetramers ) .', 'Such an unusual capsid organization has also been observed in fungal infecting partitiviruses , another family of dsRNA viruses with a bi-segmented genome [35 , 36] .', 'The CPs for PBV and partitiviruses are small in size , with a structural fold that somewhat differs from those found in larger dsRNA viruses such as reoviruses and rotaviruses .', 'Interestingly , the major capsid protein P1 of cystoviruses likely also forms tetramers , but the geometric shape of such tetramers and the structural fold of the P1 are somewhat different from those of PBV and partivirus CPs [37 , 38] .', 'To elucidate the mechanisms of RNA replication , transcription , and RdRP encapsidation by this group of largely uncharacterized , small dsRNA viruses , we have determined the structure of the hPBV RdRP and systematically characterized its biochemical and enzymatic activities .', 'The hPBV RdRP possesses a canonical polymerase fold with a 24 amino acids ( aa ) long C-terminal insertion loop structure that partially occupies the active site of the polymerase .', 'A hPBV RdRP lacking this insertion loop , ΔLOOP , was subsequently generated to determine the functional role of this structure .', 'Both the wild-type ( WT ) and ΔLOOP RdRPs are capable of RNA synthesis using both homologous and heterologous , single- and double-stranded RNA templates in the absence of the CP .', 'However , while the WT RdRP utilizes a de novo initiation mechanism for RNA synthesis , the ΔLOOP could only initiate RNA replication through back-priming , suggesting that the insertion loop serves as a platform for initiation .', 'For transcription the hPBV polymerase uses a semi-conservative mechanism in which the positive-strand of the template dsRNA is dislodged from the duplex on the RdRP surface as the negative-strands enters into the template tunnel and the newly produced transcript forms a duplex with the negative-sense RNA strand .', 'We also demonstrated terminal nucleotidyl transferase ( TNTase ) activity for hPBV polymerase being the second polymerase among dsRNA viruses reported with TNTase activity .', 'Co-expression of the hPBV RdRP and CP resulted in the formation of ~35 nm VLPs that were incapable of sequestering the RdRP molecules .', 'Results from gel shift assays indicate that the hPBV RdRP has a strong preference for the 5’-terminal untranslated region of the positive-sense genomic RNA ( i . e . 5’ ( + ) UTR ) .', 'Our results thus suggest that PBV most likely has its RdRP molecules incorporated into viral particles through direct interactions with the genomic RNAs , which are selectively packaged through specific interactions with the viral CP .'], ['Recombinant hPBV RdRP ( strain Hy005102 ) was overexpressed in Escherichia coli as a soluble protein .', 'The purified hPBV RdRP ( 534 aa , ~62 kDa with a N-terminal 6xHis tag ) exists in solution as a monomer , based on its gel filtration chromatography elution profile ( S1 Fig ) .', 'hPBV RdRP was crystallized in the space group of P21 with two molecules in each asymmetric unit .', 'The crystal structure was solved to 2 . 4 Å resolution by single-wavelength anomalous dispersion ( SAD ) using Se-Met derivatized crystals ( Table 1 ) .', 'In the final model of the hPBV RdRP , one molecule is comprised of residues 1–494 , 500–511 , and 518–534 ( Fig 1A ) , while the other molecule contains residues 1–495 and 520–534 .', 'The hPBV RdRP has an overall oval shape and is ~50 x 60 x 60 Å3 in size ( Figs 1 and 2 ) .', 'There are a total of 24 α-helices and 14 β-strands in each molecule .', 'The polypeptide can be divided into three domains based on their function: an N-terminal domain ( aa 1–84 ) , a core polymerase domain ( aa 85–470 ) , and a C-terminal domain ( aa 471–534 ) ( Fig 1A and 1C; S2 Fig ) .', 'The polymerase domain , which is structurally highly conserved amongst RNA viruses [39] , has a right-hand configuration with three subdomains: the fingers ( aa 85–230 , 268–324 ) , palm ( aa 231–267 , 325–414 ) , and thumb ( aa 415–470 ) ( Fig 1A and 1C ) .', 'The palm subdomain hosts the three key aspartic acid residues , D261 , D359 , and D360 , of the active site ( Fig 1A ) .', 'The hPBV core polymerase domain is rather compact with only 386 aa in total ( Fig 1B ) .', 'The palm subdomain , which hosts the catalytic active site , is composed of five α-helices and six β-strands ( i . e . α11–13 , 16 , 17 and β6 , 9–13 ) and contains the polymerase motifs A-E that are conserved among all RdRPs ( Fig 1B and 1C; red ) [40] .', 'The most noticeable structural feature of the palm is a central , four-stranded β-sheet consisting of a β-hairpin ( i . e . β9 , β10 ) and two anti-parallel β-strands ( i . e . β6 , β11 ) .', 'The polymerase motif C , which contains the highly conserved “-GDD-” sequence , is mapped to the β-hairpin .', 'The two other β-strands ( β6 , β11 ) of the central β-sheet contain motifs A and D , respectively .', 'The motif A has the conserved sequence of “DXXXXD” whereas motif D is more variable in sequence .', 'The first aspartate from motif A ( i . e . D261 ) and the two aspartates from motif C ( i . e . D359 and D360 ) constitute the active site as they help to coordinate two divalent metal ions for charge relay and intermediate stabilization during catalysis .', 'The motif D mediates the binding of the incoming nucleotide substrate and plays an important role in determining the efficiency and fidelity of nucleotide addition [41] .', 'Motif B , which folds into a strand-turn-helix structure at the interface between the fingers and palm subdomains , has been found to interact with the RNA template to guide it into the active site of the polymerase [42] .', 'Motif E folds into a β-hairpin at the interface between the palm and the thumb subdomains , and forms a part of the “primer grip” as discussed below [40] .', 'Close inspection shows that the fingers subdomain of the hPBV RdRP is composed of eight α-helices and seven β-strands ( Fig 1A and 1C; blue ) .', 'At the top of the fingers subdomain is a twisted , four-stranded β-sheet ( β4 , β5 , β7 , and β8 ) that forms the fingertip structure with an extended loop , which contains the rNTP binding sequence ( residues 182–199 ) denoted as the RdRP motif F ( Fig 1B and 1C ) [40] .', 'Structural studies on ϕ6 and hepatitis C virus ( HCV ) RdRPs have revealed that the basic residues in the rNTP binding loop interact with the phosphates of the incoming nucleotide [18 , 43] .', 'The fingers subdomain also contains the RdRP motif G [44] that is located near a three-stranded antiparallel β-sheet ( β1 , β2 , and β3; Fig 1B and 1C ) .', 'The structures of the ϕ6 and reovirus RdRPs have revealed that the residues of motif G interact with the entering RNA template [18 , 21] .', 'Situated at the other side of the polymerase palm across from the fingers subdomain is the thumb subdomain , which is comprised of one β-strand ( β14 ) followed by three α-helices ( α18 - α20; Fig 1A and 1C; green ) .', 'The initial β14-strand forms a part of the “primer grip” along with a β-hairpin ( β12 and β13 ) from the palm subdomain ( Fig 1A ) .', 'The “primer grip” motif is commonly observed in viral RdRPs and has been shown to interact with the nascent/primer strand during RNA synthesis [40] .', 'The hPBV N-terminal domain is made of the first 84 residues of the polypeptide and contains four α-helices ( α1–4 ) .', 'With an overall L-shape , the N-terminal domain wraps around the fingers and thumb subdomains with its long and short arms , respectively ( Fig 1A; yellow ) .', 'This interaction allows the RdRP to maintain its active site in a closed conformation despite that there is very little direct contact between the hPBV fingertip and the thumb subdomain .', 'The N-terminal domain in the RdRPs from infectious bursal disease virus , reovirus , and the rabbit hemorrhagic disease viruses also helps to encircle the polymerase active site although the size of these N-terminal domains can vary substantially [20 , 21 , 45] .', 'The C-terminal domain of hPBV is rather short with four α-helices .', 'It lies adjacent to the thumb subdomain at the front end of the polymerase palm ( Fig 1A; magenta ) .', 'The hPBV RdRP contains three channels leading to the active site of the protein that are believed to allow for dsRNA product export , template entry and NTP uptake ( Fig 2 ) .', 'The dsRNA product channel , which locates in the front of the molecule , is the largest of all three with a diameter of ~18–20 Å , comparable to the diameter of a dsRNA helix ( Fig 2A–2C ) .', 'Both the template and NTP channels are located near the interface between the fingers and thumb subdomains , and are heavily lined with positively charged residues ( Fig 2B , 2C and 2D ) .', 'The distance from the surface of the RdRP to the active site along the putative template entry channel could be spanned by 5 to 6 nucleotides as observed in the ϕ6 RdRP [46] .', 'A patch of positively charged residues is found near the putative template entry channel ( Fig 2C , dotted oval ) .', 'A positively charged plough has been previously noted in the structure of ϕ6 RdRP , and is believed to play a role in separating the two strands of a dsRNA molecule allowing the template RNA to enter the template entry channel , while the non-template RNA slides over the positively charged patch and is directed away from the RdRP [18] .', 'Based upon a pairwise comparison using the program Dali [47] , the structure of the hPBV RdRP closely resembles that of the RdRPs from the members of the Caliciviridae ( i . e . Z = 26 . 7 for the rabbit hemorrhagic disease virus and Z = 26 . 5 for the Norwalk virus ) [45 , 48] , Flaviviridae ( i . e . Z = 26 . 4 for the HCV and Z = 24 . 2 for the bovine viral diarrhea virus ( BVDV ) ) [43 , 49–51] and Picornaviridae ( i . e . Z = 25 . 3 for the poliovirus ) [52] families , suggesting a potential evolutionary relationship between the RdRP of PBV and the RdRPs of these three viral families having positive-sense ssRNA genomes .', 'By contrast , the correlation of the hPBV polymerase to the RdRPs from other dsRNA virus families appears to be more distant , with a Z = 18 . 8 for phage ϕ6 [18] , Z = 12 . 8 for rotavirus [19] , Z = 12 . 1 for infectious bursal disease virus [20] , and Z = 9 . 3 for reovirus [21] .', 'The hPBV RdRP possesses a highly flexible , 24-aa insertion loop structure ( aa 495–518 ) formed by an internal sequence from the C-terminal domain .', 'This loop structure , which is associated with higher than average temperature factor values , extends from the C-terminal domain towards the catalytic site ( Fig 1A ) .', 'The location of the insertion loop resembles the structure that functions as the initiation platform of the bacteriophage ϕ6 RdRP [23 , 24] .', 'The structure of the ϕ6 RdRP in complex with an oligonucleotide template ( PDB ID 1HI0 ) was superimposed onto the hPBV RdRP structure in order to model RNA binding by the hPBV RdRP .', 'In this model , the 3’-end of the template collides with the hPBV insertion loop structure , suggesting that the insertion loop in its current position would sterically prevent the template from binding to the active site of the RdRP as expected in the assembly of a productive initiation complex ( Fig 1A , right ) .', 'Therefore , the hPBV insertion loop structure must undergo a significant conformational change in order for RNA binding and replication to take place .', 'Similar structural rearrangement is probably required for dsRNA egress during both transcription and genome replication .', 'Interestingly , such structural arrangements were indeed observed in the β-hairpin structure that serves as an initiation platform for the HCV RdRP following RNA binding [53] .', 'In the ϕ6 polymerase , the loop that serves as the initiation platform remains in place during the assembly of the initiation complex , but undergoes a conformational change at the onset of the elongation process [54] .', 'Based on our structural modeling and comparison to both the ϕ6 and HCV polymerases , we speculate that the insertion loop of the hPBV RdRP may have two possible functions .', 'First , the loop may play an important role during the de novo initiation of RNA synthesis .', 'Like in the case of the ϕ6 , HCV and several other flavivirus polymerases , the loop could function as a platform to support the assembly of an initiation complex using a single nucleotide as a primer .', 'Another attractive hypothesis is that the insertion loop may function as a gatekeeper to regulate RNA synthesis during the PBV virion assembly .', 'The insertion loop in its observed structural form would inhibit an RNA template from binding , but upon the RdRP binding with the CP ( i . e . in an assembled capsid ) , the insertion loop would adopt an alternative conformation to allow efficient RNA synthesis in the fully/partially assembled hPBV particles .', 'To determine the exact biological function of the insertion loop structure , a hPBV RdRP lacking the loop structure ( i . e . ΔLOOP ) was synthesized and expressed .', 'Gel filtration chromatography showed that ΔLOOP was eluted at a similar position as the WT protein ( S1 Fig ) .', 'The ΔLOOP RdRP was crystallized and its structure solved by molecular replacement using the WT RdRP structure as the phasing model ( Fig 3A , Table 1 ) .', 'The overall structure of ΔLOOP appears to be essentially the same as the WT RdRP ( root-mean-square deviation in distance = 0 . 4 Å for 3275 common atoms ) , except for the deleted insertion loop which became unstructured in the ΔLOOP RdRP .', 'Given this structural information , we are confident that the removal of the insertion loop should not affect the overall folding of the polymerase , and that the ΔLOOP RdRP should provide an excellent tool to study the function of the insertion loop structure using in vitro assays .', 'Polymerase activity assays were performed to determine if the WT and ΔLOOP RdRPs could synthesize dsRNA from an ssRNA template .', 'Both RdRPs were found to replicate the positive- and negative-strands of the PBV genome segment 2 ( PBV2+ and PBV2- , respectively ) as well as ϕ6-specific ssRNA template s+ ( i . e . the positive-strand of the small , S , genome segment ) with similar efficiency ( Fig 3B ) .', 'However , the polymerase encountered some processivity issues , as it produced also dsRNA products which were shorter than the expected full length dsRNAs ( Fig 3B ) .', 'Further enzymatic assays showed that the WT RdRP is able to use dsRNA templates to carry out transcription but the transcription activity of the ΔLOOP RdRP was compromised ( Fig 3B ) .', 'Transcription activity was observed whenever homologous ( i . e . PBV2 ) or heterologous dsRNAs ( i . e . ϕ6 genomic RNA composed of segments S , M and L ) were used as templates ( Fig 3B , S1 Table ) .', 'This indicates that the hPBV RdRP is enzymatically active , can efficiently replicate and transcribe both homologous and heterologous templates , and does not require the presence of the viral CP as a cofactor for RNA synthesis .', 'Our findings thus rule out the scenario where the loop structure functions as a regulatory element to prevent premature dsRNA synthesis by the capsid-free polymerase .', 'Terminal nucleotidyl transferase ( TNTase ) activity was observed for both the WT and ΔLOOP RdRPs ( Fig 3C and 3D ) .', 'TNTase activity involves the addition of one or several nucleotide ( s ) to the 3’-end of a nucleic acid molecule ( Fig 3C ) .', 'While there was no clear preference for the hPBV-specific RNA substrate , both the WT and ΔLOOP RdRPs showed a strong preference for ssRNA over dsRNA ( Fig 3D ) .', 'Additionally , it was noted that the removal of the insertion loop significantly increased the TNTase activity of the RdRP ( Fig 3D ) .', 'Given our TNTase activity data and the fact that the insertion loop is located near the C-terminus of the hPBV RdRP , we propose that the removal of the insertion loop structure leaves the dsRNA exit channel of the protein permanently open ( Fig 2B ) , thus allowing the 3’-end of the RNA molecules to reach the active site for nucleotidyl addition in an orientation that is compatible for nucleotide addition .', 'To rule out any histidine-tag ( His-tag ) associated artifacts , three versions of the hPBV RdRP , an N-terminally and C-terminally His-tagged as well as a non-tagged RdRP , were tested for RNA synthesis activity using the PBV2+ and PBV2- ssRNA templates as well as the PBV2 dsRNA template ( S3 Fig ) .', 'Our results indicated that there was no detectable difference in the enzymatic activity between these three versions of the polymerase .', 'Therefore , all of the polymerase activity assays described in this paper has been performed using the N-terminally His-tagged protein .', 'To evaluate the potential role of the insertion loop in the initiation of dsRNA synthesis ( i . e . replication ) , aliquots of the dsRNA products were heat denatured before being analyzed by electrophoresis in a native agarose gel ( Fig 4A and 4B ) .', 'Due to the heat denaturation , the dsRNA products generated by the WT RdRP were converted to ssRNA , while the dsRNA products of the ΔLOOP RdRP retained the same mobility as the original dsRNA ( Fig 4A and 4B ) .', 'This result indicates that back-priming was taking place during dsRNA synthesis by the ΔLOOP RdRP , thus producing a product that was covalently linked to the ssRNA template ( Fig 4A , right panel and b ) .', 'Additionally , isotope incorporation from ( γ-32P ) GTP into the dsRNA product was only detected for reactions containing the WT RdRP , further indicating that this protein utilizes the de novo initiation mechanism ( Fig 4C ) .', 'Taken together , these results show that the insertion loop structure of the hPBV RdRP can effectively block template back-priming and facilitates initiation via a primer-independent mechanism , possibly by providing a docking site for the 3’-end of the RNA template and a binding site for the priming nucleotide .', 'This finding is consistent with the results obtained for the ϕ6 RdRP whenever its equivalent stabilizing platform was removed [23 , 24] .', 'There are two major ways for the transcription of the dsRNA genome: conservative and semi-conservative transcription mechanism ( Fig 5A ) .', 'The isotope labeling of the dsRNA molecules as an outcome of the transcription reaction indicated that the hPBV polymerase uses a semi-conservative transcription mechanism ( Figs 3B , 5A and 5B ) .', 'The ability of the hPBV RdRP to incorporate radioactivity from [γ-32P]-labeled GTP into the dsRNA product also confirmed that the observation of radiolabeled dsRNAs was due to de novo RNA synthesis instead of terminal nucleotidyl transfer catalyzed by the polymerase ( Fig 5B , right panel ) .', 'To get further support for the semi-conservative mechanism a time-course study using the ϕ6 genomic dsRNA as a template was performed ( S4 Fig ) .', 'Indeed , 33P-label was first incorporated in dsRNA molecules before any labeled ssRNA could be observed ( S4 Fig ) , confirming that semi-conservative transcription was taken place ( not replication of ssRNA transcripts produced by conservative mechanism ) .', 'The ϕ6 L segment was transcribed at the highest efficiency , resulting in a strong dsRNA band that started to appear at around 30 minutes with increasing intensity till 120 minutes after the experiment began ( S4 Fig ) .', 'Considering that the L segment of ϕ6 is ~6 . 4kb long , we estimate that the rate of transcription by the hPBV RdRP is ~210 bases per minute .', 'The ( - ) strands of the two hPBV genome segments both start with 3’-CAU ( S1 Table ) , so we set forth to test the transcription specificity of the hPBV RdRP using dsRNA template with different terminal sequences .', 'Coincidentally , the ( - ) strand of the ϕ6 L segment also starts with 3’-CAU , while the ( - ) strands of the ϕ6 S and M segments begin with 3’-CCU ( S1 Table ) .', 'We found that , under the applied reaction conditions , the hPBV RdRP has a preference for dsRNA templates in which the ( - ) strand starts with 3’-CAU over those starting with 3’-CCU ( S4 Fig , Figs 3B and 5B ) .', 'The preference for the ϕ6 L segment over the M and S segments was lost when the terminal sequence of L was mutated to 3’-CCU ( S5 Fig ) .', 'On the other hand , when an opposite change was introduced in the ( - ) strands of the ϕ6 genome segments M and S ( 3’-CCU to 3’-CAU ) , the amount of product synthesized by the hPBV RdRP increased considerably ( S5 Fig ) .', 'The template preference of the hPBV RdRP was also evident when the usage of the PBV2 and ϕ6 S or ϕ6 L dsRNAs was compared in a competition experiment or side-by-side ( S6B Fig ) .', 'We then tested the ability of hPBV RdRP to transcribe full-length PBV2 and its two deletion mutants , one with the first 33 nucleotides removed and the other with the first 645 nucleotides removed ( Fig 5C ) .', 'As a result of the deletions , the 3’-ends of the ( - ) strands of the two truncation mutants , Δ1–33 and Δ1–645 , became 3’-CCU and 3’-CCC , respectively .', 'Transcription assays showed that out of the three dsRNA templates , only the WT PBV2 , which has a 3’-CAU termini at the ( - ) strand , was efficiently transcribed ( Fig 5C ) .', 'Taking together the results with the PBV2- and ϕ6-specific dsRNA constructs ( S5 Fig and Fig 5C ) sharing identical 3’-terminal sequences in ( + ) strands ( S1 Table ) but variable in ( - ) strands , it appears that PBV RdRP has strong preference on dsRNA templates having 3’-CAU .', 'This data also suggest that the RdRP predominantly produces ( + ) strands of the PBV2 and ϕ6 genome segments during transcription reaction .', 'There are two possible explanations for the stronger transcription activity exhibited by the hPBV RdRP towards native PBV2 ( - ) strand sequence ending with 3’-CAU .', 'First , the lower melting temperature associated with the 3’-CAU sequence likely facilitates the initiation of RNA transcription .', 'Second , it is possible that the 3’-CAU sequence makes specific interaction with the hPBV RdRP , which would facilitate the binding of the viral RNA template and thus helps to enhance its transcription activity .', 'We have not observed any preference of the hPBV RdRP towards ssRNA template ( Figs 3B and S6A ) .', 'Indeed , the fact that virus-specific ssRNA was not replicated to a higher level when mixed with non-specific ssRNA in a competition experiment argues against the second hypothesis .', 'Therefore , we believe that the preference for virus-specific dsRNA templates by the hPBV RdRP during transcription is largely due to the lower melting temperature of the template terminal sequence .', 'Upon finding that the hPBV RdRP was capable of synthesizing dsRNA and ssRNA in the absence of the CP ( Fig 3B ) , the hPBV RdRP and CP were co-expressed to determine if the encapsidation of the RdRP is mediated through direct protein-protein interactions with the CP , as has been previously observed for the majority of dsRNA viruses characterized so far [6 , 8 , 9] .', 'Overexpression of the hPBV CP in E . coli resulted in the spontaneous formation of VLPs .', 'Such VLPs could be purified by ultracentrifugation using a CsCl gradient and have a density of ~1 . 3 g/ml according to their migration behavior .', 'Transmission electron microscopy ( TEM ) images of the hPBV VLPs determined that they have a diameter of ~35 nm ( Fig 6A ) , similar to the size of the rabbit PBV VLPs previously reported [34] .', 'While the N-terminus of the rabbit PBV CP is proteolytically removed by self-cleavage after particle assembly , this does not seem to be the case for the hPBV CP .', 'We found that the hPBV CP without the N-terminal peptide ( Δ45 ) was also capable of self-assembly , and that the N-terminally truncated CP migrated faster than the full-length protein in a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) ( Fig 6A and 6B ) .', 'The hPBV RdRP and the CP were co-expressed in E . coli using two expression vectors with different antibiotic selectors .', 'Any unpackaged His-tagged RdRP molecules that may have been present in the clarified cell lysate were removed by Ni-NTA affinity chromatography .', 'After ultracentrifugation , the VLP fraction was collected , denatured , and a Western blot was performed to test for the presence of the RdRP utilizing an antibody directed against the 6xHis-tag located at the N-terminus of the protein ( Fig 6C ) .', 'While the RdRP could be clearly detected in the clarified lysate , no RdRP could be detected in the sample of the isolated VLPs .', 'Therefore , our results suggest that direct interactions between the hPBV RdRP and CP are weak .', 'We hypothesize that viral genomic material must be present for RdRP incorporation to occur and that the interplay between the CP , RdRP , and the viral genome is needed for the encapsidation of the RdRP during virion assembly .', 'The fact that the constructs used to express the CP and RdRP contained only the protein-coding sequence of the two open reading frames suggests that the region of the hPBV genome required for RdRP encapsidation is potentially located in the untranslated regions ( UTRs ) of the genome segments .', 'Gel shift assays were conducted to further examine the interaction between the RdRP and the hPBV genome .', 'Three 20-nt long RNA oligonucleotides were probed , one containing a non-specific CA-repeat and the other two bearing the terminal sequences of the 5’- and 3’-UTRs of the ( + ) strand of the hPBV genome segment 2 ( i . e . 5’- ( + ) UTR and 3’- ( + ) UTR ) .', 'It was determined that the WT RdRP has approximately a 10-fold higher affinity for the first 20 nucleotides of the 5’- ( + ) UTR of the hPBV genome as compared to the last 20 nucleotides of the 3’- ( + ) UTR or to a nonsensical CA-repeat ( Fig 7A ) .', 'Additionally , deleting the insertion loop appeared to have a minimal effect on the overall RNA binding of the RdRP ( Fig 7B ) .', 'Notably , both segments of the hPBV genome have an AU-rich ( ~80% for nucleotides 1 to 20 ) sequence at the 5’-end of their ( + ) strands that begins with a conserved 5 nucleotide motif 5’-GUAAA- .', 'The AU-rich terminal sequence is predicted to form an RNA stem-loop structure ( S7 Fig ) according to the program ViennaRNA Package 2 . 0 [55] .', 'To test the binding affinity of the hPBV RdRP towards authentic viral RNAs , we synthesized by in vitro transcription the full-length PBV2+ ssRNA and a truncated version PBV2+ ( Δ1–645 ) ssRNA with the first 645 nucleotides removed .', 'Results from our gel shift assays show that the binding affinity of hPBV RdRP for full-length PBV2+ is ~10 times stronger than PBV2+ ( Δ1–645 ) , thus confirming that the terminal sequence at the 5’-end of the PBV2+ binds specifically to the polymerase ( Fig 7C ) .', 'The same trend was observed for the ΔLOOP RdRP , suggesting that the insertion loop is not directly involved in the 5’- ( + ) UTR binding ( Fig 7D ) .'], ['The high level of sequence conservation among RdRPs from various mammalian and avian PBV strains indicates similar three-dimensional structures ( S2A Fig ) .', 'Five of the six variable regions are located in either the N-terminal domain or the fingers subdomain , and all are mapped to the surface of the protein ( S2B Fig ) .', 'The core polymerase domain of hPBV RdRP closely resembles those from the members of the Caliciviridae , Flaviviridae , and Picornaviridae families all having ( + ) sense ssRNA genomes .', 'An interesting structural feature of this RdRP is the presence of a 24-aa loop structure that extends from near the C-terminus of the protein to just above the active site ( Fig 1A ) .', 'Through site-directed mutagenesis , we determined that the loop structure most likely functions as a priming platform to support the binding of a single priming nucleotide ( Fig 4 ) .', 'Loop structures that perform a similar function have been observed in the viral RdRPs of ϕ6 , reovirus , rotavirus , and HCV [18 , 19 , 21 , 23 , 24 , 53] .', 'In four-tunnel RdRPs such as those from reovirus and rotavirus , the internal priming loops are formed by sequences located between the fingers and palm domain .', 'However , in three-tunnel polymerases that catalyze semi-conservative RNA synth
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['During the replication cycle of double-stranded ( ds ) RNA viruses , the viral RNA-dependent RNA polymerase ( RdRP ) replicates and transcribes the viral genome from within the viral capsid .', 'How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families .', 'In this study , we have determined a 2 . 4 Å resolution structure of an RdRP from the human picobirnavirus ( hPBV ) .', 'In addition to the conserved polymerase fold , the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site .', 'In vitro RNA polymerization assays and site-directed mutagenesis showed that: ( 1 ) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; ( 2 ) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; ( 3 ) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and ( 4 ) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences .', 'Co-expression of the hPBV RdRP and the capsid protein ( CP ) indicated that , under the conditions used , the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome .', 'Additionally , the hPBV RdRP exhibited higher affinity towards the conserved 5’-terminal sequence of the viral RNA , suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly .']
['Viral polymerases replicate the genome of the virus , which is essential for the synthesis of the progeny .', 'All double-stranded ( dsRNA ) viruses have virion-associated polymerases that catalyze RNA synthesis within an intact capsid .', 'Picobirnavirus ( PBV ) is a small dsRNA virus , and it has been shown that the capsid of PBV possesses an unusual architecture suggesting an uncommon assembly pathway that it could potentially share with another group of small dsRNA viruses called partitiviruses .', 'We have performed both structural and functional studies to look at how the PBV polymerase performs its function and how these molecules are placed within the capsid .', 'The PBV RdRP structure was solved and revealed that the RdRP has an interesting loop structure in its interior .', 'Additionally , activity assays showed that the RdRP possesses catalytic activity in the absence of other viral proteins .', 'Removing the loop structure was found to change the way that the RdRP initiates RNA replication .', 'Further experiments also showed that the RdRP did not interact with the viral capsid protein ( CP ) but had a strong affinity for a conserved terminal sequence of the PBV genome .', 'This suggests that in PBV the RdRP may be encapsidated based upon both the RdRP and CP co-interacting with the viral genome .']
['sequencing techniques', 'nucleic acid synthesis', 'dna-binding proteins', 'microbiology', 'viral structure', 'viruses', 'polymerases', 'rna viruses', 'sequence motif analysis', 'molecular biology techniques', 'rna synthesis', 'microbial genomics', 'chemical synthesis', 'research and analysis methods', 'sequence analysis', 'viral genomics', 'rna structure', 'proteins', 'viral packaging', 'viral replication', 'molecular biology', 'biosynthetic techniques', 'biochemistry', 'rna', 'nucleic acids', 'virology', 'genetics', 'biology and life sciences', 'dsrna viruses', 'genomics', 'organisms', 'macromolecular structure analysis']