RNA-dependent RNA polymerases play a vital role in the growth of RNA viruses where they are responsible for genome replication, but do so with rather low fidelity that allows for the rapid adaptation to different host cell environments. These polymerases are also a target for antiviral drug development. However, both drug discovery efforts and our understanding of fidelity determinants have been hampered by a lack of detailed structural information about functional polymerase-RNA complexes and the structural changes that take place during the elongation cycle. Many of the molecular details associated with nucleotide selection and catalysis were revealed in our recent structure of the poliovirus polymerase-RNA complex solved by first purifying and then crystallizing stalled elongation complexes. In the work presented here we extend that basic methodology to determine nine new structures of poliovirus, coxsackievirus, and rhinovirus elongation complexes at 2.2–2.9 Å resolution. The structures highlight conserved features of picornaviral polymerases and the interactions they make with the template and product RNA strands, including a tight grip on eight basepairs of the nascent duplex, a fully pre-positioned templating nucleotide, and a conserved binding pocket for the +2 position template strand base. At the active site we see a pre-bound magnesium ion and there is conservation of a non-standard backbone conformation of the template strand in an interaction that may aid in triggering RNA translocation via contact with the conserved polymerase motif B. Moreover, by engineering plasticity into RNA-RNA contacts, we obtain crystal forms that are capable of multiple rounds of in-crystal catalysis and RNA translocation. Together, the data demonstrate that engineering flexible RNA contacts to promote crystal lattice formation is a versatile platform that can be used to solve the structures of viral RdRP elongation complexes and their catalytic cycle intermediates. However, they do so with the fairly low fidelity of one error in every 10pol) elongation complex The molecular basis for nucleotide selection and catalysis by these viral RdRPs was demonstrated by a series of crystal structures of the poliovirus polymerase  and fully poised for NTP binding to the active site, and second the active site itself is not structured for catalysis because the motif A is not hydrogen bonded to the motif C, disrupting the palm domain RRM-fold common to polymerase active sites. NTP then enters the polymerase complex, where it base pairs with the templating base and base stacks on the priming base, creating a State 2 structure wherein the active site RRM fold is still not fully formed. Next, State 3 is formed by a movement of motif A in the palm domain that fully structures the RRM-fold, brings in the magnesium ions and coordinating carboxylic acids needed for catalysis, and slightly repositions the bound nucleotide for catalysis. The result is a closed active site that has been captured at the pre-catalysis State 3 in a norovirus polymerase-RNA-CTP complex obtained by cocrystallization 4 in the poliovirus elongation complex after CTP soaking. Outside of the changes in covalent bonding, the structural arrangement of the polymerase-NTP-metal complex atoms at the active site appears to be essentially unchanged between a pre-catalysis state 3 and the post-catalysis state 4. This is followed by state 5 where the active site has returned to the open conformation because motifs A and C of the RRM fold are no longer hydrogen bonded to each other, but the newly incorporated nucleotide and pyrophosphate product are still in the active site. The final step in the model is then State 6 that accounts for translocation of the RNA by one base pair to position the next templating nucleotide in the active site. Thus far there are no structures of translocation intermediates and it is not yet known if translocation in these RdRPs is a power-stroke type movement to rapidly translocate the RNA in a single step or a series of Brownian ratchet type sub-steps that more gradually move the RNA into its final translocated register.The catalytic cycle model starts with State open conformation active site has been observed in all the RdRP alone and several RdRP-RNA and RdRP-RNA-NTP complex structures solved thus far, while the closed conformation has only been seen in the presence of RNA and cognate NTP bound at the active site, where it was trapped in the pre-catalytic state 3 in norovirus polymerase 4 in the poliovirus elongation complex in vivo virus mutation rates obtained by sequencing progeny virus genomes and the in vitro ability of the viral polymerases to discrimination between CTP and 2′-deoxy-CTP pol such that residues located far from the active site can have significant effects on catalysis.Based on RdRP structure comparisons, this palm-domain based active site rearrangement appears to be unique to positive-strand RNA virus polymerases: The 5 structure wherein the post-catalysis active site had returned to the open conformation in the absence of translocation. This is intriguing because these two events, opening the active site and translocating the nucleic acid, are usually tightly coupled in replicative polymerases, such at T7 RNA polymerase and Pol I polymerase, via interactions involving swinging motions of a major α-helix motif within the fingers domain The original crystallization of the poliovirus EC appeared to be in large part driven by the coaxial stacking of the upstream RNA duplexes emerging from two different complexes. This resulted in a “dumbbell” shaped minimal crystallization unit composed of a bipartite central RNA helix segment with polymerase molecules facing away from each other at each end of the RNA helix . The ECsopen conformation active site in the absence of added NTP.To examine this in further detail and to validate the polymerase–RNA interactions seen in the initial poliovirus EC structures, we here present nine new coxsackievirus, rhinovirus, and poliovirus elongation complex structures at resolutions ranging from 2.1 to 2.9 Å. The crystals were obtained by expanding on our initial EC purification methodology to systematically vary the length of the upstream RNA and the flexibility of the RNA-RNA junction during crystallization screening. The RNA-RNA contacts of the resulting structures exhibit a wide range of geometries and some of them form lattices that allow for multiple rounds of in-crystal catalysis and translocation while retaining diffraction. A comparison of the RNA structure in the resulting picornaviral polymerase elongation complexes shows a non-canonical RNA backbone conformation in the active site itself and that the conformation of the upstream duplex is essentially identical for 7–8 base pairs that make intimate contact with the picornaviral polymerases, but beyond this point the RNA adopts a wide range of crystal packing arrangements. Similarly, a comparison of the three different polymerases in these ECs highlight conserved interactions with the downstream RNA template, a consistent opening of the RNA exit channel to accommodate the duplex, and an pol) with C-terminal GSSS-His6 tags were produced in E. coli using a ubiquitin fusion construct that is post-translationally cleaved in the expression strain to generate 3Dpol molecules with the required glycine as the first residue of the protein Poliovirus (PV) and coxsackievirus B3 (CV) polymerases (3DE. coli optimized gene synthesized by Blue Heron Biotech (www.blueheronbiotech.com) that was cloned into the NcoI and XhoI sites of the arabinose-inducible pBADb vector from Invitrogen (www.invitrogen.com), verified by sequencing, and then transformed into TOP10 cells. Optimal arabinose levels, induction temperature (15–18°C), and growth time (15–18 hours) were determined empirically. Protein production scale cultures were done in 4-liter flasks with 800 ml of terrific broth at 250-rpm agitation. Cultures were induced with 0.2% arabinose at an OD600 of 1.5 and grown for 15–18 hours at 15°C. Cells were spun down and resuspended in 3 volumes of 50 mM HEPES pH 7.4, 200 mM NaCl, 5% glycerol, 1 mM EDTA, 5 mM DTT and flash-frozen in liquid nitrogen. Upon thawing, 0.1 mg lysozyme per milliliter of slurry was added and incubated on ice for 45 minutes. Lysate was sonicated briefly to decrease viscosity and a 10% solution of PEI (pH 7.8) was stirred in over 15 minutes to a final concentration of 0.4%. Cell debris and nucleic acids were pelleted by centrifugation at 15,000×g for 30 minutes. PEI and lysozyme were removed by passing clarified extract through fast flow SP sepharose. The flow-through fraction was diluted with three volumes of Buffer A , loaded onto a fresh fast flow SP sepharose column, washed with five volumes of BufferA+50 mM NaCl, and subsequently eluted with BufferA+200 mM NaCl. Eluted RV16 polymerase was diluted with an equal volume BufferA and loaded onto a HiTrap Heparin column for buffer change. The heparin column was washed with BufferB-50as . RV 3Dpol was eluted in a linear gradient of increasing ammonium sulfate, peak fractions were pooled and diluted to 50 mM ammonium sulfate in BufferB-0, loaded onto a HiTrap Q column, and eluted in a linear gradient of increasing ammonium sulfate. Pooled fractions were concentrated to 10 mg/ml and loaded onto a Superdex 75 column pre-equilibrated with BufferB-200as. Eluted monomeric 3Dpol was concentrated to 20–25 mg/ml and flash frozen in aliquots.Human Rhinovirus 16 polymerase (RV) was expressed from an pol, 30 µM pre-annealed primer-template RNA, and 300–450 µM each ATP and GTP were incubated at room temperature for 20–45 minutes to form stable ECs. CV 3Dpol is less soluble than its PV counterpart and assembly reactions where therefore done with 20 µM CV 3Dpol and 15 µM RNA in 50 mM HEPES, pH 6.5, 75 mM NaCl, 5 mM MgCl2, 2 mM TCEP, and 450 µM each ATP and GTP. Optimized conditions for the RV EC involved the addition of 10% glycerol to increase solubility of the polymerase under low salt conditions and substitution of Tris pH 7.8 and KCl for HEPES pH 6.5 and NaCl, respectively, in both the primer/template annealing reaction and EC assembly buffers. RV EC assembly was done with 20 µM 3Dpol and 10 µM RNA in a total volume of 9 ml and after incorporation of the first four nucleotides (GAGA), the pH of the reaction was adjusted to 6.5 by adding 1 M MES pH 6.0 as needed  before the following purification steps.ECs for crystallization were generated and purified using minor variations of the methods previously described for poliovirus polymerase 2, 2 mM TCEP reducing agent, and then concentrated in Amicon Ultra-4 centrifugal filters to ≈10 mg/ml for PV and ≈8 mg/ml for CV and RV complexes. Crystallization screens were carried out using the Qiagen Cryos Suite at 16°C. If needed, promising initial conditions were optimized, resulting in final crystallization complexes for the various constructs as outlined below. Unless otherwise noted, NTP or deoxy-NTP soaking experiments were done under the final cryo stabilizer conditions using 5 mM NTP or deoxy-NTP and 10 mM MgCl2 for 15–20 hours.The assembled EC’s were purified by ion exchange chromatography on a MonoQ column , buffer exchanged into 20 mM HEPES, pH 6.5, 100 mM NaCl, 2 mM MgCl2, 0.08 M Tris-HCl 8.5, 24% (w/v) PEG 4000, 20% (v/v) glycerol and were directly frozen for data collection. The PV_r5 crystals were obtained using 4.8%(v/v) isopropanol, 0.096 M tacsimate (Hampton Research), pH 8.5–9.0, 1.92–1.95 M ammonium sulfate, and 10–11%(v/v) glycerol and then gradually exchanged into a cryo stabilizer solution containing 4.8%(v/v) isopropanol, 0.096 M tacsimate, pH 8.5–9.0, 1.95 M ammonium sulfate and 19–27%(v/v) xylitol prior to freezing. The coxsackievirus CV_r2 crystals grew from 0.17 M sodium acetate, 0.085 M Tris pH 8.5, 25.5% PEG 4000, and 15% glycerol and were directly frozen. Coxsackievirus CV_r2_Mg crystals grew from 0.21 M magnesium acetate, 0.08 M sodium cacodylate pH 6.5, 20% glycerol, and 14% PEG 8000 and were directly frozen. Finally, the rhinovirus RV_r1 crystals grew in 2–3 days from 0.16 M ammonium sulfate, 0.08 M sodium acetate, pH 4.6, 20% (w/v) PEG 4000, and 20% (v/v) glycerol and were directly frozen. Note that the PV polymerase used to assemble the PV_r5 complex is with a C290M mutation that produced higher quality crystals than the wild type polymerase.Poliovirus PV_r3 and PV_r4 crystals grew in 0.16 M MgClI = A–D, ECII = E–H, …). The residue numbering used for the nucleic acid in all the complexes is always indexed such that the priming −1 position base pair is composed of nucleotide 601 on the template strand, 701 on the product strand, and 702 for any incoming nucleotide in the catalytic site, regardless of nucleotide sequence, pre- or post-catalysis state, or in-crystal translocation status.Diffraction data were obtained using beamline 4.2.2 at the Advanced Light Source . 180–185° of oscillation data were typically collected at 0.5–1° steps and the data were integrated, merged, and scaled using d*Trek To crystallize elongation complexes with coxsackievirus B3 (CV), human rhinovirus 16 (RV), and poliovirus (PV) polymerases we sought to further explore the use of RNA-RNA stacking as a mediator of lattice contacts by systematically varying the length of the RNA and weakening the RNA-RNA contacts. These alterations to the RNA would change both the overall length between the predicted pairs of ECs in the lattice and the flexibility of the central region where the two RNAs interact by coaxial stacking. Changing the viral polymerase, on the other hand, is effectively another crystallization screening parameter that explores differences in the surface residues of the proteins to facilitate crystallization. Together, these two parameters allowed us to readily crystallize all three ECs tested. The resulting structures of these highly homologous polymerases sample a wide variety of crystallization conditions, allowing us to determine what parts of the EC structures are universally conserved and what parts may be due to crystal packing effects.2–3 repeat sequence onto a synthetic RNA primer strand in order to generate stably “locked” ECs that could be purified for crystallization All ECs were formed using the previously described polymerase initiation protocol based on the addition of a (GA)r1 RNA used to crystallize and solve the initial PV polymerase EC structure (i.e. PV_r1), we were able to also crystallize the RV polymerase EC (RV_r1). The RV EC structure retained the coaxially stacked EC pairs seen with the original poliovirus structure, but note that the two RV EC pairs are in a very different spatial arrangement than previously observed. Next, we sought to slightly weaken the energetics of the coaxial stacking interactions that held two complexes together in the dumbbell geometry by replacing the 5′ terminal GC dinucleotide overhang with a UG sequence. Replacing two G-C pairs with more flexible G-U pairs weakens, but does not eliminate, the potential for base-pairing interactions at the coaxial junction. Using this r2 RNA, we were able to obtain crystals of the CV polymerase EC under two conditions but in the same crystal form, resulting in a 2.4 Å resolution structure (CV_r2) and a 2.2 Å resolution structure (CV_r2_Mg) that clearly showed a Mg2+ ion pre-bound to the coxsackievirus polymerase EC in the absence of an added NTP. Expanding on the r2 construct, the length of the upstream RNA duplex between the two ECs was shortened from 26 to 24 (r3) and 22 (r4) base pairs, and both these RNAs resulted in poliovirus EC crystals that diffracted to 2.4 Å (PV_r3) and 2.8 Å (PV_r4) resolution. Last, we further extended the length of the RNA duplex to 30 base pairs with a longer (GA)3 locking sequence in the r5 RNA and were able to obtain a third structure of the PV polymerase EC at 2.9 Å resolution (PV_r5). As further discussed below, in some cases NTP or deoxy-NTP soaks of these crystals resulted in catalysis and translocation, providing three additional structures of the PV and CV complexes.ECs were assembled on five different primer-template RNA sequences designed to systematically alter their potential for mediating crystallization contacts . Using tAll of the complexes crystallized with the anticipated coaxial stacking of the upstream RNA helices, demonstrating that the formation of the “dumbbell” orientation within EC pairs plays a critical role in establishing the crystal contacts. Despite this common lattice organization, the different dumbbell pairs and their RNA-RNA junctions displayed a multitude of geometries in the different crystal forms; for example, the junctions in the PV EC structures range from near perfect coaxial stacking to highly disordered structures that could not be fully modeled into the electron density maps . A compaThe structures were all solved in the space group P1 and non-crystallographic symmetry (NCS) restraints were applied to related complexes during refinement (see Methods). This approach was build on our experience with the earlier PV EC structures where the unit cell dimensions were very close to C2 symmetry, but NTP soaking experiments revealed differences in the catalytic abilities of the different EC pairs due to differences in crystal packing environments ′ overhang strand is not long enough to form any well-defined interactions with the polymerase surface in any of the EC structures, suggesting that its orientation is to some extent influenced by crystal packing interactions. In fact, the PV_r5 structures shows the downstream template RNA helix rotated by ≈180° such that the 5′ end of the non-template strand sits atop the fingers/thumb domain junction, but again there appear to be no well-defined interactions locking the RNA into this alternate orientation.The RV_r1 EC is the only structure where we observe significant contacts involving the downstream template duplex, but these appear to be non-specific interactions with other polymerase molecules in the lattice and not a biologically relevant interface . The conpol protein structures are by themselves highly homologous in the absence of RNA, allowing us to use the polymerase portion of the ECs to overlay the structures.The ten total EC structures containing three different viral polymerases show a number of common protein-RNA interactions, indicating that these are highly conserved features of picornaviral RdRP complexes. All the complexes feature an upstream RNA duplex that is pinched between the pinky finger and thumb domains such that the RNA exit channel is opened by ≈4 Å as compared to the polymerase alone structures . This tiTheseuswww.theseus3d.org) to obtain maximum likelihood based superpositions of the protein portion of the different EC structures, and then we carried out a careful comparative analysis of the resulting RNA alignments. The advantage of the Theseus approach to structure alignments is that highly homologous regions of the protein structure are given more weight in the superpositioning while regions of the proteins that differ in structure (including single amino acid insertions and deletions) are down weighted, resulting in a tight alignment dominated by the most homologous elements of the polymerase structures. We used the program pol α-helix composed of residues 406–420 lines the minor groove of the RNA duplex. It is only after this point that the structures diverge as a result of crystal packing contacts. Importantly, the great variation observed in the RNA-RNA junctions themselves does not affect the observed contacts between 3Dpol and the upstream duplex within the EC itself.The analysis of the upstream RNA duplex structures shows that the first seven base pairs of the RNA helix are in essentially the same conformation in all seven crystal lattices that represent the EC structures of three different viral polymerases . The RNANucleotide soaking experiments show that two of the lattices allow for catalysis and in-crystal translocation of the RNA. A key element of the resulting post-translocation structures is that the overall crystal lattice remains intact due to the plasticity of the RNA-RNA junctions . In the 2 of the catalytic cycle because the primer no longer has a reactive 3′-hydroxyl group following the first ddCTP incorporation event. The two polymerase molecules at opposite ends of the dumbbell remain essentially fixed and the translocation is accommodated by “peeling” nucleotides away from the end of the duplexes at the RNA-RNA junction so that they become disordered in solvent channels and are no longer resolved in the structure. There are minor perturbations at the end of the RNA helices as new non-canonical base stacking and hydrogen bonding interactions are formed by the nucleotides that have now been translocated into the junction regions.Soaking the CV_r2 crystals with CTP results in cracking and a low resolution dataset (≈3.4 Å) that is not of sufficient quality to elucidate structural details, but soaking with 2′,3′-dideoxy-CTP (ddCTP) leads to a 2.7 Å resolution structure (CV_r2+1) reflecting one translocation event and a second ddCTP bound in the active site. This complex is stalled at the pre-catalysis state 1 structure without bound NTP, while addition of CTP results in two translocation steps utilizing both guanosines in the template strand. The original junction in the PV_r5 lattice is interesting because while there is density for the nucleic acid phosphate backbone, the bases themselves are only weakly ordered and appear to be loosely associated in multiple conformations. We thus consider this junction to be “sticky” without being highly structured, and it is this stickiness that presumably allows it to accommodate both translation and rotation of the polymerases into a new relative arrangement without destroying the crystal lattice altogether.The nearly opposite behavior is seen in the PV_r5 lattice, where one and two translocation steps in each EC leads to a systematic movement of one polymerase relative to the other that is accommodated by twisting and sliding movements at the RNA-RNA junction point. Soaking with 2′-deoxy-CTP (2′-dCTP) results in a single incorporation and translocation event and a state Within the active sites themselves, the templating +1 nucleotide is always found fully stacked on the upstream RNA duplex and prepositioned for hydrogen bonding interactions with the incoming NTP. The next templating nucleotide, i.e. the downstream +2 position, is found above the active site where its base moiety is bound in a pocket formed between Pro20 and Lys22 from the index finger . At the open conformation for the active site wherein the β-strands of motifs A and C are not hydrogen bonded together to fully form the RMM-fold that is commonly utilized by polymerase active sites (All of the complexes show the ve sites . In the ve sites . This isve sites .The initial structure of the poliovirus polymerase EC provided insights into the path of the template and product RNA strands as they threaded through the active site and showed that viral RdRPs use a unique palm-based structural change to close their active site for catalysis Using a small library of different length RNA constructs, we were able to crystallize all three polymerases tested in multiple crystal forms. In all cases, the crystal lattices were dominated by the RNA-RNA interactions that resulted in pairs of ECs arranged into dumbbell type structures. Overall, the RNA-RNA packing interfaces showed structural heterogeneity, divergence from a canonical A-form helix, and weakened electron density as one approaches their junction regions. Thus, the coaxial stacking interactions exhibit significant plasticity, providing for a loose association of ECs that is critical for nucleating crystallization while at the same time providing enough flexibility for long-range crystal lattice formation via additional protein-protein contacts. This flexibility is further emphasized by the finding that two of the lattices allow for in-crystal RNA translocation in NTP soaking experiments, resulting in new structures with the potential to highlight intermediates in the polymerase elongation cycle.′ end of the helix is closer to the thumb domain than the fingers domain (The resulting set of ten different EC structures allows us to make direct comparisons of the different enzymes and further distinguish between conserved protein-RNA interactions found within all the picornaviral ECs and other conformations that are likely the result of crystal packing effects. This comparison can also be expanded to include similar structures from related viruses, allowing us to further highlight commonalities of viral polymerase-RNA complexes. The comparison shows that the RdRPs tightly associate with 7–8 base pairs of the product RNA duplex in an A-form helix that is held in place by an upstream clamp between the pinky finger and an α-helix from the thumb domain that packs into the minor groove. All three picornaviral polymerases exhibit a ≈4 Å opening of this channel in their EC structures as compared to their structures without RNA, indicating that the clamp structure holding the RNA product is a common feature of these polymerases . On the s domain , but aga1 of the previously described open conformation active site where motif A is not fully participating in the core RRM-fold of the palm domain and its aspartic acid Asp233 residue that is responsible for coordinating both Mg2+ ions during catalysis is pointed away from the catalytic center. In the CV_r2_Mg structure we see clear density for a bound magnesium ion that is coordinated by Asp330 in motif C and five water molecules in a six-fold coordination geometry, with Asp233 from motif A being involved in the network (The EC structures also allow for extensive comparisons of the RdRP active sites and the geometry of the RNA as it passes through the polymerase. All the structures were captured in state  network . This ma network , two respol PV_r5 crystal resulted in two nucleotide incorporation events and two translocation steps, giving a final state 1 structure that is essentially identical to the starting point, only with sequence variation in the RNA strands. The RNA-mediated lattice contacts of this crystal form allowed for significant twisting of one EC relative to the other within the dumbbell pair, but the structure within each EC was essentially unchanged. In contrast, the coxsackievirus 3Dpol CV_r2 lattice accommodated one translocation event upon 2′,3′-ddCTP addition by keeping the two ECs within a dumbbell pair essentially fixed while unraveling the ends of the two RNA duplexes at the junction point. These structures are both static snapshots of a post-translocation state that is essentially in the same conformation as the starting structure and therefore do not provide any insights into the details of how translocation is achieved in these viral polymerases. However, the structures do suggest that a straightforward NTP soaking experiment should always be employed as a simple method for sampling additional conformation states that may yield structures for such translocation intermediates in the future.The core RNA sequence used to assemble the stable ECs contains a pair of templating guanosines such that adding CTP can potentially result in elongation of the primer by at most two bases. In our prior work on the poliovirus polymerase EC structure the CTP crystal soaking experiment resulted in a single round of catalysis but no translocation of the RNA took place and the polymerase was trapped with its active site in the closed conformation. We hypothesized that the lack of translocation was due to crystal lattice constraints, and consistent with this we now observe translocation in two of the new lattices that are formed using weaker G-U base pairs in the coaxial stacking junction . A CTP sde novo initiating hepatitis C virus polymerase de novo initiation In summary, we have used a small set of five different RNA constructs to solve nine different picornaviral polymerase elongation complex structures. These structure show conservation of active site interactions between the polymerase and the RNA, such as the pre-positioned templating +1 base, the index finger binding pocked for the +2 nucleotide, the unique conformation of the template strand −1 to −2 phosphate linkage, a lack of significant interactions with the downstream template, and a consistent ≈4 Å opening of the RNA exit channel as compared to the polymerases in the absence of RNA. The structures presented here also demonstrate that engineering flexibly stacked RNA helices is a powerful experimental tool for the crystallization of polymerase ECs. The coaxial stack RNA-RNA interactions provide for a key association of complexes into dumbbell type structures that likely promote lattice nucleation, but they retain enough flexibility to allow a crystal lattice to grow via protein-protein contacts. The assembly and purification of the stable ECs themselves is straightforward for the primer dependent picornaviral polymerases, and the recent development of a method for generating similar complexes with the


The genome of Bunyamwera virus (BUNV) comprises three RNA segments that are encapsidated by the virus-encoded nucleocapsid (N) protein to form ribonucleoprotein (RNP) complexes. These RNPs are the functional templates for RNA synthesis by the virus-encoded RNA-dependent RNA polymerase (RdRp). We investigated the roles of conserved positively charged N-protein amino acids in RNA binding, in oligomerization to form model RNPs and in generating RNP templates active for both RNA replication and mRNA transcription. We identified several residues that performed important roles in RNA binding, and furthermore showed that a single amino acid change can differentially affect the ability of the resulting RNP templates to regulate the transcription and replication activities of the RdRp. These results indicate that the BUNV N protein possesses functions outside of its primary role of RNA encapsidation. Bunyaviridae of segmented negative-strand (SNS) RNA viruses contains more than 350 members categorized into five genera: Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. The prototype of the genus Orthobunyavirus is Bunyamwera virus (BUNV), which serves as a model for studying important pathogens within this family. As with all bunyaviruses, the BUNV genome comprises three segments, named small (S), medium (M) and large (L) (The family arge (L) . These sSeveral studies have investigated the specificity of this interaction for bunyaviruses. They indicate that, while there is no obligatory encapsidation sequence, there is evidence of preferred binding for viral sequences . It has Previous structural studies with human respiratory syncytial virus, rabies virus and vesicular stomatitis virus (VSV) show that the mechanism of RNA binding within the non-segmented negative-strand (NNS) RNA-virus RNP relies on multiple interactions, mediated by a variety of residues within specialized RNA-binding grooves . We selected 15 of these that were best conserved in terms of identity or positive charge for further analysis. We analysed the role of these conserved residues in RNA binding by exploiting our previously described ability to express and purify soluble N protein from bacterial cells as an N–RNA complex . The estPreviously described plasmid pET-BUN-N , and so we concluded that none of these residues play a critical role in N protein oligomerization.33P-end-labelled oligonucleotide REN-PAGE, which was designed to anneal to positive-sense products of both transcription and replication. To assess the role of the 15 charged residues in the formation and activity of RNPs, the plasmid expressing the BUNV N-protein ORF was modified to incorporate each amino acid change individually.We analysed the ability of all 15 N mutants to support the assembly of BUNV RNP templates, which were active for both transcription and replication. This was achieved by using a previously described assay (Primer-extension analysis detected both mRNAs and anti-genomic RNAs generated from assembled RNPs. By using densitometry to measure the abundance of their corresponding products, we quantitatively assessed the role of each altered residue on transcription and replication activities Fig. . Most mucis-acting signals for replication versus transcription. BUNV-replication and -transcription signals comprise interacting nucleotides from both ends of each active RNP template, although the critical nucleotides that comprise each signal are distinct (We are curious as to how the N protein is able to impart differential effects to the RdRp functions of transcription and replication. Presumably, any mechanism responsible is unlikely to involve activities that are common to both these RNA synthesis modes. One possibility is that the R94A mutation somehow affects access of the RdRp to distinct , 2005. PThe concept that the N protein of a negative-stranded RNA virus can differentially modulate the template activity of the RNP is not new, having been established with the polR1 mutant of the NNS RNA virus VSV . More re


Apis mellifera) have recently experienced higher than normal overwintering colony losses. Many factors have been evoked to explain the losses, among which are the presence of residues of pesticides and veterinary products in hives. Multiple residues are present at the same time, though most often in low concentrations so that no single product has yet been associated with losses. Involvement of a combination of residues to losses may however not be excluded. To understand the impact of an exposure to combined residues on honey bees, we propose a mechanism-based strategy, focusing here on Multi-Drug Resistance (MDR) transporters as mediators of those interactions.Honey bees (Using whole-animal bioassays, we demonstrate through inhibition by verapamil that the widely used organophosphate and pyrethroid acaricides coumaphos and τ-fluvalinate, and three neonicotinoid insecticides: imidacloprid, acetamiprid and thiacloprid are substrates of one or more MDR transporters. Among the candidate inhibitors of honey bee MDR transporters is the in-hive antibiotic oxytetracycline. Bees prefed oxytetracycline were significantly sensitized to the acaricides coumaphos and τ-fluvalinate, suggesting that the antibiotic may interfere with the normal excretion or metabolism of these pesticides.Many bee hives receive regular treatments of oxytetracycline and acaricides for prevention and treatment of disease and parasites. Our results suggest that seasonal co-application of these medicines to bee hives could increase the adverse effects of these and perhaps other pesticides. Our results also demonstrate the utility of a mechanism-based strategy. By identifying pesticides and apicultural medicines that are substrates and inhibitors of xenobiotic transporters we prioritize the testing of those chemical combinations most likely to result in adverse interactions. Honey bees are in trouble. Widespread depopulation of colonies often characterized by high overwintering losses has occurred since at least 2006 in the United States, threatening the sustainability of North American apiculture. Despite considerable effort, no single cause of the phenomenon called colony collapse disorder (CCD) has been identified, though associations of several pathogens and parasites appear to increase the risk of colony collapse Although pesticide drift and overdosing cause accidental bee kills no single pesticide has been directly implicated with widespread overwintering losses or CCD The problem, of course, is the large number of potentially adverse pesticide combinations which prevents evaluation of all, or even most, combinations of them. This problem challenges our ability to anticipate the risks associated with bee's exposure to a novel pesticide or to identify combinations of toxins contributing to a colony collapse. If we could identify mechanisms of the honey bee xenobiotic metabolism and excretion systems that systematically mediate multiple-toxin interactions, we could reduce the overwhelming number of candidate pesticide interactions to a smaller set of compounds that are substrates or inhibitors of the most predictive mechanisms.The membrane-bound transporter proteins from the ABC transporter family of proteins are found in all phyla Drosophila melanogasterHeliothis virescens (tobacco budworm) Manduca sexta (tomato hornworm) MDR transporters are relatively unstudied in insects, and completely neglected in honey bee toxicology. These transporters act in several insect tissues, including the cuticle The most well studied MDR transporter, p-glycoprotein (p-gp), has both a diverse range of substrates and is inhibited by an array of drugs, pesticides and plant compounds Interaction of neonicotinoid insecticides with insect MDR transporters has not yet been reported. Because of the likelihood of exposure of bees to these insecticides we ask if the neonicotinoid insecticides imidacloprid, acetamiprid and thiacloprid are substrates of honey bee MDR transporters. Evidence of neonicotinoid processing by MDR transporters would be significant because inhibition of those transporters could cause mortality at lower doses than normally expected for individual compounds.When fed to bees verapamil significantly increased the toxicity of all 5 acaricides/insecticides. Mean mortality of young worker bees topically treated with the acaricides coumaphos or τ-fluvalinate was significantly higher when bees were pretreated with verapamil , Table 1p = 0.002). Mean mortality of cohorts fed OTC alone were below 10% and were not significantly different from those fed sucrose alone  inhibited by verapamil play a role in protecting honey bees from pesticides, and that the acaricides coumaphos and τ-fluvalinate, and 3 neonicotinoid insecticides are substrates of these transporters in insects. The observation that coumaphos and τ-fluvalinate are substrates of honey bee p-gp or another MDR transporter was anticipated from previous study of mouse cells, and suggests that insect and mammalian MDR transporters share substrates. Clearly, the abundance of these pesticides found in the wax and pollen of bee hives 5050 of neonicotinoids possibly amplifying acute and chronic effects to bees at lower concentrations.This is the first report that neonicotinoid insecticides are substrates of insect MDR transporters. In efforts to protect honey bees, energetic opposition to the neonicotinoids has arisen in North America and Europe, but direct implication of them in overwintering losses has not been sustained by recent research The large increases in sensitivity to pesticides by inhibition of MDR transporters and the chemical diversity of the synergized pesticides suggest that these transporters may mediate adverse synergisms of diverse toxins in bees. Because of its clinical importance in human health, proven and candidate p-gp substrates and inhibitors of many types have been identified Bees were collected for laboratory bioassays from newly established colonies reared on new frames and freshly drawn comb. Colonies were not treated with apicultural medications or pesticides. Frames with emerging workers were taken from hives and placed into dark growth chambers maintained at 33±2°C and (70–80%) RH. Newly-emerged bees were collected from the frames daily and maintained in groups of 20–30 in 80×100 mm metal mesh cages capped at each end by standard polystyrene petri dishes. Bees were fed sucrose solution  through 1 mm holes from a 2.0 ml microfuge tube.Terramycin  was purchased from Dadant and Sons . Coumaphos, τ-fluvalinate  and verapamil were purchased from Sigma-Aldrich Inc. . Commercial formulations of imidacloprid (Admire Pro) and thiacloprid  were provided by Bayer CropScience , and acetamiprid (Assail) was provided by United Phosphorous Inc. .Verapamil (1 mM) and oxytetracycline  were incorporated into 50% sucrose solutions for oral dosing of 1–3 day old workers. Preliminary feeding trials of 1 mM solutions of oral verapamil revealed no toxicity. The 1.4 mM concentration of OTC provides a per-bee exposure comparable to that of the label-recommended dosage of 600 mg applied to a hive containing 12,000 bees—a typical colony size entering winter 2, and 1 ul of coumaphos (2 ug/ul)or τ-fluvalinate (3 ug/ul) in acetone  was applied to the dorsal thorax of each bee using an ISCO microapplicator driving a 1/4 cc tuberculin syringe. After application, bees were returned to cages containing the sucrose-drug or sucrose-only solution. Mortality of bees in each cage was recorded at 24 and 48 h. 5–10 replicate cohorts of 25 bees were tested for each acaricide - pretreatment combination.Cohorts of 3–6 day old workers pretreated by feeding with the two sucrose-drug solutions were anesthetized with COPre-fed cohorts of 3–6 day old workers were fed sucrose syrup containing one of the neonicotinoids. Mortality of each cage was recorded at 24 and 48 hours. Imidacloprid was tested at 5 and 50 ng/ul, acetamiprid at 25 and 100 ng/ul, and thiacloprid at 25, 100 and 500 ng/ul. These concentrations caused low-intermediate mortality of bees fed sucrose-only solution in preliminary range-finding experiments. 2–13 replicate cohorts of 25 bees were tested for each toxin concentration - pretreatment combination.The effects of verapamil or OTC pretreatment on insecticide/acaricide mortality were tested using a repeated measures analysis of variance . Following transformation (arcsine square-root), mortality was analyzed with a model that included pretreatment, insecticide concentration if multiple levels were used, and time endpoints (24 and 48 h) as fixed factors to assess the main effects and their interactions. Because only mortality at 24 h was available, analysis of τ-fluvalinate combined with OTC, was performed using a simple t-test, comparing the τ-fluvalinate and the τ-fluvalinate+OTC treatments.


Paragorgia arborea and Primnoa resedaeformis, in relation to terrain variables at multiple scales of 30 m, 90 m and 170 m were investigated at Røst Reef, Traena Reef and Sotbakken Reef on the Norwegian margin, with Ecological Niche Factor Analysis applied. To date, there have been few published studies investigating this aspect of gorgonian distribution. A similar correlation between the distribution of P. arborea and P. resedaeformis and each particular terrain variable was found at each study site, but the strength of the correlation between each variable and distribution differed by reef. The terrain variables of bathymetric position index (BPI) and curvature at analysis scales of 90 m or 170 m were most strongly linked to the distribution of both species at the three geographically distinct study sites. Both gorgonian species tended to inhabit local topographic highs across all three sites, particularly at Sotbakken Reef and Traena Reef, with both species observed almost exclusively on such topographic highs. The tendency for observed P. arborea to inhabit ridge crests at Røst Reef was much greater than was indicated for P. resedaeformis. This investigation identifies the terrain variables which most closely correlate with distribution of these two gorgonian species, and analyzes their terrain habitat selection; further development of predictive habitat models may be considered essential for effective management of these species.Investigating the relationship between deep-water coral distribution and seabed topography is important for understanding the terrain habitat selection of these species and for the development of predictive habitat models. In this study, the distribution of the deep-water gorgonians,  Paragorgia arborea and Primnoa resedaeformis are amongst the largest deep-water gorgonians, with colonies reaching heights of 50–250 cm Deep-water gorgonian corals are found throughout the world’s oceans and Mediterranean Sea, with the majority reported at depths of 200–1,000 m Deep-water corals (DWCs) are typically observed in abundance in areas of pronounced topographic relief Information on the distribution and population densities of deep-water gorgonians is required for conservation and management of these species, but such data is scarce given the high costs associated with sampling and surveying in deep sea. Predictive habitat suitability may be used as proxies for species distribution, or for optimizing mapping strategies P. arborea and P. resedaeformis, in relation to seabed topography at Røst Reef and Traena Reef, and P. arborea at Sotbakken Reef on the Norwegian margin. These three reef complexes vary greatly in topography and environmental conditions, with Røst Reef located on the shelf margin, Sotbakken Reef at the northern tip of the Norwegian Shelf and Traena Reef on the inner shelf  what, if any, trends exist in habitat selection of P. arborea and P. resedaeformis at Røst Reef and Traena Reef, and of P. arborea at Sotbakken Reef.This study investigated the distribution of the deep-sea gorgonians, er shelf . This stThe survey of the reefs presented in this study was permitted by the Institute of Marine Research, Norway.Røst Reef, Traena Reef and Sotbakken Reef are three reef complexes located on the Norwegian margin, at depths of 250–400 m , with liThe Røst Reef was discovered off the SW tip of the Lofoten islands by the Institute of Marine Research (IMR), Norway in May 2002 The Sotbakken Reef is located on a triangular plateau on the upper part of a large submarine slide (∼30 km in width) situated in the cross-shelf trough Håkjerringdjupet off Tromsø between Fugløybanken and Nordvestbanken The Traena Reef complex is located in a sheltered embayment on the northern edge of the cross-shelf trough Traenajupet The majority of the data used in this study was acquired during the Polarstern ARK XXII/1a expedition (2007) −1. The data was further cleaned using a Gaussian smoothing technique (used software package Adelie-GIS 1.8) to decrease the random error in the positioning data.The multibeam bathymetry data was logged by a Hydrosweep DS-3 multibeam sonar data acquisition system at Røst Reef and Sotbakken Reef and further processed using the open source software MB-system. The processed data was projected from longitude and latitude on the WGS84 reference ellipsoid to UTM Zone 32N coordinates at the Røst Reef, and to UTM Zone 34N coordinates at the Sotbakken Reef, and gridded to 10 m resolution raster. The bathymetry data at Traena Reef was provided in fully processed format with 10 m resolution and WGS 84 UTM Zone 32N coordinates. The positioning data for the JAGO dives was cleaned of erroneous readings by removing outliers, assuming a maximum JAGO survey speed of ∼0.8 m sP. arborea and P. resedaeformis colonies was logged using the software package OFOP 3.2.0j (Ocean Floor Observation Protocol) throughout each dive video, with locations determined by comparison with the associated, cleaned and timecode-stamped JAGO submersible positioning data. In addition, the substrate was characterized throughout each dive video, with the following categories used: coral rubble, exposed dead coral framework, live isolated coral framework, dense live coral, coral terrace, soft sediment, hardground, pebbles/cobbles, boulders  were calculated at scales of 30 m, 90 m and 170 m, corresponding to moving window sizes of 3×3, 9×9 and 17×17 cells, from the gridded multibeam bathymetry data for each of the study sites . The scaP. arborea and P. resedaeformis. Certain species (such as P. arborea and P. resedaeformis) are most numerous in complex habitats with strong structural components , whereas other species tend to occupy soft sediment on flat terrain P. arborea and P. resedaeformis.In the current study, slope has relevance in that it influences current flow amplification Ecological Niche Factor Analysis (ENFA) is a multivariate statistical method, which transforms a number of possibly correlated variables into the same number of uncorrelated factors. But unlike the similar method of Principal Component Analysis, the factors have ecological meaning, with the first factor accounting for the marginality information and part of the specialization information of species distribution, and the following factors accounting for the remaining specialization information P. arborea at Sotbakken Reef by using the 20 terrain variables outlined in In this study, the terrain variables were first normalized by applying the Box-Cox algorithm P. arborea and P. resedaeformis colonies, respectively, observed at the Røst Reef  was found to be closely related with the distribution of both species (particularly P. arborea) at Røst Reef  , and of the relief structure situated on the upper slide (Dive3)  traversed the ridges, with the third (Dive3) surveying the upper slide . Dense l (Dive3) . The L. ressions .P. arborea at Sotbakken Reef. P. arborea tended to inhabit positions with higher values of BPI, mean curvature, profile curvature, and lower values of plan curvature than mean available habitat, indicated by the coefficients of the marginality factor  to the NW . A largeL. pertusa frameworks were observed throughout the high relief structure on the western crest , similar close relationship may also exist. Additionally, the degree to which the distribution of P. arborea and P. resedaeformis correlated with terrain variables is scale-dependent. The selection of suitable analysis scales of terrain variables is therefore important in habitat prediction, to ensure features which most influence distribution of a target species are captured.The terrain variables of BPI and curvature have been reported to be strongly correlated with P. arborea and P. resedaeformis in relation to terrain variables is scale-dependent. At Røst Reef, the BPI, mean curvature, plan curvature and profile curvature at analysis scales of 90 m and 170 m, and ASP9 proved to be most relevant to the observed distribution of both species  than the mean available habitat, shown by ENFA , whilst P. resedaeformis was abundant on the east-facing side of the ridges and the small relief structures on the relatively smooth upper slide with west-facing direction   . The twoP. arborea and P. resedaeformis and terrain variables at locations on the Norwegian continental shelf using the multivariate statistics approach ENFA. A similar correlation between species distribution and terrain variables was found at each particular study site of Røst Reef and Traena Reef. The variables of curvature and BPI at analysis scales of 90 m or 170 m, particularly BPI, mean curvature and plan curvature, were most closely related to the distribution of P. arborea and P. resedaeformis at Røst Reef and Traena Reef, and P. arborea at Sotbakken Reef.This study investigated the relationship between the distribution of L. pertusa framework may play in influencing food supply, the distribution of soft sediment around the western relief structure on the crest of the mound at Sotbakken Reef and around the reefs at Traena Reef is also likely to play a role in limiting the distribution of both species locally.Both species tended to inhabit regions of local topographic highs with positive BPI at each study site, with both species observed at Sotbakken Reef and Traena Reef almost exclusively on local topographic highs. A percentage of both gorgonian species were observed on the lower sections of the ridges or the ridge-like features, or in the bathymetric depressions between these structures at Røst Reef. In addition to the role terrain features and dense Further investigation of additional environmental variables influencing coral distribution, such as the hydrodynamic conditions at both local and regional scales, would further improve our understanding of the ecological niches required by these gorgonian species.


Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3′-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3′-end of the TBSV (−)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (−)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of  Tomato bushy stunt virus (TBSV) into its replicase complex. They also show that Ded1p binds to the viral (−)RNA and promotes (+)-strand TBSV synthesis when added to a yeast-based cell-free extract depleted for Ded1p. An ATPase defective Ded1p mutant failed to promote TBSV replication in vitro, suggesting that the helicase activity of Ded1p is essential for its function during TBSV replication. In addition, the authors also show that another host protein, which also binds to the (−)RNA, namely glyceraldehyde-3-phosphate dehydrogenase (GAPDH), further enhances TBSV (+)RNA when added together with Ded1p to yeast-based cell-free extract. In summary, the authors show that the major functions of Ded1p and GAPDH host proteins are to promote TBSV replication via selectively enhancing (+)-strand synthesis.Subverted host factors play a role in plus-strand RNA virus replication. Small RNA viruses do not code for their own helicases and they might recruit host RNA helicases to aid their replication in infected cells. In this paper, the authors show that the Ded1p DEAD-box helicase, which is an essential translation factor in yeast, is recruited by  All eukaryotic plus-stranded (+)RNA viruses have similar replication cycles in infected cells. After translation of their mRNA-sense genomic RNA(s), the viral RNA and the viral replication proteins are recruited to the site of viral replication in membranous compartments. After the assembly of the membrane-bound viral replicase complexes (VRC), the viral replicase uses the viral RNA as a template to produce complementary (−)RNA. This is then followed by (+)-strand synthesis in an asymmetric manner, producing excess amounts of (+)-strand progeny, which is released from replication for other viral processes. For efficient replication, (+)RNA viruses recruit numerous host proteins Tomato bushy stunt virus (TBSV) is a model plant RNA virus that is used intensively for identification and characterization of host factors pol, which are sufficient to support TBSV replicon (rep)RNA replication in yeast (Saccharomyces cerevisiae) model host pol are components of the membrane-bound VRC that also requires the tombusviral (+)repRNA as a platform during its assembly and activation TDH2 and TDH3 in yeast) The highly purified tombusvirus VRC is known to contain at least six permanent resident host proteins, including the heat shock protein 70 chaperones  DEAD-box proteins constitute the largest family of RNA helicases that are involved in all aspects of cellular metabolism and perform RNA duplex unwinding and remodeling of RNA-protein complexes in cells In this paper, a yeast-based cell-free TBSV replication assay and recombinant Ded1p was used to test the role of Ded1p in TBSV replication. We define that Ded1p plays a role in enhancing plus-strand (+)RNA synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus VRC and binds to the 3′-end of the viral minus-stranded (−)RNA. The obtained data support the model that Ded1p unwinds the 3′-end of the TBSV (−)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and GAPDH play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (−)RNA and the replication proteins.pol replication proteins are provided Since Ded1p is an essential translation factor for all yeast mRNAs in vitro (not shown), contained Ded1p  To further test what step(s) Ded1p promotes during TBSV replication, we performed a two-step replication assay based on yeast CFE. In this assay, the first step includes the assembly of the replicase complex on the endogenous membranes present in CFE in the presence of the viral (+)repRNA, the p33/p92 replication proteins and ATP/GTP Addition of purified Ded1p to the CFE during the first step did not increase TBSV replication . InteresTo further test the direct effect of Ded1p on RNA synthesis, we utilized detergent-solubilized and affinity-purified tombusvirus replicase from Ded1p-depleted yeast . This pude novo initiation on the (−)RNA template by the tombusvirus replicase.We found that addition of purified recombinant Ded1p to the purified tombusvirus replicase programmed with the (−)repRNA stimulated (+)-strand synthesis by ∼2.5–3-fold  decreasein vitroThe ATPase inactive mutant D1 , did facin vitroin vitro.To test if Ded1p can also stimulate the activity of the tombusvirus replicase on (+)-stranded RNA templates, we used DI-72(+) RNA in the purified tombusvirus replicase assay . The TBS32P-labeled TBSV (+) and (−)RNAs in binding to purified recombinant Ded1p revealed that (−)RNA bound more readily to Ded1p in vitro than (+)RNA (32P-labeled TBSV (−)repRNA in binding to Ded1p (To identify the region(s) of the TBSV RNA bound by the recombinant Ded1p, we performed electrophoresis mobility shift assay (EMSA) with purified components. Comparison of n (+)RNA  as unlabFurther testing of Ded1p binding to (+)repRNA regions revealed that RIV(+), representing the 3′ noncoding region in TBSV RNA, was bound more efficiently by Ded1p , lane 11in vitro -strand RNA synthesis on the (−)-stranded FHV template by ∼3-fold (in vitro (To test if Flock house virus (FHV) replicase is affected by Ded1p, first we have developed an  ∼3-fold , lane 3, ∼3-fold  or FHV RNA synthesis initiated from the 3′ end of the (−)repRNA up to ∼3-fold (de novo (+)RNA synthesis and 3′TEX by ∼2- and ∼3-fold, respectively (de novo initiation from the 3′ end of the (−)RNA template.GAPDH (Tdh2p in yeast) RNA binding protein is also a host factor stimulating (+)RNA synthesis by the tombusvirus replicase paration . While D ∼3-fold , lane 6,ectively , lane 7.We also performed similar experiments with a short partial RNA/DNA duplex as a template. Both Ded1p and Tdh2p alone could enhance (+)RNA synthesis on the short RNA/DNA duplex , lane 4.RNA viruses form several different ribonucleic acid-protein complexes (RNP) in infected cells during various steps of infection It is not yet known if Ded1p is the only host helicase needed for TBSV replication, since genome-wide screens and global proteomics approaches with TBSV have identified additional host helicases as well in vivo and in vitro approaches might be necessary to dissect the function of Ded1p in RNA virus replication as shown in this paper. By using a Ded1p-depleted yeast CFE in combination with recombinant Ded1p allowed us to define that the ATPase (helicase) activity of Ded1p is required for efficient TBSV (+)-strand synthesis (see below). Ded1p also increased the RdRp activity of the FHV replicase, suggesting that recruitment of DEAD-box helicases might also be useful for additional small RNA viruses (see below).The essential nature of Ded1p for host mRNA translation, however, makes characterization of Ded1p function as a host factor difficult. Therefore, the combined use of in vitro -strand synthesis, while its effect on (−)RNA synthesis is less pronounced. The ATPase activity of Ded1p is required for this stimulatory effect, suggesting that the helicase function of Ded1p is likely important for unwinding the secondary structure of (−)RNA template with the purified tombusvirus replicase or destabilizing the replication intermediate with the membrane-bound VRC, which might contain dsRNA structure. This function of Ded1p can explain why yeast with down-regulated Ded1p level produced small amount of (+)-stranded RNA progeny, albeit it has also shown that depleted Ded1p reduced the level of p33/p92 in yeast in vitroThe Based on the available data, it seems that TBSV (+)RNA synthesis is not only affected by the p33/p92 replication proteins, but by GAPDH and Ded1p host proteins as well. Since the viral replication proteins were shown to bind to TBSV (−)RNA nonspecifically Overall, host DEAD-box or related RNA helicases have been shown to affect many different aspects of virus infections, including translation of viral proteins Saccharomyces cerevisiae strain BY4741 (MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and TET::DED1 yeast strain  was obtained from Open Biosystems . The plasmid pESC-HIS-Gal-His33/Gal-DI-72 expressing Cucumber necrosis virus (CNV) 6×His-tagged p33 and the TBSV DI-72 repRNA was described earlier E. coli as GST fusion using plasmid pGEX-TDH2, described earlier CCAGCTGCAGTCACCACCAAGAAGAGTTG)/ #3957(CCAGGAATTCATGGCTGAACTGAGCGAACAAG) and the yeast genomic DNA as a template. The plasmids pMal-Dx, expressing different Ded1p mutants, were prepared by PCR using #3956/ #3957 primers and plasmids containing mutated DED1 sequences DED1 wt and mutant sequences were digested with EcoRI and PstI and inserted between EcoRI and PstI sites in pMalc-2× (New England Biolab).GGAGGTTCCGGGGGCTCTGGAGGTTACCCATACGATGTTCCAGATTACGCTTCCGG-GGATTACAAGGACGATGACGATAAGTAACTCGAGCTCC (coding for G G S G G S G G Y P Y D V P D Y A S G D Y K D D D D K amino acids). The PCR product was amplified with oligos #3629 (CCGATCATGACTCTAAAAGTTATTCTTGGAG) and #3716 (GGAGCTCGAGTTACTTATCGTCATCGTC) followed by digestion with PagI and XhoI. It was cloned into NcoI and XhoI digested pCupHis92 To express recombinant FHV protein A in yeast, we generated plasmid pGAD/Cup/FHV/protein A/C-term/HA/FLAG. The following sequence was fused downstream to the full length FHV protein A coding region by repeated PCR and cloning steps: E. coli were carried out as described earlier with modifications E. coli strain BL21(DE3) CodonPlus. Protein expression was induced using isopropyl β-D-thiogalactopyranoside (IPTG) for 8 h at 23°C in the case of host proteins and at 16°C in the case of p33 and p92, then the cells were collected by centrifugation . The cells were suspended and sonicated in MBP column buffer containing 30 mM HEPES-KOH pH 7.4, 25 mM NaCl, 1 mM EDTA, 10 mM β-mercaptoethanol. The extract was then centrifuged at 16,000 g for 10 min, followed by incubation with amylose resin (NEB) for 15 min at 4°C. After washing the resin 2 times with the column buffer, the proteins were eluted with column buffer containing 0.18% (V/W) maltose. Purification of GST-tagged TDH2 (pGEX-TDH2) in vitroExpression and purification of the recombinant MBP-tagged host proteins and the MBP-tagged TBSV p33 and p92 replication proteins and Ded1p helicase from 32P-labeled or unlabeled full-length DI-72 (+) and (−)RNAs and the four separate regions (RI-IV), were generated as described GTAATACGACTCACTATAGTAAACAATTCCAAGTTCCAAAATGG) and #3509 (ACCTTAGTCTGTTGACTTAAACTGG) or #3519 (GTAAACAATTCCAAGTTCC) and #3527 (GTAATACGACTCACTATAGGGAACCTTAGTCTGTTGACTTAAAC) for (+) or (−)DI-634 RNAs, respectively, and pDI634 The in vitro were prepared as described earlier in vitro TBSV replication assays were performed in 20-µl total volume containing 2 µl of CFE, 0.15 µg DI-72 (+)repRNA transcript, 200 ng purified MBP-p33, 200 ng purified MBP-p92pol (both recombinant proteins were purified from E. coli), 30 mM HEPES-KOH, pH 7.4, 150 mM potassium acetate, 5 mM magnesium acetate, 0.13 M sorbitol, 0.4 µl actinomycin D (5 mg/ml), 2 µl of 150 mM creatine phosphate, 0.2 µl of 10 mg/ml creatine kinase, 0.2 µl of RNase inhibitor, 0.2 µl of 1 M dithiothreitol (DTT), 2 µl of 10 mM ATP, CTP, and GTP and 0.25 mM UTP and 0.1 µl of [32P]UTP 32P-labeled RNA products were separated by electrophoresis in a 5% polyacrylamide gel (PAGE) containing 0.5× Tris-borate-EDTA (TBE) buffer with 8 M urea. To detect the double-stranded RNA (dsRNA) in the cell-free replication assay, the 32P-labeled RNA samples were divided into two aliquotes: one half was loaded onto the gel without heat treatment in the presence of 25% formamide, while the other half was heat denatured at 85°C for 5 min in the presence of 50% formamide CFEs from BY4741 or TET::DED1 strains capable of supporting TBSV replication In vitro TBSV replication in the fractions was performed as described Fractionation of the whole cell extract was done according to ADH1 promoter and DI-72 repRNA was under the Gal1 promoter. Transformed yeast were pre-grown on SC-ULH− media containing 2% glucose at 29°C. After centrifugation at 2,000 rpm for 3 min and washing pellet with selective media containing 2% galactose and 1 mg/ml Doxycycline, yeast were grown for 24 hours in SC-ULH− media containing 2% galactose at 23°C. The replicase purification was done according to a previously described procedure 2, 10 mM KCl, 0.5 M NaCl, , and 1% [V/V] yeast protease inhibitor cocktail (Ypic)] by glass beads using FastPrep Homogenizer . The membrane fraction containing the viral replicase complex was solubilized with 1 ml TG buffer containing 1% Triton X-100, 1% [V/V] Ypic as described 2, 10 mM KCl, 50 mM NaCl, 1% Triton X-100, and 0.15 mg/ml Flag peptide (sigma)].Yeast strains (BY4741 and TET::DED1) were transformed with plasmids pGBK-HisFlagp33 (or pGBKHisp33 as a control), pGAD-HisFlagp92 (or pGAD-Hisp92 as a control) and pYC-DI72. The 6×His-Flag double-tagged HF-p33 and p92 were expressed from the In vitro RdRp activity assay was performed by using DI-72(−) or (+) RNA template transcribed in vitro by T7 transcription 32P-labeled RNA was performed at 37°C for 30 min in a 1× RNase ONE buffer containing 0.1 µl of RNase ONE (Promega).− plates, yeast were pre-grown overnight in selective media containing 2% glucose at 29°C. After centrifugation at 2,000 rpm for 3 min and washing pellet with selective media containing 2% galactose, yeast were grown 36 hours at 29°C in SC-LH− media containing 2% galactose and 50 µM CuSO4 to induce FHV RNA replication. Affinity-purification was done similarly as for tombusvirus, except for using different buffers: homogenization buffer consisted of 50 mM Tris–HCl [pH 8.0], 0.4 M sorbitol, 5 mM MgCl2, 50 mM KCl, 0.03% β -mercaptoethanol and 1% [V/V] yeast protease inhibitor cocktail (Ypic)] and solubilization (washing) and elution buffers contained 50 mM Tris–HCl [pH 8.0], 0.4 M sorbitol, 5 mM MgCl2, 50 mM KCl, 0.5 M NaCl, 0.03% β-mercaptoethanol, 1% Triton X-100 and 1% [V/V] yeast protease inhibitor cocktail (Ypic)]. Elution buffer also contained 0.15 mg/ml Flag peptide (Sigma)].To obtain FHV replicase preparation, BY4741 yeast strain was transformed with plasmid pGAD/Cup/FHV/proteinA/C-term/HA/FLAG and pESC-His-GAL1::FHVRNA1framshift. After selection of transformed yeast on SC-LHCCAGCTGCAGTCACCACCAAGAAGAGTTG) / #4602 (CCAGCCATGGCCACCAAGAAGAGTTG) followed by digestion with EcoR1 and Nco1 for DED1 and #4312(CCAGGGATCCATGACTTACGGTGGTAGAG) / #4603 (CCAGCCATGGATAGTTTGAACGACCTC), #4318 (CCAGGGATCCATGAGTCGCTACGATAGCCG) / #4604 (CCAGCCATGGGCTCCACCCTCTTCTGCTC) followed by digestion with BamH1 and Nco1 in the case of RH20 gene, respectively. Digested PCR products were fused to NubG at either the 5′- or 3′- termini (NubG-x and x-NubG) by cloning into pPRN-N-RE or pPRN-C-RE vectors −/Leu− synthetic minimal medium plates. Transformed colonies were picked with a loop, re-suspended in water, and streaked onto TLHA− (Trp−/Leu−/His−/Ade−) plates to test for p33–helicase protein interactions as described The split-ubiquitin assay was based on the Dualmembrane kit3 . The bait construct, pGAD-BT2-N-His33, expressing the CNV p33 replication protein has been described earlier S. cerevisiae strain DED1::6×HA-hphNT1 was generated by homologous recombination using strain BY4741. PCR was performed using plasmid pYM-16 (EUROSCARF) GCAGAAAACGAAGAATCCTCACCCTAGTTTGTCTGAAATCAATCGATGAATTCGAGCTCG) / #2494 (GGCTGGGGTAACAGCGGTGGTTCAAACAACTCTTCTTGGTGGCGTACGCTGCAGGTCGAC). The PCR products were transformed to BY4741 and recombinant yeast colonies were selected in YPD plates supplemented with hygromycin. Recombinant yeast strains were transformed with plasmids pGBK-HisFlagp33, pGAD-HisFlagp92 and pYC-DI72 ADH1 promoter and DI-72 transcript was under GAL1 promoter. After selection of transformed yeast on SC-ULH− plates, yeast were pre-grown overnight in selective media containing 2% glucose at 29°C. After centrifugation at 2,000 rpm for 3 min and washing the pellet with selective media containing 2% galactose, yeast were grown for 36 hours in SC-ULH− media containing 2% galactose at 23°C. 200 µl of pelleted yeast were used to affinity-purify HF-p33 and HF-p92 with anti-FLAG M2 agarose as described previously  and AP-conjugated anti-mouse antibody . DED1-6×HA protein was detected with anti-HA antibody from rabbit  and AP-conjugated anti-rabbit  followed by NBT-BCIP detection.see also  [14], .Co-purification of the p33 replication protein with Ded1p from yeast. (A) 6×His/ FLAG-tagged Ded1p was purified from yeast extracts containing 6×His/FLAG -Ded1p (lane 1) or not containing 6×His/FLAG-tagged Ded1p (lane 2) using a FLAG-affinity column Top panel: Western blot analysis of p33 tagged with 6×HA with anti-HA antibody in the purified Ded1p preparations. Bottom panel: Western blot analysis of 6×His/FLAG-tagged Ded1p with anti-His antibody. Each experiment was repeated three times. (B) Co-purification of tombusvirus replicase activity with Ded1p from yeast. FLAG-tagged Ded1p was purified from yeast expressing FLAG-Ded1p (lanes 2–4) or from yeast not expressing FLAG-Ded1p (lane 5) using a FLAG-affinity column. Yeast also co-expressed p33/p92/DI-72 repRNA as indicated by “+”. External DI-72(−)RNA template was given to samples as shown. Representative denaturing gel of (EPS)Click here for additional data file.Figure S3pol replication proteins and DI-72 (+)repRNA were used to affinity-purify the RNA-free tombusvirus replicase. The in vitro assays were programmed with DI-72 (−)repRNA, and they also contained purified recombinant Ded1p. (B) Representative denaturing gel of 32P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 1.0 µg of purified recombinant Ded1p. The level of complementary RNA synthesis producing “repRNA”  in each sample was compared to that of the replicase activity obtained in the absence of added recombinant protein. Note that this replicase preparation also synthesizes internal initiation products (“ii”) and 3′-terminal extension products (“3′TEX”). RNAse One digestion was used to confirm the replicase products: the de novo products are insensitive, while the 3′TEX product changes migration after RNase treatment (marked with an open arrowhead in lanes 2 and 4). Each experiment was repeated three times. (C) The ATPase activities of the Ded1p mutants based on REF 38. (D) Representative denaturing gel of 32P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 1.0 µg of purified recombinant Ded1p mutants. See Panel B for further details.Ded1p mutants with ATPase activity promote plus-strand synthesis by the affinity-purified tombusvirus replicase. (A) Scheme of the tombusvirus replicase assay. Yeast with depleted Ded1p co-expressing p33 and p92(EPS)Click here for additional data file.Figure S4pol replication proteins and DI-72 (+)repRNA were used to affinity-purify the RNA-free tombusvirus replicase. (B) Representative denaturing gel of 32P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro in the presence of 0.5 µg of purified recombinant Ded1p or the D1 mutant is shown. The in vitro assays were programmed with DI-72 (+)repRNA, and they also contained purified recombinant Ded1p (lane 3) or the D1 mutant (lane 4) in addition to ATP/CTP/GTP and 32P-UTP. (C) Representative denaturing gel of 32P-labeled RNA products synthesized by the purified FHV replicase in vitro in the presence of 1.0 µg of purified recombinant Ded1p or D1 mutant on the TBSV DI-72 (+)RNA template. (D) Representative denaturing gel of 32P-labeled RNA products synthesized by the purified FHV replicase in vitro in the presence of 1.0 µg of purified recombinant Ded1p or D1 mutant on the FHV DI-634 (+)RNA template.Ded1p helicase does not promote minus-strand synthesis by the affinity-purified tombusvirus and FHV replicases. (A) Scheme of the procedure. Yeast with depleted Ded1p co-expressing p33 and p92(EPS)Click here for additional data file.Figure S5in vitro. (A) In vitro binding assay with purified Ded1p using 32P-labeled ssRNA templates representing the four regions of DI-72(+) RNA. The assay contained ∼0.1 pmol labeled RNA plus 1.0 µg of purified recombinant Ded1p. The unlabeled competitor was DI-72(+) RNA (3 pmol). The free or Ded1p-bound ssRNA was separated on nondenaturing 5% acrylamide gels.Ded1p binds to various regions of TBSV (+)RNA (EPS)Click here for additional data file.Figure S6In vitro pull-down assay of GST-His-tagged Ded1p with MBP-p33 or MBP-p92 using amylose resin. Top panel: Western blot analysis with anti-His antibody of GST-His-tagged Ded1p co-purified with MBP-p33 and MBP-p92. MBP was used as a negative control. Bottom panel: Coomasie stained SDS-PAGE gel, showing quality and quantity of purified MBP, MBP-p33 and MBP-p92 proteins. (C) In vitro reverse pull-down assay of MBP-p33 or MBP-p92 with GST-His-tagged Ded1p using glutathione resin. Western blot analysis with anti-MBP antibody of MBP-p33 and MBP-p92 co-purified with GST-His-tagged Ded1p. GST-His was used as a negative control. Bottom panel: Coomasie stained SDS-PAGE gel, showing quality and quantity of purified proteins GST-HisDED1 and GST-His.Interaction between Ded1p and the TBSV replication proteins. (A) Split ubiquitin assay was used to test binding between p33 and the shown full-length proteins. The bait p33 was co-expressed with the prey proteins in yeast. Ssa1p (HSP70 chaperone), and the empty prey vector (NubG) were used as positive and negative controls, respectively. The image shows 10-fold serial dilutions of yeast cultures. (B) (EPS)Click here for additional data file.Figure S7Arabidopsis RH20 helicases.Comparison of the amino acid sequence of the yeast Ded1p (top) and the (PDF)Click here for additional data file.Figure S8in vitro. (A) In vitro binding assay with purified Ded1p using 32P-labeled FHV and TBSV RNA templates representing (+) and (−)RNAs. The assay contained ∼0.1 pmol labeled RNA plus 0.4 µg  or 1.0 µg  of purified recombinant Ded1p. The free or Ded1p-bound ssRNAs were separated on nondenaturing 5% acrylamide gels. (B) In vitro pull-down of GST-His-tagged Ded1p or GST-His with MBP-protA of FHV using amylose resin. Top panel: Western blot analysis with anti-His antibody of GST-His-tagged Ded1p co-purified with MBP-protA. MBP and GST-His were used as negative controls. Bottom panel: Coomasie stained SDS-PAGE gel, showing quality and quantity of purified MBP, and MBP-protA proteins.Ded1p binds to FHV RNA (EPS)Click here for additional data file.Text S1(DOC)Click here for additional data file.