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The current work shows that the extreme N-terminal region of bunya-, tenui- and arenavirus L-proteins functionally corresponds to the N-terminal region of the PA subunit of orthomyxoviruses. Given that the three influenza A polymerase subunits total residues, very similar to the size of many bunyavirus complete L-proteins and all these viral enzymes have common mechanisms of transcription cap-snatching and replication, a natural hypothesis that follows is that the L-proteins might be architecturally, structurally and functionally equivalent to a concatemer of the three influenza polymerase subunits in the order PA-PB1-PB2 Figure 8.

Some indirect support for the functional concatenation of the influenza polymerase subunits comes from the fact that the inter-subunit interactions are dominated by contacts between the C and N-terminal extremities of respectively PA and PB1 and PB1 and PB2 as visualised by recent crystal structures reviewed in [11]. The most significant implication of this hypothesis is that the C-terminal third of the L-protein might be structurally and functionally equivalent to PB2, which contains the cap-binding domain required for cap-snatching. Unfortunately, this region of the L-protein is the least well conserved and there are no obvious cross-genera conserved motifs that could point to a putative cap-binding site similar to that described for influenza A PB2 subunit [12].

This is perhaps not surprising as the PB2-like subunits of, for instance, salmon anaemia and Quaranfil viruses, two non-influenza orthomyxoviruses, are highly diverged from influenza [31] , [32] , even though both these viruses appear to possess an endonuclease at the N-terminus of the PA subunit Figure 7. In fact, there is no clear proof that any L-protein directly binds capped RNAs and even some evidence that in hantaviruses the viral N-protein may play this role [33]. Clearly more experimental work is required to elucidate the complete mechanism of cap-snatching in bunya-, tenui- and arenaviruses and to validate or otherwise the hypothesis that L-proteins are architecturally equivalent to the concatenation of PA-PB1-PB2.

Finally it is important to note that for nearly two decades, influenza virus endonuclease has been targeted for anti-viral drug discovery and a number of specific endonuclease inhibitors have been described [17] , [34] , [35] , [36] , [37]. Most of these compounds implicitly target the two metal binding site of the endonuclease, which is also the target for many HIV integrase inhibitors [38] including the currently approved raltegravir [39] , [40].

The recent structure determination of the endonuclease of influenza virus polymerase [13] , [14] gives new impetus to structure-based optimisation of these inhibitors.

Bishop, David H. L.

The results described here show that bunyaviruses and arenaviruses, amongst which are several dangerous and emerging pathogens, contain a very similar endonuclease to influenza virus, which is also therefore a good target for anti-viral drug design. Indeed, the close similarities between influenza and bunyavirus endonucleases suggests that compounds targeting a broad spectrum of segmented negative strand RNA viruses could be envisaged. In addition this structure provides the first concrete proof that these compounds do indeed chelate the two divalent cations in the endonuclease active site.

Mutagenesis of the proteins expressed in E. Labelled protein was obtained by expressing LC protein in E. The protein from the soluble fraction was loaded onto a 5 ml Nickel column, washed with 10 volumes of lysis buffer with 50 mM imidazol and eluted with 5 volumes of mM imidazol. After TEV cleavage all proteins have an additional glycine at the N-terminus. A second nickel column step was performed to remove unwanted material.

The resulting untagged proteins were concentrated and purified by gel filtration chromatography using a SD75 column Pharmacia with lysis buffer for in vitro experiments or 20 mM HEPES pH 7. Purified LC proteins are contaminated with a small percentage of a degradation fragment of size LC Products were characterized by N-terminal sequencing and mass spectrometry. The resulting papain resistant fragments had molecular weights between Proteins LC, , , and were subsequently produced. Finally, the protein construct LC was used for all in vitro biochemical experiments and LC for structural studies.

Divalent cations were added to 2 mM final concentration. For data analysis the heat produced by the metal ion dilution into the buffer was subtracted from the heat obtained in the presence of protein. The same procedure was performed with up to 12 mM of MgCl 2 but gave no interaction signal. The binding isotherms were analyzed by non-linear least squares fitting Microcal Origin software using models corresponding to a single site or two independent sites for the D52A and the wt respectively.

Thermodynamic values given are the average and standard deviation of at least two experiments. Proteins LC, , , and were expressed and tested for crystallization using a Cartesian nanovolume robotic system for screening.

The seleno-methionine LC crystals were obtained with a reservoir composition of 3. Crystals are of space-group P 6 1 22 with four molecules in the asymmetric unit. Native and DPBA data were collected to 2. Statistics of data collection and refinement are given in Table 1. The structure solution was obtained by the SAD method using autoSHARP [42] which found 16 anomalous sites, four including a manganese site for each of the four chains in the asymmetric unit.

The loop containing Asp52 is either in the open position or partially open and intermediate. The structure of the complex with the inhibitor was solved by molecular replacement using PHASER [45] and the previously obtained model. The loop containing Asp52 is in the closed position. Sub-confluent monolayers of Huh7 cells in well plates were transfected with 0. An additional 0. Structure figures were drawn with Molscript [46] or Bobscript [47] and rendered with Raster3d [48]. Metal ion and inhibitor binding in the active site of LC Blue: final 2fo-fc electron density at 0.

Anomalous difference map contoured at 4. Manganese ions and water molecules are represented by pink and blue spheres respectively. Mn1 has anomalous peak heights of between 7. DPDA is in purple stick representation. Colours are as in a with in addition, the unbiased fo-fc positive difference electron density at 0. Mn1 and Mn2 have anomalous peak heights of respectively between Surface representation of the LACV top, cyan and Influenza bottom, violet endonuclease structures in two orientations after superposition.

The conserved active site residues are coloured in yellow and the two metal ions are red spheres. The LC active site is in an open channel formed between two lobes of the protein into which double-stranded nucleic acid might fit. N- and C-termini of the proteins are marked. Nuclease assays of LC in the presence magnesium. Significant RNA digestion is only observed above Even at the highest concentrations the activity with magnesium is far less than that with 2 mM manganese.

The maximum activity is achieved with manganese ions for both proteins, although the influenza endonuclease has some activity in the presence of magnesium ions, whereas LC does not. Neither have activity in the presence of calcium ions. Single Thermofluor experiment of LC in the presence of 2 mM of various divalent cations. Anal Biochem — The apparent melting temperature is derived from the inflexion point of the curve and the value given represents the average of three separate experiments. All the curves show similar denaturation patterns. For cobalt, the fluorescence was quenched by the metal but interpretable curves were still obtained off-set.

Manganese ion binding to LC In each case, the upper plot shows the binding isotherm and the lower plot shoes the integrated values of each corresponding isotherm after subtracting the heat produced by the manganese dilution. For wild-type A the data were fitted with a model comprising two independent sites yielding Kds of 7. For the D52A mutant B the data were fitted with a model comprising a single site giving a Kd of The red points were not used for the curve fitting. The lack of the second binding site explains the lower molar ratio needed for saturation.

Values for the thermodynamic parameters derived from the model fitting are shown in Supplementary Table S1. Schematic of the procedure used to reconstitute recombinant LACV nucleocapsids in vivo. Cells were transfected with expression plasmids for the viral polymerase genes N, L and a minireplicon construct encoding a Renilla luciferase Ren-Luc gene flanked by viral promoter sequences vRen. A co-transfected firefly luciferase FF-Luc serves as transfection control not shown. At 24 h post-transfection, cells were fixed, permeabilised, and immune-stained using a rabbit polyclonal antiserum raised against recombinant LC B and C.

Luciferase counts were normalized to L activities with cotransfected pTM1. We acknowledge the Partnership for Structural Biology for an integrated structural biology environment, notably the high-throughput crystallisation facility. Work in the Weber laboratory benefited from the excellent technical assistance of Valentina Wagner. We are indebted to Luis Carrasco for rapidly providing the polio virus 2A constructs and for helpful advice.

The authors have declared that no competing interests exist. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Viral RNA synthesis

National Center for Biotechnology Information , U. PLoS Pathog. Published online Sep Rey, Editor. Author information Article notes Copyright and License information Disclaimer. Received Apr 7; Accepted Aug Copyright Reguera et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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This article has been cited by other articles in PMC. Figure S3: Nuclease assays of LC in the presence magnesium. Figure S6: Manganese ion binding to LC Abstract Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase L-protein to transcribe viral mRNAs.

Author Summary Bunyaviruses are a large family of RNA viruses that include serious human, animal and plant pathogens. Introduction Bunyaviridae is the largest single family of mostly animal viruses comprising more than species, divided into five genera: Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus and Tospovirus, the latter infecting plants.

Open in a separate window. Figure 1. Structure based alignment of the N-terminal domain of Orthobunya red species names and Tospovirus blue species names L-proteins. Results Crystal structure of the LACV polymerase N-terminal domain The original 1— residue construct of LACV L-protein LC was truncated on the basis of partial proteolysis with papain in order to identify a minimal active and stable fragment that was well expressed and soluble.

Figure 2. Figure 3. Divalent cation dependent nuclease activity and thermal stability of the LACV polymerase N-terminal domain Biochemical characterisation of the nuclease activity of LC was investigated using RNA and DNA digestion assays in the presence of a variety of divalent metal ions.

Associated Data

Figure 4. Divalent cation-dependent nuclease activity and thermal stability of LC Figure 5. Mutational analysis of the in vitro nuclease activity and thermal stability of the LACV polymerase N-terminal domain We made a series of alanine point mutants of key conserved residues in the active site of LC in order to assess their importance for activity. Figure 6. Mutational analysis of the in vitro nuclease activity and thermal stability of LC Mutational analysis of the transcriptional activity of the full-length LACV polymerase To test the effect of the nuclease inactivating mutants in the context of the full-length LACV L-protein we used a previously described in vivo RNP reconstitution system in which a Renilla Luciferase REN-Luc reporter gene is used as a readout of cap-dependent transcription by the viral polymerase [21] For a schematic outline of this assay see Supplementary Figure S7.

Discussion Cap-snatching as a method of priming transcription is uniquely restricted to segmented negative strand viruses, notably orthomyxoviruses influenza , bunyaviruses and arenaviruses. Figure 7. Structure based multiple alignment of the endonuclease of four genera of bunyavirus L-proteins together with the endonuclease of the PA subunit of selected orthomyxoviruses.

Figure 8. Schematic diagram of the polymerase architecture of negative strand segmented viruses. Crystallization Proteins LC, , , and were expressed and tested for crystallization using a Cartesian nanovolume robotic system for screening. Crystallography Crystals are of space-group P 6 1 22 with four molecules in the asymmetric unit.

Table 1 Data collection and refinement statistics. Software Structure figures were drawn with Molscript [46] or Bobscript [47] and rendered with Raster3d [48]. Co-ordinates and structure factor deposition The native structure of LC has wwPDB ID 2xi5 for the coordinate entry and r2xi5sf for the structure factors. Figure S3 Nuclease assays of LC in the presence magnesium. Figure S6 Manganese ion binding to LC Acknowledgments We acknowledge the Partnership for Structural Biology for an integrated structural biology environment, notably the high-throughput crystallisation facility.

Footnotes The authors have declared that no competing interests exist. References 1. Nucleic Acids Res. La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease. J Virol. Jin H, Elliott RM. J Gen Virol. In vivo analysis of the TSWV cap-snatching mechanism: single base complementarity and primer length requirements. Embo J. Rift Valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases.

Sequence determination of the Crimean-Congo hemorrhagic fever virus L segment. Towards an atomic resolution understanding of the influenza virus replication machinery. Curr Opin Struct Biol. The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Crystal structure of an avian influenza polymerase PA N reveals an endonuclease active site. BMC Struct Biol. Arch Virol. Inhibition of cap m7GpppXm -dependent endonuclease of influenza virus by 4-substituted 2,4-dioxobutanoic acid compounds.

Antimicrob Agents Chemother. Nucleoside monophosphate complex structures of the endonuclease domain from the influenza virus polymerase PA subunit reveal the substrate binding site inside the catalytic center. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem. Mutational and metal binding analysis of the endonuclease domain of the influenza virus polymerase PA subunit.

Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids. Mutational analysis of poliovirus 2Apro. Distinct inhibitory functions of 2apro on translation and transcription. J Biol Chem. Biology and molecular biology of viruses in the genus Tenuivirus. Annu Rev Phytopathol. A novel superfamily of predicted cysteine proteases from eukaryotes, viruses and Chlamydia pneumoniae. Trends Biochem Sci. Genomic analysis of rice stripe virus Zhejiang isolate shows the presence of an OTU-like domain in the RNA1 protein and a novel sequence motif conserved within the intergenic regions of ambisense segments of tenuiviruses.

Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISGdependent innate immune responses. Cell Host Microbe. An N-terminal region of Lassa virus L protein plays a critical role in transcription but not replication of the virus genome. Mielke N, Muehlbach HP. A novel, multipartite, negative-strand RNA virus is associated with the ringspot disease of European mountain ash Sorbus aucuparia L. Complete nucleotide sequence of four RNA segments of fig mosaic virus.

Isolation and characterisation of segment 1 of the infectious salmon anaemia virus genome. Virus Res. A novel antiviral agent which inhibits the endonuclease of influenza viruses. Use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors. J Med Chem. PA subunit of RNA polymerase as a promising target for anti-influenza virus agents. Antiviral Res. Green tea catechins inhibit the endonuclease activity of influenza A virus RNA polymerase.

PLoS Curr Influenza. Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes. Retroviral intasome assembly and inhibition of DNA strand transfer. Kabsch W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Cryst. Methods Mol Biol. Automated protein model building combined with iterative structure refinement. Nat Struct Biol. Murshudov GN. Refinement of macromolecular structures by the maximum-likelihood method.

Acta Crystallogr D Biol Crystallogr. Read RJ. Pushing the boundaries of molecular replacement with maximum likelihood. Kraulis PJ. Esnouf RM. Further additions to Molscript version 1. Acta Crystallogr. Raster3D Photorealistic molecular graphics. Methods Enzymol.

Segmented Negative Strand Viruses: Arenaviruses, Bunyaviruses, and Orthomyxoviruses

CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Orthomyxoviruses -- Congresses. Negative strand viruses Contents Includes bibliographical references. Includes bibliographies and index. View online Borrow Buy Freely available Show 0 more links Set up My libraries How do I set up "My libraries"?

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