Abstract
In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail1,2. Here we show that the 2 Å resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage φ6 (refs 3, 4) is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.
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References
Reinisch, K. M., Nibert, M. L. & Harrison, S. C. Structure of the reovirus core at 3. 6 Å resolution. Nature 404, 960–967 (2000).
Grimes, J. M. et al. The atomic structure of the bluetongue virus core. Nature 395, 470–478 (1998).
Makeyev, E. V. & Bamford, D. H. Replicase activity of purified recombinant protein P2 of double- stranded RNA bacteriophage φ6. EMBO J. 19, 124–133 (2000).
Butcher, S. J., Makeyev, E. V., Grimes, J. M., Stuart, D. I. & Bamford, D. H. Crystallization and preliminary X-ray crystallographic studies on the bacteriophage φ6 RNA-dependent RNA polymerase. Acta Crystallogr. D 56, 1473–1475 (2000).
Mindich, L. Reverse genetics of dsRNA bacteriophage φ6. Adv. Virus Res. 53, 341–353 (1999).
Gottlieb, P., Strassman, J., Quao, X., Frucht, A. & Mindich, L. In vitro replication, packaging, and transcription of the segmented, double-stranded RNA genome of bacteriophage φ6: studies with procapsids assembled from plasmid-encoded proteins. J. Bacteriol. 172, 5774–5782 (1990).
Mindich, L. Precise packaging of the three genomic segments of the double-stranded-RNA bacteriophage φ6. Microbiol. Mol. Biol. Rev. 63, 149–160 (1999).
Makeyev, E. V. & Bamford, D. H. The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism. EMBO J. 19, 124–133 (2000).
Ollis, D. L., Kline, C. & Steitz, T. A. Domain of E. coli DNA polymerase I showing sequence homology to T7 DNA polymerase. Nature 313, 818–819 (1985).
Delarue, M., Poch, O., Tordo, N., Moras, D. & Argos, P. An attempt to unify the structure of polymerases. Protein Eng. 3, 461–467 (1990).
Lesburg, C. A. et al. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nature Struct. Biol. 6, 937–943 (1999).
Ago, H. et al. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Struct. Fold. Des. 7, 1417–1426 (1999).
Bressanelli, S. et al. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc. Natl Acad. Sci. USA 96, 13034–13039 (1999).
Stuart, D. I., Levine, M., Muirhead, H. & Stammers, D. K. Crystal structure of cat muscle pyruvate kinase at resolution of 2. 6Å. J. Mol. Biol. 134, 109–142 (1979).
Oh, J. W., Ito, T. & Lai, M. M. A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA. J. Virol. 73, 7694–7702 (1999).
Lohmann, V., Overton, H. & Bartenschlager, R. Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP. J. Biol. Chem. 274, 10807–10815 (1999).
Frilander, M., Poranen, M. & Bamford, D. H. The large genome segment of dsRNA bacteriophage φ6 is the key regulator in the in vitro minus and plus strand synthesis. RNA 1, 510–518 (1995).
van Dijk, A. A., Frilander, M. & Bamford, D. H. Differentitation between minus- and plus-strand synthesis: polymerase activity of dsRNA bacteriophage φ6 in an in vitro packaging and replication system. Virology 211, 320–323 (1995).
Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282, 1669–1675 (1998).
Zhong, W., Uss, A. S., Ferrari, E., Lau, J. Y. & Hong, Z. De novo initiation of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase. J. Virol. 74, 2017–2022 (2000).
Yazaki, K. & Miura, K. Relation of the structure of cytoplasmic polyhedrosis virus and the synthesis of its messenger RNA. Virology 105, 467–479 (1980).
Hendrickson, W. A. Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science 254, 51–58 (1991).
Brunger, A. T. et al. Crystallography and NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).
Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 164–182 (1994).
Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).
Esnouf, R. M. An extensively modified version of MolScript that includes greatly enhanced colouring capabilities. J. Mol. Graph. 15, 132–134 (1997).
Merritt, E. A. & Bacon, D. J. in Macromolecular Crystallography (eds Carter, J. W. Jr & Sweet, R. M.) 505–524 (Academic, San Diego, 1997).
Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).
Acknowledgements
J. Diprose and G. Sutton helped with synchrotron data collection; J. Diprose and S. Ikemizu with calculations; and R. Esnouf and K. Harlos with computing and in-house data collection. We thank the staff at the beamlines of the ESRF, SRS and APS, in particular Sergey Korolev at the APS for help with the MAD experiment. S.J.B. is a Marie Curie Fellow. J.M.G. is funded by the Royal Society and D.I.S. by the Medical Research Council. The work was supported by the Academy of Finland, the Medical Research Council and the European Union.
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Butcher, S., Grimes, J., Makeyev, E. et al. A mechanism for initiating RNA-dependent RNA polymerization. Nature 410, 235–240 (2001). https://doi.org/10.1038/35065653
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DOI: https://doi.org/10.1038/35065653
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