To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

Astbury Centre for Structural Molecular Biology University of Leeds Leeds UK

Department of Chemistry Center for NanoScience Ludwig Maximilian University of Munich Munich Germany

Faculty of Science University of South Bohemia Ceske Budejovice Czech Republic

Institute of Physics Faculty of Mathematics and Physics Charles University Ke Karlovu 5 CZ 12116 Prague 2 Czech Republic

School of Molecular and Cellular Biology University of Leeds Leeds UK

Zobrazit více v PubMed

Desselberger U. Rotaviruses. Virus Res. 2014; 190:75–96. PubMed

Mertens P. The dsRNA viruses. Virus Res. 2004; 101:3–13. PubMed

McDonald S.M., Nelson M.I., Turner P.E., Patton J.T.. Reassortment in segmented RNA viruses: mechanisms and outcomes. Nat. Rev. Microbiol. 2016; 14:448–460. PubMed PMC

McDonald S.M., Patton J.T.. Assortment and packaging of the segmented rotavirus genome. Trends Microbiol. 2011; 19:136–144. PubMed PMC

Sung P.Y., Roy P.. Sequential packaging of RNA genomic segments during the assembly of bluetongue virus. Nucleic Acids Res. 2014; 42:13824–13838. PubMed PMC

Lourenco S., Roy P.. In vitro reconstitution of Bluetongue virus infectious cores. Proc. Natl. Acad. Sci. U.S.A. 2011; 108:13746–13751. PubMed PMC

Anzola J. V, Xu Z.K., Asamizu T., Nuss D.L.. Segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tumor virus genome and defective interfering RNAs. Proc. Natl. Acad. Sci. U.S.A. 1987; 84:8301–8305. PubMed PMC

Tourís-Otero F., Cortez-San Martín M., Martínez-Costas J., Benavente J.. Avian reovirus morphogenesis occurs within viral factories and begins with the selective recruitment of σNS and λA to μNS inclusions. J. Mol. Biol. 2004; 341:361–374. PubMed

Netherton C.L., Wileman T.. Virus factories, double membrane vesicles and viroplasm generated in animal cells. Curr. Opin. Virol. 2011; 1:381–387. PubMed PMC

Silvestri L.S., Taraporewala Z.F., Patton J.T.. Rotavirus replication: Plus-Sense templates for Double-Stranded RNA synthesis are made in viroplasms. J. Virol. 2004; 78:7763–7774. PubMed PMC

Jiang X., Jayaram H., Kumar M., Ludtke S.J., Estes M.K., Venkataram Prasad B.V.. Cryoelectron microscopy structures of rotavirus NSP2-NSP5 and NSP2-RNA complexes: Implications for genome replication. J. Virol. 2006; 80:10829–10835. PubMed PMC

Taraporewala Z.F., Jiang X., Vasquez-Del Carpio R., Jayaram H., Prasad B.V.V., Patton J.T.. Structure-function analysis of rotavirus NSP2 octamer by using a novel complementation system. J. Virol. 2006; 80:7984–7994. PubMed PMC

Touris-Otero F., Martínez-Costas J., Vakharia V.N., Benavente J.. Avian reovirus nonstructural protein μNS forms viroplasm-like inclusions and recruits protein σNS to these structures. Virology. 2004; 319:94–106. PubMed

Miller C.L., Broering T.J., Parker J.S.L., Arnold M.M., Nibert M.L.. Reovirus σNS protein localizes to inclusions through an association requiring the μNS amino terminus. J. Virol. 2003; 77:4566–4576. PubMed PMC

Desmet E.A., Anguish L.J., Parker J.S.L.. Virus-mediated compartmentalization of the host translational machinery. Mbio. 2014; 5:1–11. PubMed PMC

Miller C.L., Arnold M.M., Broering T.J., Hastings C.E., Nibert M.L.. Localization of mammalian orthoreovirus proteins to cytoplasmic factory-like structures via nonoverlapping regions of microNS. J. Virol. 2010; 84:867–882. PubMed PMC

Borodavka A., Dykeman E.C., Schrimpf W., Lamb D.C.. Protein-mediated RNA folding governs sequence-specific interactions between rotavirus genome segments. Elife. 2017; 6:1–22. PubMed PMC

Borodavka A., Ault J., Stockley P.G., Tuma R.. Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment. Nucleic Acids Res. 2015; 43:7044–7057. PubMed PMC

Borodavka A., Singaram S.W., Stockley P.G., Gelbart W.M., Ben-Shaul A., Tuma R.. Sizes of long RNA molecules are determined by the branching patterns of their secondary structures. Biophys. J. 2016; 111:2077–2085. PubMed PMC

Konarev P. V, Volkov V. V, Sokolova A. V, Koch M.H.J., Svergun D.I.. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 2003; 36:1277–1282.

Konarev P.V., Petoukhov M.V., Volkov V.V., Svergun D.I.. ATSAS 2.1, a program package for small-angle scattering data analysis. J. Appl. Crystallogr. 2006; 39:277–286. PubMed PMC

Petoukhov M.V., Franke D., Shkumatov A.V., Tria G., Kikhney A.G., Gajda M., Gorba C., Mertens H.D.T., Konarev P.V., Svergun D.I.. New developments in the ATSAS program package for small-angle scattering data analysis. J. Appl. Crystallogr. 2012; 45:342–350. PubMed PMC

Franke D., Svergun D.I.. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Crystallogr. 2009; 42:342–346. PubMed PMC

Volkov V.V., Svergun D.I.. Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 2003; 36:860–864. PubMed PMC

Morgner N., Robinson C.V.. Massign: An assignment strategy for maximizing information from the mass spectra of heterogeneous protein assemblies. Anal. Chem. 2012; 84:2939–2948. PubMed

Ruotolo B.T., Benesch J.L.P., Sandercock A.M., Hyung S.-J., Robinson C.V.. Ion mobility–mass spectrometry analysis of large protein complexes. Nat. Protoc. 2008; 3:1139–1152. PubMed

Bush M.F., Hall Z., Giles K., Hoyes J., Robinson C.V., Ruotolo B.T.. Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Anal. Chem. 2010; 82:9557–9565. PubMed

Smith D., Knapman T., Campuzano I., Malham R., Berryman J., Radford S.E., Ashcroft A.. Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies. Eur. J. Mass Spectrom. 2009; 15:113–130. PubMed

Ruotolo B.T., Robinson C.V. Aspects of native proteins are retained in vacuum. Curr. Opin. Chem. Biol. 2006; 10:402–408. PubMed

Marklund E.G., Degiacomi M.T., Robinson C.V., Baldwin A.J., Benesch J.L.P.. Collision cross sections for structural proteomics. Structure. 2015; 23:791–799. PubMed

Davidovich C., Zheng L., Goodrich K.J., Cech T.R.. Promiscuous RNA binding by polycomb repressive complex 2. Nat. Struct. Mol. Biol. 2013; 20:1250–1257. PubMed PMC

Record M.T., Lohman T.M., de Haseth P.. Ion effects on ligand-nucleic acid interactions. J. Mol. Biol. 1976; 107:145–158. PubMed

Schuck P., Taraporewala Z., McPhie P., Patton J.T.. Rotavirus nonstructural protein NSP2 Self-assembles into octamers that undergo Ligand-induced conformational changes. J. Biol. Chem. 2001; 276:9679–9687. PubMed

Kudryavtsev V., Sikor M., Kalinin S., Mokranjac D., Seidel C.A.M., Lamb D.C.. Combining MFD and PIE for accurate single-pair förster resonance energy transfer measurements. Chemphyschem. 2012; 13:1060–1078. PubMed

Voith von Voithenberg L., Sánchez-Rico C., Kang H.-S., Madl T., Zanier K., Barth A., Warner L.R., Sattler M., Lamb D.C.. Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift. Proc. Natl. Acad. Sci. U.S.A. 2016; 113:E7169–E7175. PubMed PMC

Nir E., Michalet X., Hamadani K., Laurence T.A., Neuhauser D., Kovchegov Y., Weiss S.. Shot-noise limited single-molecule FRET histograms: comparison between theory and experiments. J. Phys. Chem. B. 2006; 110:22103–22124. PubMed PMC

Tomov T.E., Tsukanov R., Masoud R., Liber M., Plavner N., Nir E.. Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis. Biophys. J. 2012; 102:1163–1173. PubMed PMC

Eggeling C., Fries J.R., Brand L., Gunther R., Seidel C.A.M.. Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 1998; 95:1556–1561. PubMed PMC

Laurence T.A., Kwon Y., Yin E., Hollars C.W., Camarero J.A., Barsky D.. Correlation spectroscopy of minor fluorescent species: signal purification and distribution analysis. Biophys. J. 2007; 92:2184–2198. PubMed PMC

Schrimpf Waldemar. PAM: A framework for integrated analysis of imaging, Single-Molecule, and ensemble fluorescence data. Biophys. J. 2018; 114:1518–1528. PubMed PMC

Palacký J., Mojzeš P., Bok J.. SVD-based method for intensity normalization, background correction and solvent subtraction in Raman spectroscopy exploiting the properties of water stretching vibrations. J. Raman Spectrosc. 2011; 42:1528–1539.

Jayaram H., Taraporewala Z., Patton J.T., Prasad B.V.V.. Rotavirus protein involved in genome replication and packaging exhibits a HIT-like fold. Nature. 2002; 417:311–315. PubMed

Schiffrin B., Calabrese A.N., Devine P.W.A., Harris S.A., Ashcroft A.E., Brockwell D.J., Radford S.E.. Skp is a multivalent chaperone of outer-membrane proteins. Nat. Struct. Mol. Biol. 2016; 23:786–793. PubMed PMC

Ghetu A.F., Arthur D.C., Kerppola T.K., Glover J.N.M.. Probing FinO-FinP RNA interactions by site-directed protein-RNA crosslinking and in-gel FRET. RNA. 2002; 8:816–823. PubMed PMC

Radman-Livaja M., Biswas T., Mierke D., Landy A.. Architecture of recombination intermediates visualized by in-gel FRET of λ integrase–Holliday junction–arm DNA complexes. Proc. Natl. Acad. Sci. U.S.A. 2005; 102:3913–3920. PubMed PMC

Gillian A.L., Schmaechel S.C., Livny J., Schiff L.A., Nibert M.L.. Reovirus protein σNS binds in multiple copies to single stranded RNA and shares properties with single stranded DNA binding proteins. J. Virol. 2000; 74:5939–5948. PubMed PMC

Draper D.E. A guide to ions and RNA structure. RNA. 2004; 10:335–343. PubMed PMC

Misra V.K., Draper D.E.. On the role of magnesium ions in RNA stability. Biopolymers. 1998; 48:113–135. PubMed

Fajardo T., Sung P.-Y., Roy P.. Disruption of specific RNA-RNA interactions in a Double-Stranded RNA virus inhibits genome packaging and virus infectivity. PLOS Pathog. 2015; 11:e1005321. PubMed PMC

Fajardo T.J., Al Shaikhahmed K., Roy P.. Generation of infectious RNA complexes in Orbiviruses: RNA-RNA interactions of genomic segments. Oncotarget. 2016; 7:72559–72570. PubMed PMC

Russell R. RNA misfolding and the action of chaperones. Front. Biosci. 2008; 13:1–20. PubMed PMC

Rajkowitsch L., Chen D., Stampfl S., Semrad K., Waldsich C., Mayer O., Jantsch M.F., Konrat R., Bläsi U., Schroeder R.. RNA chaperones, RNA annealers and RNA helicases. RNA Biol. 2007; 4:118–130. PubMed

Müller U.F., Göringer H.U.. Mechanism of the gBP21-mediated RNA / RNA annealing reaction: matchmaking and charge reduction. Nucleic Acids Res. 2002; 30:447–455. PubMed PMC

Peng Yi, Curtis J.E., Fang X., Woodson S.A.. Structural model of an mRNA in complex with the bacterial chaperone Hfq. Proc. Natl. Acad. Sci. U.S.A. 2014; 111:17134–17139. PubMed PMC

Mayer O., Rajkowitsch L., Lorenz C., Konrat R., Schroeder R.. RNA chaperone activity and RNA-binding properties of the E. coli protein StpA. Nucleic Acids Res. 2007; 35:1257–1269. PubMed PMC

D’Souza V., Summers M.F.. Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus. Nature. 2004; 431:586–590. PubMed

Grossberger R., Mayer O., Waldsich C., Semrad K., Urschitz S., Schroeder R.. Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA. Nucleic Acids Res. 2005; 33:2280–2289. PubMed PMC

Grohman J.K., Gorelick R.J., Lickwar C.R., Lieb J.D., Bower B.D., Znosko B.M., Weeks K.M.. A guanosine-centric mechanism for RNA chaperone function. Science. 2013; 340:190–195. PubMed PMC

Milles S., Jensen M.R., Communie G., Maurin D., Schoehn G., Ruigrok R.W.H., Blackledge M.. Self-assembly of measles virus nucleocapsid-like particles: kinetics and RNA sequence-dependence. Angew. Chem. Int. Ed. 2016; 55:9356–9360. PubMed PMC

Reguera J., Cusack S., Kolakofsky D.. Segmented negative strand RNA virus nucleoprotein structure. Curr. Opin. Virol. 2014; 5:7–15. PubMed

Durham A.C., Finch J.T., Klug A.. States of aggregation of tobacco mosaic virus protein. Nat. New Biol. 1971; 229:37–42. PubMed

Butler P.J.G., Klug A.. Assembly of the particle of tobacco mosaic virus from RNA and disks of protein. Nat. New Biol. 1971; 229:47–50. PubMed

Mumtsidu E., Makhov A.M., Roessle M., Bathke A., Tucker P.A.. Structural features of the Bluetongue virus NS2 protein. J. Struct. Biol. 2007; 160:157–167. PubMed

Butan C., Tucker P.. Insights into the role of the non-structural protein 2 (NS2) in Bluetongue virus morphogenesis. Virus Res. 2010; 151:109–117. PubMed

Butan C., Van Der Zandt H., Tucker P.A.. Structure and assembly of the RNA binding domain of bluetongue virus non-structural protein 2. J. Biol. Chem. 2004; 279:37613–37621. PubMed

Gillian A.L., Nibert M.L.. Amino terminus of reovirus nonstructural protein sigma NS is important for ssRNA binding and nucleoprotein complex formation. Virology. 1998; 240:1–11. PubMed

Akita F., Higashiura A., Shimizu T., Pu Y., Suzuki M., Uehara-Ichiki T., Sasaya T., Kanamaru S., Arisaka F., Tsukihara T.. Crystallographic analysis reveals octamerization of viroplasm matrix protein P9-1 of Rice black streaked dwarf virus. J. Virol. 2012; 86:746–756. PubMed PMC

Wu J., Li J., Mao X., Wang W., Cheng Z., Zhou Y., Zhou X., Tao X.. Viroplasm protein P9-1 of Rice black-streaked dwarf virus preferentially binds to single-stranded RNA in its octamer form, and the central interior structure formed by this octamer constitutes the major RNA binding site. J. Virol. 2013; 87:12885–12899. PubMed PMC

Akita F., Miyazaki N., Hibino H., Shimizu T., Higashiura A., Uehara-Ichiki T., Sasaya T., Tsukihara T., Nakagawa A., Iwasaki K.. Viroplasm matrix protein Pns9 from rice gall dwarf virus forms an octameric cylindrical structure. J. Gen. Virol. 2011; 92:2214–2221. PubMed

Structural basis of rotavirus RNA chaperone displacement and RNA annealing