In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. It was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. Here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic RNA. These findings demonstrate that the protein sequence is not the primary cause of altered genomic and subgenomic RNA production. The synonymous codon changes affect both the structure of the frameshift signal and frameshifting efficiency. Small differences in frameshifting efficiency result in dramatic differences in genomic RNA production and TCID50 suggesting that the frameshifting frequency must stay above a certain threshold for optimal virus production. The data suggest that either the RNA sequence or the ratio of viral proteins resulting from different levels of frameshifting affects viral replication.
References
[1]
Nga, P.T.; Parquet, M.C.; Lauber, C.; Parida, M.; Nabeshima, T.; Yu, F.; Thuy, N.T.; Inoue, S.; Ito, T.; Okamoto, K.; et al. Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes. PLoS Pathog. 2011, 7, e1002215, doi:10.1371/journal.ppat.1002215.
[2]
Masters, P.S. The molecular biology of coronaviruses. Adv. Virus Res. 2006, 66, 193–292, doi:10.1016/S0065-3527(06)66005-3.
[3]
de Haan, C.A.; Rottier, P.J. Hosting the severe acute respiratory syndrome coronavirus: Specific cell factors required for infection. Cell Microbiol. 2006, 8, 1211–1218, doi:10.1111/j.1462-5822.2006.00744.x.
[4]
Sawicki, S.G.; Sawicki, D.L.; Siddell, S.G. A contemporary view of coronavirus transcription. J. Virol. 2007, 81, 20–29, doi:10.1128/JVI.01358-06.
[5]
Ziebuhr, J. The coronavirus replicase. Curr. Top. Microbiol. Immunol. 2005, 287, 57–94, doi:10.1007/3-540-26765-4_3.
[6]
Plant, E.P. Ribosomal Frameshift Signals in Viral Genomes. In Viral Genomes—Molecular Structure, Diversity, Gene Expression Mechanisms and Host-Virus Interactions; Garcia, M.L, Romanowski, V., Eds.; InTech: Rijeka, Croatia, 2012; pp. 91–122.
[7]
Plant, E.P.; Rakauskaite, R.; Taylor, D.R.; Dinman, J.D. Achieving a golden mean: Mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins. J. Virol. 2010, 84, 4330–4340, doi:10.1128/JVI.02480-09.
Plant, E.P.; Perez-Alvarado, G.C.; Jacobs, J.L.; Mukhopadhyay, B.; Hennig, M.; Dinman, J.D. A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal. PLoS Biol. 2005, 3, e172, doi:10.1371/journal.pbio.0030172.
[10]
Su, M.C.; Chang, C.T.; Chu, C.H.; Tsai, C.H.; Chang, K.Y. An atypical RNA pseudoknot stimulator and an upstream attenuation signal for -1 ribosomal frameshifting of SARS coronavirus. Nucleic Acids Res. 2005, 33, 4265–4275, doi:10.1093/nar/gki731.
[11]
Plant, E.P.; Dinman, J.D. Comparative study of the effects of heptameric slippery site composition on -1 frameshifting among different eukaryotic systems. RNA 2006, 12, 666–673, doi:10.1261/rna.2225206.
[12]
Brierley, I.; Rolley, N.J.; Jenner, A.J.; Inglis, S.C. Mutational analysis of the RNA pseudoknot component of a coronavirus ribosomal frameshifting signal. J. Mol. Biol. 1991, 220, 889–902, doi:10.1016/0022-2836(91)90361-9.
[13]
Herold, J.; Siddell, S.G. An 'elaborated' pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA. Nucleic Acids Res. 1993, 21, 5838–5842, doi:10.1093/nar/21.25.5838.
[14]
Gohlke, C.; Murchie, A.I.; Lilley, D.M.; Clegg, R.M. Kinking of DNA and RNA helices by bulged nucleotides observed by fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 1994, 91, 11660–11664.
[15]
Baril, M.; Dulude, D.; Gendron, K.; Lemay, G.; Brakier-Gingras, L. Efficiency of a programmed -1 ribosomal frameshift in the different subtypes of the human immunodeficiency virus type 1 group M. RNA 2003, 9, 1246–1253, doi:10.1261/rna.5113603.
[16]
Chen, X.; Kang, H.; Shen, L.X.; Chamorro, M.; Varmus, H.E.; Tinoco, I., Jr. A characteristic bent conformation of RNA pseudoknots promotes -1 frameshifting during translation of retroviral RNA. J. Mol. Biol. 1996, 260, 479–483, doi:10.1006/jmbi.1996.0415.
[17]
Chung, B.Y.; Firth, A.E.; Atkins, J.F. Frameshifting in alphaviruses: A diversity of 3' stimulatory structures. J. Mol. Biol. 2010, 397, 448–456, doi:10.1016/j.jmb.2010.01.044.
[18]
Brierley, I.; Digard, P.; Inglis, S.C. Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot. Cell 1989, 57, 537–547, doi:10.1016/0092-8674(89)90124-4.
[19]
Ishimaru, D.; Plant, E.P.; Sims, A.C.; Yount, B.L.; Roth, B.M.; Eldho, N.V.; Perez-Alvarado, G.C.; Armbruster, D.W.; Baric, R.S.; Dinman, J.D.; Taylor, D.R.; Hennig, M. RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus. Nucleic Acids Res.. In Press .
[20]
Yount, B.; Curtis, K.M.; Fritz, E.A.; Hensley, L.E.; Jahrling, P.B.; Prentice, E.; Denison, M.R.; Geisbert, T.W.; Baric, R.S. Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc. Natl. Acad. Sci. USA 2003, 100, 12995–13000.
[21]
Reed, L.J.; Muench, H. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 1938, 27, 493–497.
[22]
Wilkinson, K.A.; Merino, E.J.; Weeks, K.M. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat. Protoc. 2006, 1, 1610–1616, doi:10.1038/nprot.2006.249.
Sola, I.; Mateos-Gomez, P.A.; Almazan, F.; Zuniga, S.; Enjuanes, L. RNA-RNA and RNA-protein interactions in coronavirus replication and transcription. RNA Biol. 2011, 8, 237–248, doi:10.4161/rna.8.2.14991.
[25]
Nicholson, B.L.; Lee, P.K.; White, K.A. Internal RNA Replication Elements are Prevalent in Tombusviridae. Front. Microbiol. 2012, 3, 279.
[26]
Paul, V.; Rieder, E.; Kim, D.W.; van Boom, J.H.; Wimmer, E. Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg. J. Virol. 2000, 74, 10359–10370, doi:10.1128/JVI.74.22.10359-10370.2000.
[27]
You, S.; Stump, D.D.; Branch, A.D.; Rice, C.M. A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication. J. Virol. 2004, 78, 1352–1366.
[28]
Ogram, S.A.; Flanegan, J.B. Non-template functions of viral RNA in picornavirus replication. Curr. Opin. Virol. 2011, 1, 339–346, doi:10.1016/j.coviro.2011.09.005.
[29]
Chen, S.-C.; Olsthoorn, R.C. Group-specific structural features of the 5′-proximal sequences of coronavirus genomic RNAs. Virology 2010, 401, 29–41, doi:10.1016/j.virol.2010.02.007.
[30]
Chen, S.-C.; Olsthoorn, R.C. New structure model for the packaging signal in the genome of Group IIa Coronaviruses. J. Virol. 2007, 81, 6771–6774, doi:10.1128/JVI.02231-06.
[31]
Dinman, J.D.; Wickner, R.B. Ribosomal frameshifting efficiency and gag/gag-pol ratio are critical for yeast M1 double-stranded RNA virus propagation. J. Virol. 1992, 66, 3669–3676.
[32]
Imbert, I.; Guillemot, J.C.; Bourhis, J.M.; Bussetta, C.; Coutard, B.; Egloff, M.P.; Ferron, F.; Gorbalenya, A.E.; Canard, B. A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus. EMBO J. 2006, 25, 4933–4942, doi:10.1038/sj.emboj.7601368.
[33]
te Velthuis, A.J.; van den Worm, S.H.; Snijder, E.J. The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension. Nucl. Acids Res. 2012, 40, 1737–1747.
[34]
Xiao, Y.; Ma, Q.; Restle, T.; Shang, W.; Svergun, D.I.; Ponnusamy, R.; Sczakiel, G.; Hilgenfeld, R. Nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits primer-independent RNA polymerase activity. J. Virol. 2012, 86, 4444–4454.
[35]
te Velthuis, A.J.; Arnold, J.J.; Cameron, C.E.; van den Worm, S.H.; Snijder, E.J. The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent. Nucl. Acids Res. 2010, 38, 203–214, doi:10.1093/nar/gkp904.
[36]
Spencer, P.S.; Siller, E.; Anderson, J.F.; Barral, J.M. Silent substitutions predictably alter translation elongation rates and protein folding efficiencies. J. Mol. Biol. 2012, 422, 328–335, doi:10.1016/j.jmb.2012.06.010.
