Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of “self-tagged” empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines

Detection of myxoma viruses encoding a defective M135R gene from clinical cases of myxomatosis; possible implications for the role of the M135R protein as a virulence factor

<p>Abstract</p> <p>Background</p> <p>Myxoma virus is a member of the <it>Poxviridae </it>and causes disease in European rabbits. Laboratory confirmation of the clinical disease, which occurs in the autumn of most years in Denmark, has been achieved previously using antigen ELISA and electron microscopy.</p> <p>Results</p> <p>An unusually large number of clinically suspected cases of myxomatosis were observed in Denmark during 2007. Myxoma virus DNA was detected, using a new real time PCR assay which targets the M029L gene, in over 70% of the clinical samples submitted for laboratory confirmation. Unexpectedly, further analysis revealed that a high proportion of these viral DNA preparations contained a frame-shift mutation within the M135R gene that has previously been identified as a virulence factor. This frame-shift mutation results in expression of a greatly truncated product. The same frame-shift mutation has also been found recently within an avirulent strain of myxoma virus (6918). However, three other frame-shift mutations found in this strain (in the genes M009L, M036L and M148R) were not shared with the Danish viruses but a single nucleotide deletion in the M138R/M139R intergenic region was a common feature.</p> <p>Conclusions</p> <p>It appears that expression of the full-length myxoma virus M135R protein is not required for virulence in rabbits. Hence, the frame-shift mutation in the M135R gene in the nonpathogenic 6918 virus strain is not sufficient to explain the attenuation of this myxoma virus but one/some of the other frame-shift mutations alone or in conjunction with one/some of the thirty two amino acid substitutions must also contribute. The real time PCR assay for myxoma virus is a useful diagnostic tool for laboratory confirmation of suspected cases of myxomatosis.</p

Expression of Foot-and-Mouth Disease Virus Capsid Proteins in Silkworm-Baculovirus Expression System and Its Utilization as a Subunit Vaccine

Background: Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals. Although the traditional inactivated vaccine has been proved effective, it may lead to a new outbreak of FMD because of either incomplete inactivation of FMDV or the escape of live virus from vaccine production workshop. Thus, it is urgent to develop a novel FMDV vaccine that is safer, more effective and more economical than traditional vaccines. Methodology and Principal Findings: A recombinant silkworm baculovirus Bm-P12A3C which contained the intact P1-2A and 3C protease coding regions of FMDV Asia 1/HNK/CHA/05 was developed. Indirect immunofluorescence test and sandwich-ELISA were used to verify that Bm-P12A3C could express the target cassette. Expression products from silkworm were diluted to 30 folds and used as antigen to immunize cattle. Specific antibody was induced in all vaccinated animals. After challenge with virulent homologous virus, four of the five animals were completely protected, and clinical symptoms were alleviated and delayed in the remaining one. Furthermore, a PD50 (50 % bovine protective dose) test was performed to assess the bovine potency of the subunit vaccine. The result showed the subunit vaccine could achieve 6.34 PD50 per dose

Genetic characterization of slow bee paralysis virus of the honeybee (Apis mellifera L.)

Complete genome sequences were determined for two distinct strains of slow bee paralysis virus (SBPV) of honeybees (Apis mellifera). The SBPV genome is approximately 9 5 kb long and contains a single ORF flanked by 5'- and 3'-UTRs and a naturally polyadenylated 3' tail, with a genome organization typical of members of the family Iflaviridae The two strains, labelled `Rothamsted' and 'Harpenden', are 83% identical at the nucleotide level (94% identical at the amino acid level), although this variation is distributed unevenly over the genome. The two strains were found to co-exist at different proportions in two independently propagated SBPV preparations The natural prevalence of SBPV for 847 colonies in 162 apiaries across five European countries was <2%, with positive samples found only in England and Switzerland, in colonies with variable degrees of Varroa infestatio

Counteracting Quasispecies Adaptability: Extinction of a Ribavirin-Resistant Virus Mutant by an Alternative Mutagenic Treatment

[Background] Lethal mutagenesis, or virus extinction promoted by mutagen-induced elevation of mutation rates of viruses, may meet with the problem of selection of mutagen-resistant variants, as extensively documented for standard, nonmutagenic antiviral inhibitors. Previously, we characterized a mutant of foot-and-mouth disease virus that included in its RNA-dependent RNA polymerase replacement M296I that decreased the sensitivity of the virus to the mutagenic nucleoside analogue ribavirin.[Methodology and Principal Findings] Replacement M296I in the viral polymerase impedes the extinction of the mutant foot-and-mouth disease virus by elevated concentrations of ribavirin. In contrast, wild type virus was extinguished by the same ribavirin treatment and, interestingly, no mutants resistant to ribavirin were selected from the wild type populations. Decreases of infectivity and viral load of the ribavirin-resistant M296I mutant were attained with a combination of the mutagen 5-fluorouracil and the non-mutagenic inhibitor guanidine hydrocloride. However, extinction was achieved with a sequential treatment, first with ribavirin, and then with a minimal dose of 5-fluorouracil in combination with guanidine hydrochloride. Both, wild type and ribavirin-resistant mutant M296I exhibited equal sensitivity to this combination, indicating that replacement M296I in the polymerase did not confer a significant cross-resistance to 5-fluorouracil. We discuss these results in relation to antiviral designs based on lethal mutagenesis[Conclusions] (i) When dominant in the population, a mutation that confers partial resistance to a mutagenic agent can jeopardize virus extinction by elevated doses of the same mutagen. (ii) A wild type virus, subjected to identical high mutagenic treatment, need not select a mutagen-resistant variant, and the population can be extinguished. (iii) Extinction of the mutagen-resistant variant can be achieved by a sequential treatment of a high dose of the same mutagen, followed by a combination of another mutagen with an antiviral inhibitor.Work supported by grants BFU2005-00863, BFU2008-02816/BMC, Proyecto Intramural de Frontera del CSIC 200820FO191, FIPSE 36558/06, and Fundacio´n Ramo´n Areces. CIBERehd is funded by Instituto de Salud Carlos III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptPeer reviewe

An Internal Ribosome Entry Site Directs Translation of the 39-Gene from Pelargonium Flower Break Virus Genomic RNA: Implications for Infectivity

[EN] Pelargonium flower break virus (PFBV, genus Carmovirus) has a single-stranded positive-sense genomic RNA (gRNA) which contains five ORFs. The two 59-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 39-proximal ORF encodes a polypeptide (p37) which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 59-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 39-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES). Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread.This research was supported by grants BFU2006-11230 and BFU2009-11699 from the Spanish Ministerio de Ciencia e Innovacion (MICINN) and by grants ACOM/2006/210 and ACOMP/2009/040 (to CH) and GVPRE/2008/121 (to OF-M) from the Generalitat Valenciana. The latter was the recipient of an I3P postdoctoral contract from the Spanish Consejo Superior de Investigaciones Cientificas and an additional contract from MICINN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Fernandez Miragall, O.; Hernandez Fort, C. (2011). An Internal Ribosome Entry Site Directs Translation of the 39-Gene from Pelargonium Flower Break Virus Genomic RNA: Implications for Infectivity. PLoS ONE. 6(7):22617-22617. https://doi.org/10.1371/journal.pone.0022617S226172261767Gallie, D. R. (1996). Translational control of cellular and viral mRNAs. Plant Molecular Biology, 32(1-2), 145-158. doi:10.1007/bf00039381Kozak, M. (2002). Pushing the limits of the scanning mechanism for initiation of translation. Gene, 299(1-2), 1-34. doi:10.1016/s0378-1119(02)01056-9Sachs, A. B., Sarnow, P., & Hentze, M. W. (1997). Starting at the Beginning, Middle, and End: Translation Initiation in Eukaryotes. Cell, 89(6), 831-838. doi:10.1016/s0092-8674(00)80268-8Kozak, M. (1992). Regulation of Translation in Eukaryotic Systems. Annual Review of Cell Biology, 8(1), 197-225. doi:10.1146/annurev.cb.08.110192.001213Sonenberg, N., & Hinnebusch, A. G. (2009). Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets. Cell, 136(4), 731-745. doi:10.1016/j.cell.2009.01.042F�tterer, J., & Hohn, T. (1996). Translation in plants-rules and exceptions. Plant Molecular Biology, 32(1-2), 159-189. doi:10.1007/bf00039382Gale, M., Tan, S.-L., & Katze, M. G. (2000). Translational Control of Viral Gene Expression in Eukaryotes. Microbiology and Molecular Biology Reviews, 64(2), 239-280. doi:10.1128/mmbr.64.2.239-280.2000Kozak, M. (2001). Constraints on reinitiation of translation in mammals. Nucleic Acids Research, 29(24), 5226-5232. doi:10.1093/nar/29.24.5226Pelletier, J., & Sonenberg, N. (1988). Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature, 334(6180), 320-325. doi:10.1038/334320a0Mokrejš, M., Mašek, T., Vopálenský, V., Hlubuček, P., Delbos, P., & Pospíšek, M. (2009). IRESite—a tool for the examination of viral and cellular internal ribosome entry sites. Nucleic Acids Research, 38(suppl_1), D131-D136. doi:10.1093/nar/gkp981Basso, J., Dallaire, P., Charest, P. J., Devantier, Y., & Laliberte, J.-F. (1994). Evidence for an Internal Ribosome Entry Site Within the 5’ Non-translated Region of Turnip Mosaic Potyvirus RNA. Journal of General Virology, 75(11), 3157-3165. doi:10.1099/0022-1317-75-11-3157Levis, C., & Astier-Manifacier, S. (1993). The 5′ untranslated region of PVY RNA, even located in an internal position, enables initiation of translation. Virus Genes, 7(4), 367-379. doi:10.1007/bf01703392Karetnikov, A., & Lehto, K. (2007). The RNA2 5’ leader of Blackcurrant reversion virus mediates efficient in vivo translation through an internal ribosomal entry site mechanism. Journal of General Virology, 88(1), 286-297. doi:10.1099/vir.0.82307-0Ivanov, P. A., Karpova, O. V., Skulachev, M. V., Tomashevskaya, O. L., Rodionova, N. P., Dorokhov, Y. L., & Atabekov, J. G. (1997). A Tobamovirus Genome That Contains an Internal Ribosome Entry Site Functionalin Vitro. Virology, 232(1), 32-43. doi:10.1006/viro.1997.8525Skulachev, M. V., Ivanov, P. A., Karpova, O. V., Korpela, T., Rodionova, N. P., Dorokhov, Y. L., & Atabekov, J. G. (1999). Internal Initiation of Translation Directed by the 5′-Untranslated Region of the Tobamovirus Subgenomic RNA I2. Virology, 263(1), 139-154. doi:10.1006/viro.1999.9928Jaag, H. M., Kawchuk, L., Rohde, W., Fischer, R., Emans, N., & Prufer, D. (2003). An unusual internal ribosomal entry site of inverted symmetry directs expression of a potato leafroll polerovirus replication-associated protein. Proceedings of the National Academy of Sciences, 100(15), 8939-8944. doi:10.1073/pnas.1332697100Balvay, L., Rifo, R. S., Ricci, E. P., Decimo, D., & Ohlmann, T. (2009). Structural and functional diversity of viral IRESes. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1789(9-10), 542-557. doi:10.1016/j.bbagrm.2009.07.005Kneller, E. L. P., Rakotondrafara, A. M., & Miller, W. A. (2006). Cap-independent translation of plant viral RNAs. Virus Research, 119(1), 63-75. doi:10.1016/j.virusres.2005.10.010Rico, P., & Hern�ndez, C. (2004). Complete nucleotide sequence and genome organization of Pelargonium flower break virus. Archives of Virology, 149(3), 641-651. doi:10.1007/s00705-003-0231-5Martinez-Turino, S., & Hernandez, C. (2010). Identification and characterization of RNA-binding activity in the ORF1-encoded replicase protein of Pelargonium flower break virus. Journal of General Virology, 91(12), 3075-3084. doi:10.1099/vir.0.023093-0Martínez-Turiño, S., & Hernández, C. (2011). A membrane-associated movement protein of Pelargonium flower break virus shows RNA-binding activity and contains a biologically relevant leucine zipper-like motif. Virology, 413(2), 310-319. doi:10.1016/j.virol.2011.03.001Martinez-Turino, S., & Hernandez, C. (2009). Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors. Journal of General Virology, 90(2), 519-525. doi:10.1099/vir.0.006098-0Rico, P., & Hernández, C. (2009). Characterization of the subgenomic RNAs produced by Pelargonium flower break virus: Identification of two novel RNAs species. Virus Research, 142(1-2), 100-107. doi:10.1016/j.virusres.2009.01.018Koh, D. C.-Y., Wong, S.-M., & Liu, D. X. (2003). Synergism of the 3′-Untranslated Region and an Internal Ribosome Entry Site Differentially Enhances the Translation of a Plant Virus Coat Protein. Journal of Biological Chemistry, 278(23), 20565-20573. doi:10.1074/jbc.m210212200Hellen, C. U. T. (2001). Internal ribosome entry sites in eukaryotic mRNA molecules. Genes & Development, 15(13), 1593-1612. doi:10.1101/gad.891101Martínez-Salas, E. (1999). Internal ribosome entry site biology and its use in expression vectors. Current Opinion in Biotechnology, 10(5), 458-464. doi:10.1016/s0958-1669(99)00010-5Dobrikova, E., Florez, P., Bradrick, S., & Gromeier, M. (2003). Activity of a type 1 picornavirus internal ribosomal entry site is determined by sequences within the 3’ nontranslated region. Proceedings of the National Academy of Sciences, 100(25), 15125-15130. doi:10.1073/pnas.2436464100Belsham, G. J. (2009). Divergent picornavirus IRES elements. Virus Research, 139(2), 183-192. doi:10.1016/j.virusres.2008.07.001Fernández-Miragall, O., Quinto, S. L. de, & Martínez-Salas, E. (2009). Relevance of RNA structure for the activity of picornavirus IRES elements. Virus Research, 139(2), 172-182. doi:10.1016/j.virusres.2008.07.009Pestova, T. V., Kolupaeva, V. G., Lomakin, I. B., Pilipenko, E. V., Shatsky, I. N., Agol, V. I., & Hellen, C. U. T. (2001). Molecular mechanisms of translation initiation in eukaryotes. Proceedings of the National Academy of Sciences, 98(13), 7029-7036. doi:10.1073/pnas.111145798FERNANDEZ-MIRAGALL, O. (2003). Structural organization of a viral IRES depends on the integrity of the GNRA motif. RNA, 9(11), 1333-1344. doi:10.1261/rna.5950603ROBERTSON, M. E. M., SEAMONS, R. A., & BELSHAM, G. J. (1999). A selection system for functional internal ribosome entry site (IRES) elements: Analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES. RNA, 5(9), 1167-1179. doi:10.1017/s1355838299990301Dorokhov, Y. L., Skulachev, M. V., Ivanov, P. A., Zvereva, S. D., Tjulkina, L. G., Merits, A., … Atabekov, J. G. (2002). Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proceedings of the National Academy of Sciences, 99(8), 5301-5306. doi:10.1073/pnas.082107599Xia, X., & Holcik, M. (2009). Strong Eukaryotic IRESs Have Weak Secondary Structure. PLoS ONE, 4(1), e4136. doi:10.1371/journal.pone.0004136Lu, J., Zhang, J., Wang, X., Jiang, H., Liu, C., & Hu, Y. (2006). In vitro and in vivo identification of structural and sequence elements in the 5’ untranslated region of Ectropis obliqua picorna-like virus required for internal initiation. Journal of General Virology, 87(12), 3667-3677. doi:10.1099/vir.0.82090-0Yang, L. J., Hidaka, M., Sonoda, J., Masaki, H., & Uozumi, T. (1997). Mutational Analysis of the Potato Virus Y 5′ Untranslated Region for Alteration in Translational Enhancement in Tobacco Protoplasts. Bioscience, Biotechnology, and Biochemistry, 61(12), 2131-2133. doi:10.1271/bbb.61.2131BERGAMINI, G., PREISS, T., & HENTZE, M. W. (2000). Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system. RNA, 6(12), 1781-1790. doi:10.1017/s1355838200001679Bradrick, S. S. (2006). The hepatitis C virus 3’-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase. Nucleic Acids Research, 34(4), 1293-1303. doi:10.1093/nar/gkl019Lopez de Quinto, S. (2002). IRES-driven translation is stimulated separately by the FMDV 3’-NCR and poly(A) sequences. Nucleic Acids Research, 30(20), 4398-4405. doi:10.1093/nar/gkf569Song, Y., Friebe, P., Tzima, E., Junemann, C., Bartenschlager, R., & Niepmann, M. (2006). The Hepatitis C Virus RNA 3’-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site. Journal of Virology, 80(23), 11579-11588. doi:10.1128/jvi.00675-06Koh, D. C.-Y., Liu, D. X., & Wong, S.-M. (2002). A Six-Nucleotide Segment within the 3’ Untranslated Region of Hibiscus Chlorotic Ringspot Virus Plays an Essential Role in Translational Enhancement. Journal of Virology, 76(3), 1144-1153. doi:10.1128/jvi.76.3.1144-1153.2002Stupina, V. A., Meskauskas, A., McCormack, J. C., Yingling, Y. G., Shapiro, B. A., Dinman, J. D., & Simon, A. E. (2008). The 3’ proximal translational enhancer of Turnip crinkle virus binds to 60S ribosomal subunits. RNA, 14(11), 2379-2393. doi:10.1261/rna.1227808Truniger, V., Nieto, C., González-Ibeas, D., & Aranda, M. (2008). Mechanism of plant eIF4E-mediated resistance against a Carmovirus (Tombusviridae): cap-independent translation of a viral RNA controlledin cisby an (a)virulence determinant. The Plant Journal, 56(5), 716-727. doi:10.1111/j.1365-313x.2008.03630.xMiller, W. A., Wang, Z., & Treder, K. (2007). The amazing diversity of cap-independent translation elements in the 3′-untranslated regions of plant viral RNAs. Biochemical Society Transactions, 35(6), 1629-1633. doi:10.1042/bst0351629Miller, W. A., & White, K. A. (2006). Long-Distance RNA-RNA Interactions in Plant Virus Gene Expression and Replication. Annual Review of Phytopathology, 44(1), 447-467. doi:10.1146/annurev.phyto.44.070505.143353Koh, D. C.-Y., Wang, X., Wong, S.-M., & Liu, D. X. (2006). Translation initiation at an upstream CUG codon regulates the expression of Hibiscus chlorotic ringspot virus coat protein. Virus Research, 122(1-2), 35-44. doi:10.1016/j.virusres.2006.06.008Castaño, A., Ruiz, L., & Hernández, C. (2009). Insights into the translational regulation of biologically active open reading frames of Pelargonium line pattern virus. Virology, 386(2), 417-426. doi:10.1016/j.virol.2009.01.017Fraser, C. S., & Doudna, J. A. (2006). Structural and mechanistic insights into hepatitis C viral translation initiation. Nature Reviews Microbiology, 5(1), 29-38. doi:10.1038/nrmicro1558LÓPEZ-LASTRA, M., RIVAS, A., & BARRÍA, M. I. (2005). Protein synthesis in eukaryotes: The growing biological relevance of cap-independent translation initiation. Biological Research, 38(2-3). doi:10.4067/s0716-97602005000200003Pacheco, A., & Martinez-Salas, E. (2010). Insights into the Biology of IRES Elements through Riboproteomic Approaches. Journal of Biomedicine and Biotechnology, 2010, 1-12. doi:10.1155/2010/458927Bernstein, J., Sella, O., Le, S.-Y., & Elroy-Stein, O. (1997). PDGF2/c-sismRNA Leader Contains a Differentiation-linked Internal Ribosomal Entry Site (D-IRES). Journal of Biological Chemistry, 272(14), 9356-9362. doi:10.1074/jbc.272.14.9356Scheper, G. C., Voorma, H. O., & Thomas, A. A. M. (1994). Basepairing with 18S ribosomal RNA in internal initiation of translation. FEBS Letters, 352(3), 271-275. doi:10.1016/0014-5793(94)00975-9Dresios, J., Chappell, S. A., Zhou, W., & Mauro, V. P. (2005). An mRNA-rRNA base-pairing mechanism for translation initiation in eukaryotes. Nature Structural & Molecular Biology, 13(1), 30-34. doi:10.1038/nsmb1031Reigadas, S., Pacheco, A., Ramajo, J., de Quinto, S. L., & Martinez-Salas, E. (2005). Specific interference between two unrelated internal ribosome entry site elements impairs translation efficiency. FEBS Letters, 579(30), 6803-6808. doi:10.1016/j.febslet.2005.11.015Ishitani, M., Xiong, L., Stevenson, B., & Zhu, J. K. (1997). Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. The Plant Cell, 9(11), 1935-1949. doi:10.1105/tpc.9.11.1935Knoester, M., van Loon, L. C., van den Heuvel, J., Hennig, J., Bol, J. F., & Linthorst, H. J. M. (1998). Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi. Proceedings of the National Academy of Sciences, 95(4), 1933-1937. doi:10.1073/pnas.95.4.1933Mathews, D. H., Sabina, J., Zuker, M., & Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. Journal of Molecular Biology, 288(5), 911-940. doi:10.1006/jmbi.1999.2700Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31(13), 3406-3415. doi:10.1093/nar/gkg59