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

Occurrence of Polysiphonia epiphytes in Kappaphycus farms at Calaguas Is., Camarines Norte, Phillippines

This paper describes the occurrence of an epiphyte infestation of Kappaphycus farms in Calaguas Is. Camarines Norte, Philippines. In particular, percentage cover of ‘goose bump’-Polysiphonia and ‘ice-ice’ disease, and some environmental parameters that influence the thallus condition of Kappaphycus alvarezii in Calaguas Is. were assessed during 3 separate visits and are discussed. Commercial cultivation of Kappaphycus at Calaguas Is. began in the early 1990s. After five years of farming, the stock was destroyed by a strong typhoon. The area was re-planted the following year and production increased annually and reached its peak in 1998–1999. However, the following year, the first occurrence of a Polysiphonia epiphyte infestation occurred concurrently with an ‘ice-ice’ disease. Consequently, annual production and the number of seaweed planters declined rapidly, and this situation persists to the present time. This paper highlights the etiological factors and their consequences. Results show that farm-site selection is critical for the success of Kappaphycus production. Characteristics of water movement and light intensity in farming areas contributed to the occurrence and detrimental effect of the phenomenon described as ‘goose bumps’: a morphological distortion of the host seaweed due to the presence of a Polysiphonia sp. epiphyte. A strong inverse correlation was observed between the occurrence of Polysiphonia and water movement: areas with low water motion registered a higher % cover (65%) of Polysiphonia than those in more exposed areas (17%). Although ‘goose bump’-Polysiphonia infestation and ‘ice-ice’ disease pose a tremendous problem to the seaweed farmers, the results of this limited assessment provide a useful baseline for future work

Economics of cultivating Kappaphycus alvarezii using the fixed-bottom line and hanging-long line methods in Panagatan Cays, Caluya, Antique, Philippines

A socio-economic survey was conducted among the Kappaphycus alvarezii planters of Panagatan Cay, Caluya, Antique, Philippines to determine some social information, farming practices and cost and returns of farming the seaweed. Cultivation is dominated by brown and green morphotypes using the fixed-bottom and hanging-long line methods. Approximately 9.3 t d. wt ha−1 and 7.2 t d. wt ha−1 is produced from fixed-bottom and hanging-long lines methods, respectively, after 60–90 days of culture. The former method requires a working capital and total investment of P7490 and P1870, respectively, compared to the hanging-long line which requires P8455 and P25464, respectively (US$ 1 = P26). A higher total revenue (P139500), net income ((P187895), and return of investment 1002%), but a shorter pay back period (0.10 years) were obtained in fixed-bottom than in hanging-long line. A lower total expenses were incurred in fixed-bottom (P21354) than in hanging-long line (P24566). The farming of K. alvarezii in this area has brought tremendous economic impact to the marginal fishermen

Effect of short-term immersion of Kappaphycus alvarezii (Doty) Doty in high nitrogen on the growth, nitrogen assimilation, carrageenan quality, and occurrence of “ice-ice” disease

Short-term immersion of Kappaphycus alvarezii (Doty) Doty in a high-nitrogen-containing medium was tested to increase growth, improve the quality of carrageenan, and decrease “ice-ice” disease occurrence. Tank-reared Kappaphycus were used as explants. Growth, nitrogen assimilation, carrageenan quality, and occurrence of ice-ice disease of enriched (E/N) K. alvarezii were determined. E/N and un-enriched (control) K. alvarezii were planted inside net cages in the sea. Nitrogen assimilation was monitored to determine if nitrogen was incorporated in the tissues after 12 h. Total thallus nitrogen of K. alvarezii doubled after immersion in high nitrogen. Growth rate and carrageenan yield of E/N K. alvarezii were significantly higher than those of the control. Gel strengths of E/N and the control were not significantly different. Ice-ice disease occurrence was significantly higher in the control than the enriched seaweeds. Short-term immersion of K. alvarezii in a high-nitrogen medium before outplanting increased growth rate and decreased the occurrence of “ice-ice”.Acknowledgement is due to the Australian Centre for International Agricultural Research (ACIAR SMAR/2008/025)