Intra-specific variability and biological relevance of P3N-PIPO protein length in potyviruses

Background: Pipo was recently described as a new ORF encoded within the genome of the Potyviridae family members (PNAS 105: 5897-5902, 2008). It is embedded within the P3 cistron and is translated in the +2 reading frame relative to the potyviral long ORF as the P3N-PIPO fusion protein. In this work, we first collected pipo nucleotide sequences available for different isolates of 48 Potyvirus species. Second, to determine the biological implications of variation in pipo length, we measured infectivity, viral accumulation, cell-to-cell and systemic movements for two Turnip mosaic virus (TuMV) variants with pipo alleles of different length in three different susceptible host species, and tested for differences between the two variants. Results: In addition to inter-specific variation, there was high variation in the length of the PIPO protein among isolates within species (ranging from 1 to 89 amino acids). Furthermore, selection analyses on the P3 cistron did not account for the existence of stop codons in the pipo ORF, but showed that positive selection was significant in the overlapping region for Potato virus Y (PVY) and TuMV. In some cases, variability in length was associated with host species, geographic provenance and/or other strain features. We found significant empirical differences among the phenotypes associated with TuMV pipo alleles, though the magnitude and sign of the effects were host-dependent. Conclusions: The combination of computational molecular evolution analyses and experiments stemming from these analyses provide clues about the selective pressures acting upon the different-length pipo alleles and show that variation in length may be maintained by host-driven selection.We thank Jose A. Daros for providing the TuMV-GFP infectious clone, Mario A. Fares and Guillaume Lafforgue for fruitful discussion, Guillermo Rodrigo for assistance with MATLAB and for critically reading of the manuscript, Mark P. Zwart for critically reading the manuscript, and Francisca de la Iglesia for excellent technical assistance. Funding for this work was provided by the Spanish Direccion General de Investigacion Cientifica y Tecnica grant BFU2012-30805 (to SFE), a predoctoral fellowship from the Spanish Ministerio de Economia y Competitividad (to JH), and a postdoctoral contract from CSIC JAE-doc program (to JMC).Hillung, J.; Elena Fito, SF.; Cuevas Torrijos, JM. (2013). Intra-specific variability and biological relevance of P3N-PIPO protein length in potyviruses. BMC Evolutionary Biology. 13(249):1-10. https://doi.org/10.1186/1471-2148-13-24911013249Riechmann, J. L., Lain, S., & Garcia, J. A. (1992). Highlights and prospects of potyvirus molecular biology. Journal of General Virology, 73(1), 1-16. doi:10.1099/0022-1317-73-1-1Green, K. Y., Ando, T., Balayan, M. S., Berke, T., Clarke, I. N., Estes, M. K., … Thiel, H. (2000). Taxonomy of the Caliciviruses. The Journal of Infectious Diseases, 181(s2), S322-S330. doi:10.1086/315591Van der Wilk, F., Dullemans, A. M., Verbeek, M., & Van den Heuvel, J. F. J. M. (1997). Nucleotide Sequence and Genomic Organization of Acyrthosiphon Pisum Virus. Virology, 238(2), 353-362. doi:10.1006/viro.1997.8835Urcuqui-Inchima, S., Haenni, A.-L., & Bernardi, F. (2001). Potyvirus proteins: a wealth of functions. Virus Research, 74(1-2), 157-175. doi:10.1016/s0168-1702(01)00220-9Chung, B. Y.-W., Miller, W. A., Atkins, J. F., & Firth, A. E. (2008). An overlapping essential gene in the Potyviridae. Proceedings of the National Academy of Sciences, 105(15), 5897-5902. doi:10.1073/pnas.0800468105Wei, T., Zhang, C., Hong, J., Xiong, R., Kasschau, K. D., Zhou, X., … Wang, A. (2010). Formation of Complexes at Plasmodesmata for Potyvirus Intercellular Movement Is Mediated by the Viral Protein P3N-PIPO. PLoS Pathogens, 6(6), e1000962. doi:10.1371/journal.ppat.1000962Wen, R.-H., & Hajimorad, M. R. (2010). Mutational analysis of the putative pipo of soybean mosaic virus suggests disruption of PIPO protein impedes movement. Virology, 400(1), 1-7. doi:10.1016/j.virol.2010.01.022Vijayapalani, P., Maeshima, M., Nagasaki-Takekuchi, N., & Miller, W. A. (2012). Interaction of the Trans-Frame Potyvirus Protein P3N-PIPO with Host Protein PCaP1 Facilitates Potyvirus Movement. PLoS Pathogens, 8(4), e1002639. doi:10.1371/journal.ppat.1002639Lin, L., Luo, Z., Yan, F., Lu, Y., Zheng, H., & Chen, J. (2011). Interaction between potyvirus P3 and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of host plants. Virus Genes, 43(1), 90-92. doi:10.1007/s11262-011-0596-6Choi, S. H., Hagiwara-Komoda, Y., Nakahara, K. S., Atsumi, G., Shimada, R., Hisa, Y., … Uyeda, I. (2013). Quantitative and Qualitative Involvement of P3N-PIPO in Overcoming Recessive Resistance against Clover Yellow Vein Virus in Pea Carrying the cyv1 Gene. Journal of Virology, 87(13), 7326-7337. doi:10.1128/jvi.00065-13Cuevas, J. M., Delaunay, A., Visser, J. C., Bellstedt, D. U., Jacquot, E., & Elena, S. F. (2012). Phylogeography and Molecular Evolution of Potato virus Y. PLoS ONE, 7(5), e37853. doi:10.1371/journal.pone.0037853Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32(5), 1792-1797. doi:10.1093/nar/gkh340Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28(10), 2731-2739. doi:10.1093/molbev/msr121Martin, D. P., Lemey, P., Lott, M., Moulton, V., Posada, D., & Lefeuvre, P. (2010). RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics, 26(19), 2462-2463. doi:10.1093/bioinformatics/btq467Ilardi, V., & Nicola-Negri, E. D. (2011). Genetically engineered resistance to Plum pox virus infection in herbaceous and stone fruit hosts. GM Crops, 2(1), 24-33. doi:10.4161/gmcr.2.1.15096Schubert, J., Fomitcheva, V., & Sztangret-Wiśniewska, J. (2007). Differentiation of Potato virus Y strains using improved sets of diagnostic PCR-primers. Journal of Virological Methods, 140(1-2), 66-74. doi:10.1016/j.jviromet.2006.10.017Ohshima, K., Tomitaka, Y., Wood, J. T., Minematsu, Y., Kajiyama, H., Tomimura, K., & Gibbs, A. J. (2007). Patterns of recombination in turnip mosaic virus genomic sequences indicate hotspots of recombination. Journal of General Virology, 88(1), 298-315. doi:10.1099/vir.0.82335-0Kosakovsky Pond, S. L., Frost, S. D. W., Grossman, Z., Gravenor, M. B., Richman, D. D., & Brown, A. J. L. (2006). Adaptation to Different Human Populations by HIV-1 Revealed by Codon-Based Analyses. PLoS Computational Biology, 2(6), e62. doi:10.1371/journal.pcbi.0020062Kosakovsky Pond, S. L., & Frost, S. D. W. (2005). Not So Different After All: A Comparison of Methods for Detecting Amino Acid Sites Under Selection. Molecular Biology and Evolution, 22(5), 1208-1222. doi:10.1093/molbev/msi105Delport, W., Poon, A. F. Y., Frost, S. D. W., & Kosakovsky Pond, S. L. (2010). Datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics, 26(19), 2455-2457. doi:10.1093/bioinformatics/btq429Drummond, A. J., & Rambaut, A. (2007). BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7(1), 214. doi:10.1186/1471-2148-7-214Parker, J., Rambaut, A., & Pybus, O. G. (2008). Correlating viral phenotypes with phylogeny: Accounting for phylogenetic uncertainty. Infection, Genetics and Evolution, 8(3), 239-246. doi:10.1016/j.meegid.2007.08.001Wang, T. H., Donaldson, Y. K., Brettle, R. P., Bell, J. E., & Simmonds, P. (2001). Identification of Shared Populations of Human Immunodeficiency Virus Type 1 Infecting Microglia and Tissue Macrophages outside the Central Nervous System. Journal of Virology, 75(23), 11686-11699. doi:10.1128/jvi.75.23.11686-11699.2001Lafforgue, G., Tromas, N., Elena, S. F., & Zwart, M. P. (2012). Dynamics of the Establishment of Systemic Potyvirus Infection: Independent yet Cumulative Action of Primary Infection Sites. Journal of Virology, 86(23), 12912-12922. doi:10.1128/jvi.02207-12Gibbs, A. J., Ohshima, K., Phillips, M. J., & Gibbs, M. J. (2008). The Prehistory of Potyviruses: Their Initial Radiation Was during the Dawn of Agriculture. PLoS ONE, 3(6), e2523. doi:10.1371/journal.pone.0002523Glasa, M., Malinowski, T., Predajňa, L., Pupola, N., Dekena, D., Michalczuk, L., & Candresse, T. (2011). Sequence Variability, Recombination Analysis, and Specific Detection of the W Strain ofPlum pox virus. Phytopathology, 101(8), 980-985. doi:10.1094/phyto-12-10-0334Zwart, M. P., Daròs, J.-A., & Elena, S. F. (2011). One Is Enough: In Vivo Effective Population Size Is Dose-Dependent for a Plant RNA Virus. PLoS Pathogens, 7(7), e1002122. doi:10.1371/journal.ppat.1002122Zwart, M. P., Daròs, J.-A., & Elena, S. F. (2012). Effects of Potyvirus Effective Population Size in Inoculated Leaves on Viral Accumulation and the Onset of Symptoms. Journal of Virology, 86(18), 9737-9747. doi:10.1128/jvi.00909-12Agudelo-Romero, P., Carbonell, P., Perez-Amador, M. A., & Elena, S. F. (2008). Virus Adaptation by Manipulation of Host’s Gene Expression. PLoS ONE, 3(6), e2397. doi:10.1371/journal.pone.0002397Jenner, C. E., Wang, X., Tomimura, K., Ohshima, K., Ponz, F., & Walsh, J. A. (2003). The Dual Role of the Potyvirus P3 Protein ofTurnip mosaic virusas a Symptom and Avirulence Determinant in Brassicas. Molecular Plant-Microbe Interactions, 16(9), 777-784. doi:10.1094/mpmi.2003.16.9.777Suehiro, N. (2004). An important determinant of the ability of Turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein. Journal of General Virology, 85(7), 2087-2098. doi:10.1099/vir.0.79825-0Kim, B. M., Suehiro, N., Natsuaki, T., Inukai, T., & Masuta, C. (2010). The P3 Protein ofTurnip mosaic virusCan Alone Induce Hypersensitive Response-Like Cell Death inArabidopsis thalianaCarryingTuNI. Molecular Plant-Microbe Interactions, 23(2), 144-152. doi:10.1094/mpmi-23-2-0144Hjulsager, C. K., Olsen, B. S., Jensen, D. M. K., Cordea, M. I., Krath, B. N., Johansen, I. E., & Lund, O. S. (2006). Multiple determinants in the coding region of Pea seed-borne mosaic virus P3 are involved in virulence against sbm-2 resistance. Virology, 355(1), 52-61. doi:10.1016/j.virol.2006.07.016Desbiez, C., Gal-On, A., Girard, M., Wipf-Scheibel, C., & Lecoq, H. (2003). Increase inZucchini yellow mosaic virusSymptom Severity in Tolerant Zucchini Cultivars Is Related to a Point Mutation in P3 Protein and Is Associated with a Loss of Relative Fitness on Susceptible Plants. Phytopathology, 93(12), 1478-1484. doi:10.1094/phyto.2003.93.12.1478Glasa, M., Svoboda, J., & Nováková, S. (2007). Analysis of the molecular and biological variability of Zucchini yellow mosaic virus isolates from Slovakia and Czech Republic. Virus Genes, 35(2), 415-421. doi:10.1007/s11262-007-0101-4Eggenberger, A. L., Hajimorad, M. R., & Hill, J. H. (2008). Gain of Virulence onRsv1-Genotype Soybean by an AvirulentSoybean mosaic virusRequires Concurrent Mutations in Both P3 and HC-Pro. Molecular Plant-Microbe Interactions, 21(7), 931-936. doi:10.1094/mpmi-21-7-0931WEN, R.-H., MAROOF, M. A. S., & HAJIMORAD, M. R. (2011). Amino acid changes in P3, and not the overlapping pipo-encoded protein, determine virulence of Soybean mosaic virus on functionally immune Rsv1-genotype soybean. Molecular Plant Pathology, 12(8), 799-807. doi:10.1111/j.1364-3703.2011.00714.xTan, Z. (2005). Mutations in Turnip mosaic virus genomes that have adapted to Raphanus sativus. Journal of General Virology, 86(2), 501-510. doi:10.1099/vir.0.80540-0Choi, I.-R. (2005). An internal RNA element in the P3 cistron of Wheat streak mosaic virus revealed by synonymous mutations that affect both movement and replication. Journal of General Virology, 86(9), 2605-2614. doi:10.1099/vir.0.81081-