1887

Journal of General Virology header logo

Volume 95, Issue 5

The asparagine residue in the FRNK box of potyviral helper-component protease is critical for its small RNA binding and subcellular localization Free

Abstract

The multifunctional potyviral helper-component protease (HcPro) contains variable regions with some functionally conserved domains, such as the FRNK box. Natural variants occur at the FRNK box, a conserved central domain, known for its role in RNA binding and RNAi suppression activities, although no dominant natural variants for the N residue are known to occur. Here, a mutant at HcPro was developed to investigate its role in natural populations. Using studies, we found an increase in the small RNA (sRNA) binding potential of HcPro without affecting its protein–protein interaction properties, suggesting that the presence of N is critical to maintain threshold levels of sRNAs, but does not interfere in the self-interaction of HcPro. Furthermore, we found that expression of HcPro in affected plant growth. Transient expression of HcPro induced reporter gene expression in 16c GFP transgenic plants more than HcPro did, suggesting that replacement of asparagine in the FRNK box favours RNA silencing suppression. HcPro was found to be distributed in the nucleus and cytoplasm, whereas HcPro was observed only in cytoplasmic inclusion bodies in leaves, when fused to a GFP tag and expressed by agro-infiltration, suggesting mutation favours oligomerization of HcPro. These findings suggest that amino acid N of the conserved FRNK box may regulate RNA silencing mechanisms, and is required for maintenance of the subcellular localization of the protein for its multi-functionality. Hence, the N residue of the FRNK box seems to be indispensable for potyvirus infection during evolution.

  • Received:
  • Accepted:
  • Published Online:
© 2014 The Authors
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.060269-0
2014-05-01
2023-11-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/95/5/1167.html?itemId=/content/journal/jgv/10.1099/vir.0.060269-0&mimeType=html&fmt=ahah

References

  1. Anandalakshmi R., Marathe R., Ge X., Herr J. M. Jr, Mau C., Mallory A., Pruss G., Bowman L., Vance V. B. 2000; A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants. Science 290:142–144 [View Article][PubMed]
     [Google Scholar]
  2. Atreya C. D., Pirone T. P. 1993; Mutational analysis of the helper component-proteinase gene of a potyvirus: effects of amino acid substitutions, deletions, and gene replacement on virulence and aphid transmissibility. Proc Natl Acad Sci U S A 90:11919–11923 [View Article][PubMed]
     [Google Scholar]
  3. Ballut L., Drucker M., Pugnière M., Cambon F., Blanc S., Roquet F., Candresse T., Schmid H. P., Nicolas P. other authors 2005; HcPro, a multifunctional protein encoded by a plant RNA virus, targets the 20S proteasome and affects its enzymic activities. J Gen Virol 86:2595–2603 [View Article][PubMed]
     [Google Scholar]
  4. Blanc S., López-Moya J. J., Wang R., García-Lampasona S., Thornbury D. W., Pirone T. P. 1997; A specific interaction between coat protein and helper component correlates with aphid transmission of a potyvirus. Virology 231:141–147 [View Article][PubMed]
     [Google Scholar]
  5. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [View Article][PubMed]
     [Google Scholar]
  6. Canto T., Uhrig J. F., Swanson M., Wright K. M., MacFarlane S. A. 2006; Translocation of Tomato bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing suppressor activity. J Virol 80:9064–9072 [View Article][PubMed]
     [Google Scholar]
  7. Carrington J. C., Dougherty W. G. 1988; A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing. Proc Natl Acad Sci U S A 85:3391–3395 [View Article][PubMed]
     [Google Scholar]
  8. Castel S. E., Martienssen R. A. 2013; RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat Rev Genet 14:100–112 [View Article][PubMed]
     [Google Scholar]
  9. Chapman E. J., Prokhnevsky A. I., Gopinath K., Dolja V. V., Carrington J. C. 2004; Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18:1179–1186 [View Article][PubMed]
     [Google Scholar]
  10. Da G., Lenkart J., Zhao K., Shiekhattar R., Cairns B. R., Marmorstein R. 2006; Structure and function of the SWIRM domain, a conserved protein module found in chromatin regulatory complexes. Proc Natl Acad Sci U S A 103:2057–2062 [View Article][PubMed]
     [Google Scholar]
  11. Dunoyer P., Lecellier C. H., Parizotto E. A., Himber C., Voinnet O. 2004; Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. Plant Cell 16:1235–1250 [View Article][PubMed]
     [Google Scholar]
  12. Fargette D., Pinel A., Rakotomalala M., Sangu E., Traoré O., Sérémé D., Sorho F., Issaka S., Hébrard E. other authors 2008; Rice yellow mottle virus, an RNA plant virus, evolves as rapidly as most RNA animal viruses. J Virol 82:3584–3589 [View Article][PubMed]
     [Google Scholar]
  13. Fernández F. T., González I., Doblas P., Rodríguez C., Sahana N., Kaur H., Tenllado F., Praveen S., Canto T. 2013; The influence of cis-acting P1 protein and translational elements on the expression of Potato virus Y helper-component proteinase (HCPro) in heterologous systems and its suppression of silencing activity. Mol Plant Pathol 14:530–541 [View Article][PubMed]
     [Google Scholar]
  14. García-Arenal F., Fraile A., Malpica J. M. 2003; Variation and evolution of plant virus populations. Int Microbiol 6:225–232 [View Article][PubMed]
     [Google Scholar]
  15. González I., Martínez L., Rakitina D. V., Lewsey M. G., Atencio F. A., Llave C., Kalinina N. O., Carr J. P., Palukaitis P., Canto T. 2010; Cucumber mosaic virus 2b protein subcellular targets and interactions: their significance to RNA silencing suppressor activity. Mol Plant Microbe Interact 23:294–303 [View Article][PubMed]
     [Google Scholar]
  16. Govier D. A., Kassanis B., Pirone T. P. 1977; Partial purification and characterization of the potato virus Y helper component. Virology 78:306–314 [View Article][PubMed]
     [Google Scholar]
  17. Guo B., Lin J., Ye K. 2011; Structure of the autocatalytic cysteine protease domain of potyvirus helper-component proteinase. J Biol Chem 286:21937–21943 [View Article][PubMed]
     [Google Scholar]
  18. Jin Y., Ma D., Dong J., Jin J., Li D., Deng C., Wang T. 2007; HC-Pro protein of Potato virus Y can interact with three Arabidopsis 20S proteasome subunits in planta. J Virol 81:12881–12888 [View Article][PubMed]
     [Google Scholar]
  19. Kasschau K. D., Cronin S., Carrington J. C. 1997; Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase. Virology 228:251–262 [View Article][PubMed]
     [Google Scholar]
  20. Kasschau K. D., Xie Z., Allen E., Llave C., Chapman E. J., Krizan K. A., Carrington J. C. 2003; P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev Cell 4:205–217 [View Article][PubMed]
     [Google Scholar]
  21. Kelley L. A., Sternberg M. J. E. 2009; Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363–371 [View Article][PubMed]
     [Google Scholar]
  22. Lakatos L., Csorba T., Pantaleo V., Chapman E. J., Carrington J. C., Liu Y. P., Dolja V. V., Calvino L. F., López-Moya J. J., Burgyán J. 2006; Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. EMBO J 25:2768–2780 [View Article][PubMed]
     [Google Scholar]
  23. Lloyd R. E. 2012; How do viruses interact with stress-associated RNA granules?. PLoS Pathog 8:e1002741 [View Article][PubMed]
     [Google Scholar]
  24. Maia I. G., Bernardi F. 1996; Nucleic acid-binding properties of a bacterially expressed potato virus Y helper component-proteinase. J Gen Virol 77:869–877 [View Article][PubMed]
     [Google Scholar]
  25. Mallory A. C., Ely L., Smith T. H., Marathe R., Anandalakshmi R., Fagard M., Vaucheret H., Pruss G., Bowman L., Vance V. B. 2001; HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal. Plant Cell 13:571–583[PubMed] [CrossRef]
     [Google Scholar]
  26. Mangrauthia S. K., Singh Shakya V. P., Jain R. K., Praveen S. 2009; Ambient temperature perception in papaya for papaya ringspot virus interaction. Virus Genes 38:429–434 [View Article][PubMed]
     [Google Scholar]
  27. Mérai Z., Kerényi Z., Kertész S., Magna M., Lakatos L., Silhavy D. 2006; Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing. J Virol 80:5747–5756 [View Article][PubMed]
     [Google Scholar]
  28. Mlotshwa S., Schauer S. E., Smith T. H., Mallory A. C., Herr J. M. Jr, Roth B., Merchant D. S., Ray A., Bowman L. H., Vance V. B. 2005; Ectopic DICER-LIKE1 expression in P1/HC-Pro Arabidopsis rescues phenotypic anomalies but not defects in microRNA and silencing pathways. Plant Cell 17:2873–2885 [View Article][PubMed]
     [Google Scholar]
  29. Plisson C., Drucker M., Blanc S., German-Retana S., Le Gall O., Thomas D., Bron P. 2003; Structural characterization of HC-Pro, a plant virus multifunctional protein. J Biol Chem 278:23753–23761 [View Article][PubMed]
     [Google Scholar]
  30. Rakitina D. V., Yelina N. E., Kalinina N. O. 2006; Zinc ions stimulate the cooperative RNA binding of hordeiviral gammab protein. FEBS Lett 580:5077–5083 [View Article][PubMed]
     [Google Scholar]
  31. Riedel D., Lesemann D. E., Maiss E. 1998; Ultrastructural localization of nonstructural and coat proteins of 19 potyviruses using antisera to bacterially expressed proteins of plum pox potyvirus. Arch Virol 143:2133–2158 [View Article][PubMed]
     [Google Scholar]
  32. Roossinck M. J. 1997; Mechanisms of plant virus evolution. Annu Rev Phytopathol 35:191–209 [View Article][PubMed]
     [Google Scholar]
  33. Ruiz-Ferrer V., Boskovic J., Alfonso C., Rivas G., Llorca O., López-Abella D., López-Moya J. J. 2005; Structural analysis of tobacco etch potyvirus HC-pro oligomers involved in aphid transmission. J Virol 79:3758–3765 [View Article][PubMed]
     [Google Scholar]
  34. Sahana N., Kaur H., Basavaraj, Tena F., Jain R. K., Palukaitis P., Canto T., Praveen S. 2012; Inhibition of the host proteasome facilitates papaya ringspot virus accumulation and proteosomal catalytic activity is modulated by viral factor HcPro. PLoS ONE 7:e52546 [View Article][PubMed]
     [Google Scholar]
  35. Shiboleth Y. M., Haronsky E., Leibman D., Arazi T., Wassenegger M., Whitham S. A., Gaba V., Gal-On A. 2007; The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development. J Virol 81:13135–13148 [View Article][PubMed]
     [Google Scholar]
  36. Stenger D. C., Seifers D. L., French R. 2002; Patterns of polymorphism in wheat streak mosaic virus: sequence space explored by a clade of closely related viral genotypes rivals that between the most divergent strains. Virology 302:58–70 [View Article][PubMed]
     [Google Scholar]
  37. Stenger D. C., Young B. A., Qu F., Morris T. J., French R. 2007; Wheat streak mosaic virus lacking helper component-proteinase is competent to produce disease synergism in double infections with Maize chlorotic mottle virus . Phytopathology 97:1213–1221 [View Article][PubMed]
     [Google Scholar]
  38. Tena F., González I., Doblas P., Rodríguez C., Sahana N., Kaur H., Tenllado F., Praveen S., Canto T. 2013; The influence of cis-acting P1 and translational elements on the expression of Potato virus Y helper-component proteinase (HCPro) in heterologous systems and its suppression of silencing activity. Molecular Plant Path 2013: doi: 10.1111/mpp.12025 [View Article]
     [Google Scholar]
  39. Torres-Barceló C., Martín S., Daròs J. A., Elena S. F. 2008; From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of the Tobacco etch potyvirus HC-Pro. Genetics 180:1039–1049 [View Article][PubMed]
     [Google Scholar]
  40. Urcuqui-Inchima S., Walter J., Drugeon G., German-Retana S., Haenni A. L., Candresse T., Bernardi F., Le Gall O. 1999; Potyvirus helper component-proteinase self-interaction in the yeast two-hybrid system and delineation of the interaction domain involved. Virology 258:95–99 [View Article][PubMed]
     [Google Scholar]
  41. Vazquez F., Hohn T. 2013; Biogenesis and biological activity of secondary siRNAs in plants. Scientifica 2013:783253 [View Article][PubMed]
     [Google Scholar]
  42. Xiong R., Wang A. 2013; SCE1, the SUMO-conjugating enzyme in plants that interacts with NIb, the RNA-dependent RNA polymerase of Turnip mosaic virus, is required for viral infection. J Virol 87:4704–4715 [View Article][PubMed]
     [Google Scholar]
  43. Zheng H., Yan F., Lu Y., Sun L., Lin L., Cai L., Hou M., Chen J. 2011; Mapping the self-interacting domains of TuMV HC-Pro and the subcellular localization of the protein. Virus Genes 42:110–116 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.060269-0
Loading
The asparagine residue in the FRNK box of potyviral helper-component protease is critical for its small RNA binding and subcellular localization
J. Gen. Virol. 95, 1167 (2014); https://doi.org/10.1099/vir.0.060269-0
/content/journal/jgv/10.1099/vir.0.060269-0
/content/journal/jgv/10.1099/vir.0.060269-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error