Potyviruses are plant pathogens transmitted by aphids in a non-persistent manner. During transmission, the virus-encoded factor helper-component protease (HCPro) is presumed to act as a molecular bridge, mediating the reversible retention of virions to uncharacterized binding sites in the vector mouthparts. Whilst the predicted interaction between HCPro and the coat protein (CP) of virions has been confirmed experimentally, the characterization of putative HCPro-specific receptors in aphids has remained elusive, with the exception of a report that described binding of HCPro of zucchini yellow mosaic virus to several cuticle proteins. To identify other aphid components that could play a role during transmission, this study used purified HCPro of tobacco etch virus (TEV) in far-Western blotting assays as bait to select interactors among proteins extracted from aphid heads. With this approach, new HCPro-interacting proteins were found, and several were identified after mass spectrometry analysis and searches in databases dedicated to aphid sequences. Among these interactors, a ribosomal protein S2 (RPS2) was chosen for further investigation due to its homology with the laminin receptor precursor, known to act as the receptor of several viruses. The specific interaction between RPS2 and TEV HCPro was confirmed after cloning and heterologous expression of the corresponding gene. The possible involvement of RPS2 in the transmission process was further suggested by testing a variant of HCPro that was non-functional for transmission due to a mutation in the conserved KITC motif (EITC variant). This variant retained its ability to bind CP but failed to interact with RPS2.


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  1. Akache, B., Grimm, D., Pandey, K., Yant, S. R., Xu, H. & Kay, M. A.(2006). The 37/67-kilodalton laminin receptor is a receptor for adeno-associated virus serotypes 8, 2, 3, and 9. J Virol 80, 9831–9836.[CrossRef] [Google Scholar]
  2. Allison, R., Johnston, R. E. & Dougherty, W. G.(1986). The nucleotide sequence of the coding region of tobacco etch virus genomic RNA: evidence for the synthesis of a single polyprotein. Virology 154, 9–20.[CrossRef] [Google Scholar]
  3. Ammar, E. D., Jarlfors, U. & Pirone, T. P.(1994). Association of potyvirus helper component protein and virions and the cuticle lining the maxillary food canal and foregut of an aphid vector. Phytopathology 84, 1054–1060.[CrossRef] [Google Scholar]
  4. 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.[CrossRef] [Google Scholar]
  5. 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.[CrossRef] [Google Scholar]
  6. Atreya, C. D., Atreya, P. L., Thornbury, D. W. & Pirone, T. P.(1992). Site-directed mutations in the potyvirus HC-Pro gene affect helper component activity, virus accumulation, and symptom expression in infected tobacco plants. Virology 191, 106–111.[CrossRef] [Google Scholar]
  7. Ballut, L., Drucker, M., Pugniere, 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.[CrossRef] [Google Scholar]
  8. Blanc, S., Ammar, E. D., Garcia-Lampasona, S., Dolja, V. V., Llave, C., Baker, J. & Pirone, T. P.(1998). Mutations in the potyvirus helper component protein: effects on interactions with virions and aphid stylets. J Gen Virol 79, 3119–3122. [Google Scholar]
  9. Blanc, S., Dolja, V. V., Llave, C. & Pirone, T. P.(1999). Histidine-tagging and purification of tobacco etch potyvirus helper component protein. J Virol Methods 77, 11–15.[CrossRef] [Google Scholar]
  10. Carrington, J. C., Cary, S. M., Parks, T. D. & Dougherty, W. G.(1989). A second proteinase encoded by a plant potyvirus genome. EMBO J 8, 365–370. [Google Scholar]
  11. Cheng, Y. Q., Liu, Z. M., Xu, J., Zhou, T., Wang, M., Chen, Y. T., Li, H. F. & Fan, Z. F.(2008). HC-Pro protein of sugar cane mosaic virus interacts specifically with maize ferredoxin-5 in vitro and in planta. J Gen Virol 89, 2046–2054.[CrossRef] [Google Scholar]
  12. Cronin, S., Verchot, J., Haldeman-Cahill, R., Schaad, M. C. & Carrington, J. C.(1995). Long-distance movement factor: a transport function of the potyvirus helper component proteinase. Plant Cell 7, 549–559.[CrossRef] [Google Scholar]
  13. Dombrovsky, A., Gollop, N., Chen, S., Chejanovsky, N. & Raccah, B.(2007).In vitro association between the helper component-proteinase of zucchini yellow mosaic virus and cuticle proteins of Myzus persicae. J Gen Virol 88, 1602–1610.[CrossRef] [Google Scholar]
  14. Fernández-Calvino, L., López-Abella, D., López-Moya, J. J. & Fereres, A.(2006). Comparison of Potato virus Y and Plum pox virus transmission by two aphid species in relation to their probing behavior. Phytoparasitica 34, 315–324.[CrossRef] [Google Scholar]
  15. Froissart, R., Michalakis, Y. & Blanc, S.(2002). Helper component-transcomplementation in the vector transmission of plant viruses. Phytopathology 92, 576–579.[CrossRef] [Google Scholar]
  16. Gauczynski, S., Peyrin, J. M., Haik, S., Leucht, C., Hundt, C., Rieger, R., Krasemann, S., Deslys, J. P., Dormont, D. & other authors(2001). The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein. EMBO J 20, 5863–5875.[CrossRef] [Google Scholar]
  17. Gorg, A., Weiss, W. & Dunn, M. J.(2004). Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685.[CrossRef] [Google Scholar]
  18. Govier, D. A. & Kassanis, B.(1974a). A virus-induced component of plant sap needed when aphids acquire potato virus Y from purified preparations. Virology 61, 420–426.[CrossRef] [Google Scholar]
  19. Govier, D. A. & Kassanis, B.(1974b). Evidence that a component other than the virus particle is needed for aphid transmission of potato virus Y. Virology 57, 285–286.[CrossRef] [Google Scholar]
  20. Govier, D. A., Kassanis, B. & Pirone, T. P.(1977). Partial purification and characterization of the potato virus Y helper component. Virology 78, 306–314.[CrossRef] [Google Scholar]
  21. Goytia, E., Fernández-Calvino, L., Martínez-García, B., López-Abella, D. & López-Moya, J. J.(2006). Production of plum pox virus HC-Pro functionally active for aphid transmission in a transient-expression system. J Gen Virol 87, 3413–3423.[CrossRef] [Google Scholar]
  22. Gray, S. M. & Banerjee, N.(1999). Mechanisms of arthropod transmission of plant and animal viruses. Microbiol Mol Biol Rev 63, 128–148. [Google Scholar]
  23. Guo, D., Spetz, C., Saarma, M. & Valkonen, J. P.(2003). Two potato proteins, including a novel RING finger protein (HIP1), interact with the potyviral multifunctional protein HCpro. Mol Plant Microbe Interact 16, 405–410.[CrossRef] [Google Scholar]
  24. He, N., Botelho, J. M., McNall, R. J., Belozerov, V., Dunn, W. A., Mize, T., Orlando, R. & Willis, J. H.(2007). Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry. Insect Biochem Mol Biol 37, 135–146.[CrossRef] [Google Scholar]
  25. Hogenhout, S. A., Ammar, el-D., Whitfield, A. E. & Redinbaugh, M. G.(2008). Insect vector interactions with persistently transmitted viruses. Annu Rev Phytopathol 46, 327–359.[CrossRef] [Google Scholar]
  26. Hoh, F., Uzest, M., Drucker, M., Plisson-Chastang, C., Bron, P., Blanc, S. & Dumas, C.(2010). Structural insights into the molecular mechanisms of Cauliflower mosaic virus transmission by its insect vector. J Virol 84, 4706–4713.[CrossRef] [Google Scholar]
  27. Jin, Y., Ma, D., Dong, J., Jin, J., Li, D., Deng, C. & Wang, T.(2007a). HC-Pro protein of Potato virus Y can interact with three Arabidopsis 20S proteasome subunits in planta. J Virol 81, 12881–12888.[CrossRef] [Google Scholar]
  28. Jin, Y., Ma, D., Dong, J., Li, D., Deng, C., Jin, J. & Wang, T.(2007b). The HC-pro protein of potato virus Y interacts with NtMinD of tobacco. Mol Plant Microbe Interact 20, 1505–1511.[CrossRef] [Google Scholar]
  29. Kalume, D. E., Okulate, M., Zhong, J., Reddy, R., Suresh, S., Deshpande, N., Kumar, N. & Pandey, A.(2005). A proteomic analysis of salivary glands of female Anopheles gambiae mosquito. Proteomics 5, 3765–3777.[CrossRef] [Google Scholar]
  30. 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.[CrossRef] [Google Scholar]
  31. Laemmli, U. K.(1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef] [Google Scholar]
  32. 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. & Burgyan, J.(2006). Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. EMBO J 25, 2768–2780.[CrossRef] [Google Scholar]
  33. Leh, V., Jacquot, E., Geldreich, A., Hermann, T., Leclerc, D., Cerutti, M., Yot, P., Keller, M. & Blanc, S.(1999). Aphid transmission of cauliflower mosaic virus requires the viral PIII protein. EMBO J 18, 7077–7085.[CrossRef] [Google Scholar]
  34. Li, C., Cox-Foster, D., Gray, S. M. & Gildow, F.(2001). Vector specificity of barley yellow dwarf virus (BYDV) transmission: identification of potential cellular receptors binding BYDV-MAV in the aphid, Sitobion avenae. Virology 286, 125–133.[CrossRef] [Google Scholar]
  35. López-Moya, J. J., Valli, A. & Garcia, J. A.(2009). Potyviridae. In Encyclopedia of Life Sciences, pp. 1–10. Chichester. : Wiley. [Google Scholar]
  36. Martin, B., Collar, J. L., Tjallingii, W. F. & Fereres, A.(1997). Intracellular ingestion and salivation by aphids may cause the acquisition and inoculation of non-persistently transmitted plant viruses. J Gen Virol 78, 2701–2705. [Google Scholar]
  37. Martiniere, A., Gargani, D., Uzest, M., Lautredou, N., Blanc, S. & Drucker, M.(2009). A role for plant microtubules in the formation of transmission-specific inclusion bodies of Cauliflower mosaic virus. Plant J 58, 135–146.[CrossRef] [Google Scholar]
  38. Medeiros, R. B., Ullman, D. E., Sherwood, J. L. & German, T. L.(2000). Immunoprecipitation of a 50-kDa protein: a candidate receptor component for tomato spotted wilt tospovirus (Bunyaviridae) in its main vector, Frankliniella occidentalis. Virus Res 67, 109–118.[CrossRef] [Google Scholar]
  39. Moury, B., Fabre, F. & Senoussi, R.(2007). Estimation of the number of virus particles transmitted by an insect vector. Proc Natl Acad Sci U S A 104, 17891–17896.[CrossRef] [Google Scholar]
  40. Murphy, J. F., Rhoads, R. E., Hunt, A. G. & Shaw, J. G.(1990). The VPg of tobacco etch virus RNA is the 49-kDa proteinase or the N-terminal 24-kDa part of the proteinase. Virology 178, 285–288.[CrossRef] [Google Scholar]
  41. Nelson, J., McFerran, N. V., Pivato, G., Chambers, E., Doherty, C., Steele, D. & Timson, D. J.(2008). The 67 kDa laminin receptor: structure, function and role in disease. Biosci Rep 28, 33–48.[CrossRef] [Google Scholar]
  42. Ng, J. C. & Falk, B. W.(2006). Virus–vector interactions mediating nonpersistent and semipersistent transmission of plant viruses. Annu Rev Phytopathol 44, 183–212.[CrossRef] [Google Scholar]
  43. Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S.(1999). Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567.[CrossRef] [Google Scholar]
  44. Pirone, T. P. & Blanc, S.(1996). Helper-dependent vector transmission of plant viruses. Annu Rev Phytopathol 34, 227–247.[CrossRef] [Google Scholar]
  45. Pirone, T. P. & Perry, K. L.(2002). Aphids: non-persistent transmission. In Advances in Botanical Research, pp. 1–19. Edited by Plumb, R. T.. San Diego, CA. : Academic Press. [Google Scholar]
  46. 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.[CrossRef] [Google Scholar]
  47. Plisson, C., Uzest, M., Drucker, M., Froissart, R., Dumas, C., Conway, J., Thomas, D., Blanc, S. & Bron, P.(2005). Structure of the mature P3–virus particle complex of cauliflower mosaic virus revealed by cryo-electron microscopy. J Mol Biol 346, 267–277.[CrossRef] [Google Scholar]
  48. Rabilloud, T.(1998). Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis 19, 758–760.[CrossRef] [Google Scholar]
  49. Raccah, B., Huet, H. & Blanc, S.(2001). Potyviruses. In Virus–Insect–Plant Interactions, pp. 181–206. Edited by Harris, K., Smith, O. P. & Duffus, J. E.. San Diego, CA. : Academic Press. [Google Scholar]
  50. Rojas, M. R., Zerbini, F. M., Allison, R. F., Gilbertson, R. L. & Lucas, W. J.(1997). Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins. Virology 237, 283–295.[CrossRef] [Google Scholar]
  51. 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.[CrossRef] [Google Scholar]
  52. Seddas, P., Boissinot, S., Strub, J. M., Van Dorsselaer, A., Van Regenmortel, M. H. & Pattus, F.(2004). Rack-1, GAPDH3, and actin: proteins of Myzus persicae potentially involved in the transcytosis of beet western yellows virus particles in the aphid. Virology 325, 399–412.[CrossRef] [Google Scholar]
  53. Senapin, S. & Phongdara, A.(2006). Binding of shrimp cellular proteins to Taura syndrome viral capsid proteins VP1, VP2 and VP3. Virus Res 122, 69–77.[CrossRef] [Google Scholar]
  54. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M.(1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68, 850–858.[CrossRef] [Google Scholar]
  55. Suckau, D., Resemann, A., Schuerenberg, M., Hufnagel, P., Franzen, J. & Holle, A.(2003). A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal Bioanal Chem 376, 952–965.[CrossRef] [Google Scholar]
  56. Tagu, D., Klingler, J. P., Moya, A. & Simon, J. C.(2008). Early progress in aphid genomics and consequences for plant–aphid interactions studies. Mol Plant Microbe Interact 21, 701–708.[CrossRef] [Google Scholar]
  57. The International Aphid Genomics Consortium(2010). Genome sequence of the pea aphid Acyrthosiphon pisum. PLoS Biol 8, e1000313.[CrossRef] [Google Scholar]
  58. Tio, P. H., Jong, W. W. & Cardosa, M. J.(2005). Two dimensional VOPBA reveals laminin receptor (LAMR1) interaction with dengue virus serotypes 1, 2 and 3. Virol J 2, 25.[CrossRef] [Google Scholar]
  59. Uzest, M., Gargani, D., Drucker, M., Hebrard, E., Garzo, E., Candresse, T., Fereres, A. & Blanc, S.(2007). A protein key to plant virus transmission at the tip of the insect vector stylet. Proc Natl Acad Sci U S A 104, 17959–17964.[CrossRef] [Google Scholar]
  60. Uzest, M., Gargani, D., Dombrovsky, A., Cazevieille, C., Cot, D. & Blanc, S.(2010). The “acrostyle”: a newly described anatomical structure in aphid stylets. Arthropod Struct Dev 39, 221–229.[CrossRef] [Google Scholar]
  61. van den Heuvel, J. F., Bruyere, A., Hogenhout, S. A., Ziegler-Graff, V., Brault, V., Verbeek, M., van der Wilk, F. & Richards, K.(1997). The N-terminal region of the luteovirus readthrough domain determines virus binding to Buchnera GroEL and is essential for virus persistence in the aphid. J Virol 71, 7258–7265. [Google Scholar]
  62. Wang, K. S., Kuhn, R. J., Strauss, E. G., Ou, S. & Strauss, J. H.(1992). High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells. J Virol 66, 4992–5001. [Google Scholar]
  63. Wang, R. Y., Ammar, E. D., Thornbury, D. W., López-Moya, J. J. & Pirone, T. P.(1996). Loss of potyvirus transmissibility and helper-component activity correlate with non-retention of virions in aphid stylets. J Gen Virol 77, 861–867.[CrossRef] [Google Scholar]
  64. Yan, J. X., Wait, R., Berkelman, T., Harry, R. A., Westbrook, J. A., Wheeler, C. H. & Dunn, M. J.(2000). A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21, 3666–3672.[CrossRef] [Google Scholar]
  65. Yang, X., Thannhauser, T. W., Burrows, M., Cox-Foster, D., Gildow, F. E. & Gray, S. M.(2008). Coupling genetics and proteomics to identify aphid proteins associated with vector-specific transmission of polerovirus (Luteoviridae). J Virol 82, 291–299.[CrossRef] [Google Scholar]
  66. Yang, C., Zhang, C., Dittman, J. D. & Whitham, S. A.(2009). Differential requirement of ribosomal protein S6 by plant RNA viruses with different translation initiation strategies. Virology 390, 163–173.[CrossRef] [Google Scholar]

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