1887

Abstract

The putative NTP-binding protein (NTB) of (ToRSV) contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. In infected plants, the NTB–VPg polyprotein (containing the domain for the genome-linked protein) is associated with endoplasmic reticulum-derived membranes that are active in ToRSV replication. Recent results from proteinase K protection assays suggested a luminal location for the VPg domain in infected plants, providing support for the presence of a transmembrane domain at the C terminus of NTB. In this study, we have shown that NTB–VPg associates with canine microsomal membranes in the absence of other viral proteins and adopts a topology similar to that observed in that the VPg is present in the lumen. Truncated proteins containing 60 amino acids at the C terminus of NTB and the entire VPg exhibited a similar topology, confirming that this region of the protein contains a functional transmembrane domain. Deletion of portions of the C-terminal hydrophobic region of NTB by mutagenesis and introduction of glycosylation sites to map the luminal regions of the protein revealed that only the first stretch of hydrophobic amino acids traverses the membrane, while the second stretch of hydrophobic amino acids is located in the lumen. Our results provide additional evidence supporting the hypothesis that the NTB–VPg polyprotein acts as a membrane-anchor for the replication complex.

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2004-02-01
2024-04-18
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References

  1. Amberg S. M., Rice C. M. 1999; Mutagenesis of the NS2B–NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing. J Virol 73:8083–8094
    [Google Scholar]
  2. Buck K. W. 1996; Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv Virus Res 47:159–251
    [Google Scholar]
  3. Carette J. E., Stuiver M., van Lent J., Wellink J., van Kammen A. 2000; Cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not Golgi membranes and is dependent on de novo membrane synthesis. J Virol 74:6556–6563
    [Google Scholar]
  4. Carette J. E., van Lent J., MacFarlane S. A., Wellink J., van Kammen A. 2002; Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes. J Virol 76:6293–6301
    [Google Scholar]
  5. Fisher C. L., Pei G. K. 1997; Modification of a PCR-based site-directed mutagenesis method. Biotechniques 23:570–574
    [Google Scholar]
  6. Guinea R., Carrasco L. 1990; Phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena. EMBO J 9:2011–2016
    [Google Scholar]
  7. Han S., Sanfaçon H. 2003; Tomato ringspot virus proteins containing the nucleoside triphosphate binding domain are transmembrane proteins that associate with the endoplasmic reticulum and cofractionate with replication complexes. J Virol 77:523–534
    [Google Scholar]
  8. Hans F., Sanfaçon H. 1995; Tomato ringspot nepovirus protease: characterization and cleavage site specificity. J Gen Virol 76:917–927
    [Google Scholar]
  9. Hirokawa T., Boon-Chieng S., Mitaku S. 1998; SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14:378–379
    [Google Scholar]
  10. Hofmann K., Stoffel W. 1993; TMbase – a database of membrane spanning protein segments. Biol Chem Hoppe-Seyler 347:166
    [Google Scholar]
  11. Jones D. T., Taylor W. R., Thornton J. M. 1994; A model recognition approach to the prediction of all-helical membrane protein structure and topology. Biochemistry 33:3038–3049
    [Google Scholar]
  12. Kasturi L., Eshleman J. R., Wunner W. H., Shakin-Eshleman S. H. 1995; The hydroxy amino acid in an Asn-X-Ser/Thr sequon can influence N -linked core glycosylation efficiency and the level of expression of a cell surface glycoprotein. J Biol Chem 270:14756–14761
    [Google Scholar]
  13. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  14. Lee E., Stocks C. E., Amberg S. M., Rice C. M., Lobigs M. 2000; Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage in vitro is lethal for virus production. J Virol 74:24–32
    [Google Scholar]
  15. Mayo M. A., Robinson D. J. 1996; Nepoviruses: molecular biology and replication. In The Plant Viruses vol 5 Polyhedral Virions and Bipartite RNA Genomes pp 139–185 Edited by Harrison B. D., Murrant A. F. New York: Plenum;
    [Google Scholar]
  16. Monne M., von Heijne G. 2001; Effects of ‘hydrophobic mismatch’ on the location of transmembrane helices in the ER membrane. FEBS Lett 496:96–100
    [Google Scholar]
  17. Nielsen H., Engelbrecht J., Brunak S., von Heijne G. 1997; Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–9
    [Google Scholar]
  18. Nilsson I. M., von Heijne G. 1993; Determination of the distance between the oligosaccharyltransferase active site and the endoplasmic reticulum membrane. J Biol Chem 268:5798–5801
    [Google Scholar]
  19. Nilsson I., Johnson A. E., von Heijne G. 2002; Cleavage of a tail-anchored protein by signal peptidase. FEBS Lett 516:106–108
    [Google Scholar]
  20. Paul A. V., van Boom J. H., Filippov D., Wimmer E. 1998; Protein-primed RNA synthesis by purified poliovirus RNA polymerase. Nature 393:280–284
    [Google Scholar]
  21. Porter A. G. 1993; Picornavirus nonstructural proteins: emerging roles in virus replication and inhibition of host cell functions. J Virol 67:6917–6921
    [Google Scholar]
  22. Ritzenthaler C., Laporte C., Gaire F. 8 other authors 2002; Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes. J Virol 76:8808–8819
    [Google Scholar]
  23. Rost B., Casadio R., Fariselli P. 1996; Refining neural network predictions for helical transmembrane proteins by dynamic programming. ISMB 4:192–200
    [Google Scholar]
  24. Rott M. E., Gilchrist A., Lee L., Rochon D. 1995; Nucleotide sequence of tomato ringspot virus RNA1. J Gen Virol 76:465–473
    [Google Scholar]
  25. Sanfaçon H. 1995; Nepoviruses. . In Pathogenesis and Host Specificity in Plant Diseases ,vol. III, Viruses and Viroids pp  129–141 Edited by Singh R. P., Singh U. S., Kohmoto K. Oxford: Pergamon Press;
    [Google Scholar]
  26. Schaad M. C., Jensen P. E., Carrington J. C. 1997; Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein. EMBO J 16:4049–4059
    [Google Scholar]
  27. Sonnhammer E. L., von Heijne G., Krogh A. 1998; A hidden Markov model for predicting transmembrane helices in protein sequences. ISMB 6:175–182
    [Google Scholar]
  28. Suhy D. A., Giddings T. H. Jr., Kirkegaard K. 2000; Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles. J Virol 74:8953–8965
    [Google Scholar]
  29. Tusnady G. E., Simon I. 1998; Principles governing amino acid composition of integral membrane proteins: applications to topology prediction. J Mol Biol 283:489–506
    [Google Scholar]
  30. von Heijne G. 1992; Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol 225:487–494
    [Google Scholar]
  31. Wang A., Sanfaçon H. 2000a; Diversity in the coding regions for the coat protein, VPg, protease and putative RNA-dependent RNA polymerase among tomato ringspot nepovirus isolates. Can J Plant Pathol 22:145–149
    [Google Scholar]
  32. Wang A., Sanfaçon H. 2000b; Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro . J Gen Virol 81:2771–2781
    [Google Scholar]
  33. Wang A., Carrier K., Chisholm J., Wieczorek A., Huguenot C., Sanfaçon H. 1999; Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the VPg, protease and putative RNA-dependent RNA polymerase. J Gen Virol 80:799–809
    [Google Scholar]
  34. Xiang W., Paul A. V., Wimmer E. 1997; RNA signals in entero- and rhinovirus genome replication. Semin Virol 8:256–273
    [Google Scholar]
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