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. 1997 Jul 1;16(13):4049–4059. doi: 10.1093/emboj/16.13.4049

Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein.

M C Schaad 1, P E Jensen 1, J C Carrington 1
PMCID: PMC1170028  PMID: 9233814

Abstract

The mechanisms that direct positive-stranded RNA virus replication complexes to plant and animal cellular membranes are poorly understood. We describe a specific interaction between a replication protein of an RNA plant virus and membranes in vitro and in live cells. The tobacco etch virus (TEV) 6 kDa protein associated with membranes as an integral protein via a central 19 amino acid hydrophobic domain. In the presence or absence of other viral proteins, fluorescent fusion proteins containing the 6 kDa protein associated with large vesicular compartments derived from the endoplasmic reticulum (ER). Infection by TEV was associated with a collapse of the ER network into a series of discrete aggregated structures. Viral RNA replication complexes from infected cells were also associated with ER-like membranes. Targeting of TEV RNA replication complexes to membranous sites of replication is proposed to involve post-translational interactions between the 6 kDa protein and the ER.

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Selected References

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  1. Bienz K., Egger D., Pasamontes L. Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography. Virology. 1987 Sep;160(1):220–226. doi: 10.1016/0042-6822(87)90063-8. [DOI] [PubMed] [Google Scholar]
  2. Carrington J. C., Haldeman R., Dolja V. V., Restrepo-Hartwig M. A. Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo. J Virol. 1993 Dec;67(12):6995–7000. doi: 10.1128/jvi.67.12.6995-7000.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cho M. W., Teterina N., Egger D., Bienz K., Ehrenfeld E. Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2BC in human cells. Virology. 1994 Jul;202(1):129–145. doi: 10.1006/viro.1994.1329. [DOI] [PubMed] [Google Scholar]
  4. Doedens J. R., Kirkegaard K. Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A. EMBO J. 1995 Mar 1;14(5):894–907. doi: 10.1002/j.1460-2075.1995.tb07071.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dolja V. V., Haldeman R., Robertson N. L., Dougherty W. G., Carrington J. C. Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants. EMBO J. 1994 Mar 15;13(6):1482–1491. doi: 10.1002/j.1460-2075.1994.tb06403.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dolja V. V., McBride H. J., Carrington J. C. Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10208–10212. doi: 10.1073/pnas.89.21.10208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dougherty W. G., Semler B. L. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes. Microbiol Rev. 1993 Dec;57(4):781–822. doi: 10.1128/mr.57.4.781-822.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Echeverri A. C., Dasgupta A. Amino terminal regions of poliovirus 2C protein mediate membrane binding. Virology. 1995 Apr 20;208(2):540–553. doi: 10.1006/viro.1995.1185. [DOI] [PubMed] [Google Scholar]
  9. Frangioni J. V., Beahm P. H., Shifrin V., Jost C. A., Neel B. G. The nontransmembrane tyrosine phosphatase PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence. Cell. 1992 Feb 7;68(3):545–560. doi: 10.1016/0092-8674(92)90190-n. [DOI] [PubMed] [Google Scholar]
  10. García J. A., Riechmann J. L., Laín S. Proteolytic activity of the plum pox potyvirus NIa-like protein in Escherichia coli. Virology. 1989 Jun;170(2):362–369. doi: 10.1016/0042-6822(89)90426-1. [DOI] [PubMed] [Google Scholar]
  11. Gardiner M., Chrispeels M. J. Involvement of the Golgi Apparatus in the Synthesis and Secretion of Hydroxyproline-rich Cell Wall Glycoproteins. Plant Physiol. 1975 Mar;55(3):536–541. doi: 10.1104/pp.55.3.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hellmann G. M., Shaw J. G., Rhoads R. E. In vitro analysis of tobacco vein mottling virus NIa cistron: evidence for a virus-encoded protease. Virology. 1988 Apr;163(2):554–562. doi: 10.1016/0042-6822(88)90296-6. [DOI] [PubMed] [Google Scholar]
  13. Hong Y., Levay K., Murphy J. F., Klein P. G., Shaw J. G., Hunt A. G. A potyvirus polymerase interacts with the viral coat protein and VPg in yeast cells. Virology. 1995 Dec 1;214(1):159–166. doi: 10.1006/viro.1995.9944. [DOI] [PubMed] [Google Scholar]
  14. Klein P. G., Klein R. R., Rodríguez-Cerezo E., Hunt A. G., Shaw J. G. Mutational analysis of the tobacco vein mottling virus genome. Virology. 1994 Nov 1;204(2):759–769. doi: 10.1006/viro.1994.1591. [DOI] [PubMed] [Google Scholar]
  15. Kutay U., Hartmann E., Rapoport T. A. A class of membrane proteins with a C-terminal anchor. Trends Cell Biol. 1993 Mar;3(3):72–75. doi: 10.1016/0962-8924(93)90066-a. [DOI] [PubMed] [Google Scholar]
  16. Laurière M., Laurière C., Chrispeels M. J., Johnson K. D., Sturm A. Characterization of a xylose-specific antiserum that reacts with the complex asparagine-linked glycans of extracellular and vacuolar glycoproteins. Plant Physiol. 1989 Jul;90(3):1182–1188. doi: 10.1104/pp.90.3.1182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li X. H., Carrington J. C. Complementation of tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):457–461. doi: 10.1073/pnas.92.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li X. H., Valdez P., Olvera R. E., Carrington J. C. Functions of the tobacco etch virus RNA polymerase (NIb): subcellular transport and protein-protein interaction with VPg/proteinase (NIa). J Virol. 1997 Feb;71(2):1598–1607. doi: 10.1128/jvi.71.2.1598-1607.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Masaki R., Yamamoto A., Tashiro Y. Microsomal aldehyde dehydrogenase is localized to the endoplasmic reticulum via its carboxyl-terminal 35 amino acids. J Cell Biol. 1994 Sep;126(6):1407–1420. doi: 10.1083/jcb.126.6.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mitoma J., Ito A. The carboxy-terminal 10 amino acid residues of cytochrome b5 are necessary for its targeting to the endoplasmic reticulum. EMBO J. 1992 Nov;11(11):4197–4203. doi: 10.1002/j.1460-2075.1992.tb05513.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Murphy J. F., Klein P. G., Hunt A. G., Shaw J. G. Replacement of the tyrosine residue that links a potyviral VPg to the viral RNA is lethal. Virology. 1996 Jun 15;220(2):535–538. doi: 10.1006/viro.1996.0344. [DOI] [PubMed] [Google Scholar]
  22. Murphy J. F., Rhoads R. E., Hunt A. G., Shaw J. G. The VPg of tobacco etch virus RNA is the 49-kDa proteinase or the N-terminal 24-kDa part of the proteinase. Virology. 1990 Sep;178(1):285–288. doi: 10.1016/0042-6822(90)90405-g. [DOI] [PubMed] [Google Scholar]
  23. Murphy J. F., Rychlik W., Rhoads R. E., Hunt A. G., Shaw J. G. A tyrosine residue in the small nuclear inclusion protein of tobacco vein mottling virus links the VPg to the viral RNA. J Virol. 1991 Jan;65(1):511–513. doi: 10.1128/jvi.65.1.511-513.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Picot D., Loll P. J., Garavito R. M. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature. 1994 Jan 20;367(6460):243–249. doi: 10.1038/367243a0. [DOI] [PubMed] [Google Scholar]
  25. Quadt R., Rosdorff H. J., Hunt T. W., Jaspars E. M. Analysis of the protein composition of alfalfa mosaic virus RNA-dependent RNA polymerase. Virology. 1991 May;182(1):309–315. doi: 10.1016/0042-6822(91)90674-z. [DOI] [PubMed] [Google Scholar]
  26. Restrepo-Hartwig M. A., Carrington J. C. Regulation of nuclear transport of a plant potyvirus protein by autoproteolysis. J Virol. 1992 Sep;66(9):5662–5666. doi: 10.1128/jvi.66.9.5662-5666.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Restrepo-Hartwig M. A., Carrington J. C. The tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication. J Virol. 1994 Apr;68(4):2388–2397. doi: 10.1128/jvi.68.4.2388-2397.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Restrepo M. A., Freed D. D., Carrington J. C. Nuclear transport of plant potyviral proteins. Plant Cell. 1990 Oct;2(10):987–998. doi: 10.1105/tpc.2.10.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sawicki S. G., Sawicki D. L. Coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis. J Virol. 1990 Mar;64(3):1050–1056. doi: 10.1128/jvi.64.3.1050-1056.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schaad M. C., Haldeman-Cahill R., Cronin S., Carrington J. C. Analysis of the VPg-proteinase (NIa) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing, and genome amplification. J Virol. 1996 Oct;70(10):7039–7048. doi: 10.1128/jvi.70.10.7039-7048.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Semler B. L., Anderson C. W., Hanecak R., Dorner L. F., Wimmer E. A membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide. Cell. 1982 Feb;28(2):405–412. doi: 10.1016/0092-8674(82)90358-0. [DOI] [PubMed] [Google Scholar]
  32. Shahabuddin M., Shaw J. G., Rhoads R. E. Mapping of the tobacco vein mottling virus VPg cistron. Virology. 1988 Apr;163(2):635–637. doi: 10.1016/0042-6822(88)90307-8. [DOI] [PubMed] [Google Scholar]
  33. Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Takeda N., Kuhn R. J., Yang C. F., Takegami T., Wimmer E. Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells. J Virol. 1986 Oct;60(1):43–53. doi: 10.1128/jvi.60.1.43-53.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Takegami T., Semler B. L., Anderson C. W., Wimmer E. Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides. Virology. 1983 Jul 15;128(1):33–47. doi: 10.1016/0042-6822(83)90316-1. [DOI] [PubMed] [Google Scholar]
  36. Towner J. S., Ho T. V., Semler B. L. Determinants of membrane association for poliovirus protein 3AB. J Biol Chem. 1996 Oct 25;271(43):26810–26818. doi: 10.1074/jbc.271.43.26810. [DOI] [PubMed] [Google Scholar]
  37. Troxler M., Egger D., Pfister T., Bienz K. Intracellular localization of poliovirus RNA by in situ hybridization at the ultrastructural level using single-stranded riboprobes. Virology. 1992 Dec;191(2):687–697. doi: 10.1016/0042-6822(92)90244-j. [DOI] [PubMed] [Google Scholar]
  38. Whitley P., Grahn E., Kutay U., Rapoport T. A., von Heijne G. A 12-residue-long polyleucine tail is sufficient to anchor synaptobrevin to the endoplasmic reticulum membrane. J Biol Chem. 1996 Mar 29;271(13):7583–7586. doi: 10.1074/jbc.271.13.7583. [DOI] [PubMed] [Google Scholar]
  39. Wimmer E., Hellen C. U., Cao X. Genetics of poliovirus. Annu Rev Genet. 1993;27:353–436. doi: 10.1146/annurev.ge.27.120193.002033. [DOI] [PubMed] [Google Scholar]
  40. van Bokhoven H., van Lent J. W., Custers R., Vlak J. M., Wellink J., van Kammen A. Synthesis of the complete 200K polyprotein encoded by cowpea mosaic virus B-RNA in insect cells. J Gen Virol. 1992 Nov;73(Pt 11):2775–2784. doi: 10.1099/0022-1317-73-11-2775. [DOI] [PubMed] [Google Scholar]

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