1887

Abstract

The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191–4344) could be expressed in and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 10 copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L–EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L–EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

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2004-05-01
2024-12-07
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References

  1. Adkins S., Quadt R., Choi T. J., Ahlquist P., German T. 1995; An RNA-dependent RNA polymerase activity associated with virions of tomato spotted wilt virus, a plant- and insect-infecting bunyavirus. Virology 207:308–311
    [Google Scholar]
  2. Akkina R. K., Chambers T. M., Londo D. R., Nayak D. P. 1987; Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB1 protein from cloned cDNA. J Virol 61:2217–2224
    [Google Scholar]
  3. Andersson I., Simon M., Lundkvist Å., Nilsson M., Holmström A., Elgh F., Mirazimi A. 2004; Role of actin filaments in targeting of Crimean Congo hemorrhagic fever virus nucleocapsid protein to perinuclear regions of mammalian cells. J Med Virol 72:83–93
    [Google Scholar]
  4. Betenbaugh M., Yu M., Kuehl K., White J., Pennock D., Spik K., Schmaljohn C. 1995; Nucleocapsid- and virus-like particles assemble in cells infected with recombinant baculoviruses or vaccinia viruses expressing the M and the S segments of Hantaan virus. Virus Res 38:111–124
    [Google Scholar]
  5. Bridgen A., Elliott R. M. 1996; Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proc Natl Acad Sci U S A 93:15400–15404
    [Google Scholar]
  6. Brisson M., He Y., Li S., Yang J. P., Huang L. 1999; A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes. Gene Ther 6:263–270
    [Google Scholar]
  7. Cevik B., Smallwood S., Moyer S. A. 2003; The L-L oligomerization domain resides at the very N-terminus of the sendai virus L RNA polymerase protein. Virology 313:525–536
    [Google Scholar]
  8. den Boon J. A., Chen J., Ahlquist P. 2001; Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes. J Virol 75:12370–12381
    [Google Scholar]
  9. Dunn E. F., Pritlove D. C., Jin H., Elliott R. M. 1995; Transcription of a recombinant bunyavirus RNA template by transiently expressed bunyavirus proteins. Virology 211:133–143
    [Google Scholar]
  10. Duprex W. P., Collins F. M., Rima B. K. 2002; Modulating the function of the measles virus RNA-dependent RNA polymerase by insertion of green fluorescent protein into the open reading frame. J Virol 76:7322–7328
    [Google Scholar]
  11. Elliott L. H., Kiley M. P., McCormick J. B. 1984; Hantaan virus: identification of virion proteins. J Gen Virol 65:1285–1293
    [Google Scholar]
  12. Elliott R. M., Bouloy M., Calisher C. H., Goldbach R., Moyer J. T., Nichol S. T., Pettersson R., Plyusnin A., Schmaljohn C. S. 2000; Bunyaviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses pp  599–621 Edited by van Regenmortel M. H. V., Fauquet C. M., Bishop D. H. L., Carstens E. B., Estes M. K., Lemon S. M., Maniloff J., Mayo M. A., McGeoch D. J., Pringle C. R., Wickner R. B. San Diego: Academic Press;
    [Google Scholar]
  13. Flick K., Hooper J. W., Schmaljohn C. S., Pettersson R. F., Feldmann H., Flick R. 2003; Rescue of hantaan virus minigenomes. Virology 306:219–224
    [Google Scholar]
  14. Fuerst T. R., Earl P. L., Moss B. 1987; Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. Mol Cell Biol 7:2538–2544
    [Google Scholar]
  15. Jin H., Elliott R. M. 1992; Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies. J Gen Virol 73:2235–2244
    [Google Scholar]
  16. Kariwa H., Tanabe H., Mizutani T. 9 other authors 2003; Synthesis of Seoul virus RNA and structural proteins in cultured cells. Arch Virol 148:1671–1685
    [Google Scholar]
  17. Kukkonen S. K., Vaheri A., Plyusnin A. 1998; Completion of the Tula hantavirus genome sequence: properties of the L segment and heterogeneity found in the 3′ termini of S and L genome RNAs. J Gen Virol 79:2615–2622
    [Google Scholar]
  18. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  19. Lopez N., Müller R., Prehaud C., Bouloy M. 1995; The L protein of Rift Valley fever virus can rescue viral ribonucleoproteins and transcribe synthetic genome-like RNA molecules. J Virol 69:3972–3979
    [Google Scholar]
  20. Lukashevich I. S., Djavani M., Shapiro K., Sanchez A., Ravkov E., Nichol S. T., Salvato M. S. 1997; The Lassa fever virus L gene: nucleotide sequence, comparison, and precipitation of a predicted 250 kDa protein with monospecific antiserum. J Gen Virol 78:547–551
    [Google Scholar]
  21. Lundkvist Å., Vapalahti O., Plyusnin A., Sjölander K. B., Niklasson B., Vaheri A. 1996; Characterization of Tula virus antigenic determinants defined by monoclonal antibodies raised against baculovirus-expressed nucleocapsid protein. Virus Res 45:29–44
    [Google Scholar]
  22. Lyle J. M., Bullitt E., Bienz K., Kirkegaard K. 2002; Visualization and functional analysis of RNA-dependent RNA polymerase lattices. Science 296:2218–2222
    [Google Scholar]
  23. Määttä J., Hallikas O., Welti S., Hilden P., Schröder J., Kuismanen E. 2000; Limited caspase cleavage of human BAP31. FEBS Lett 484:202–206
    [Google Scholar]
  24. Meyer B. J., Schmaljohn C. 2000; Accumulation of terminally deleted RNAs may play a role in Seoul virus persistence. J Virol 74:1321–1331
    [Google Scholar]
  25. Moss B., Elroy-Stein O., Mizukami T., Alexander W. A., Fuerst T. R. 1990; Product review. New mammalian expression vectors. Nature 348:91–92
    [Google Scholar]
  26. Ng F. W., Nguyen M., Kwan T., Branton P. E., Nicholson D. W., Cromlish J. A., Shore G. C. 1997; p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum. J Cell Biol 139:327–338
    [Google Scholar]
  27. Padula P. J., Sanchez A. J., Edelstein A., Nichol S. T. 2002; Complete nucleotide sequence of the M RNA segment of Andes virus and analysis of the variability of the termini of the virus S, M and L RNA segments. J Gen Virol 83:2117–2122
    [Google Scholar]
  28. Pensiero M. N., Jennings G. B., Schmaljohn C. S., Hay J. 1988; Expression of the Hantaan virus M genome segment by using a vaccinia virus recombinant. J Virol 62:696–702
    [Google Scholar]
  29. Peränen J., Rikkonen M., Kääriäinen L. 1993; A method for exposing hidden antigenic sites in paraformaldehyde-fixed cultured cells, applied to initially unreactive antibodies. J Histochem Cytochem 41:447–454
    [Google Scholar]
  30. Plyusnin A., Vapalahti O., Lankinen H. 9 other authors 1994; Tula virus: a newly detected hantavirus carried by European common voles. J Virol 68:7833–7839
    [Google Scholar]
  31. Plyusnin A., Kukkonen S. K., Plyusnina A., Vapalahti O., Vaheri A. 2002; Transfection-mediated generation of functionally competent Tula hantavirus with recombinant S RNA segment. EMBO J 21:1497–1503
    [Google Scholar]
  32. Ravkov E. V., Compans R. W. 2001; Hantavirus nucleocapsid protein is expressed as a membrane-associated protein in the perinuclear region. J Virol 75:1808–1815
    [Google Scholar]
  33. Ravkov E. V., Nichol S. T., Peters C. J., Compans R. W. 1998; Role of actin microfilaments in Black Creek Canal virus morphogenesis. J Virol 72:2865–2870
    [Google Scholar]
  34. Rossier C., Patterson J., Kolakofsky D. 1986; La Crosse virus small genome mRNA is made in the cytoplasm. J Virol 58:647–650
    [Google Scholar]
  35. Ruusala A., Persson R., Schmaljohn C. S., Pettersson R. F. 1992; Coexpression of the membrane glycoproteins G1 and G2 of Hantaan virus is required for targeting to the Golgi complex. Virology 186:53–64
    [Google Scholar]
  36. Schwartz M., Chen J., Janda M., Sullivan M., den Boon J., Ahlquist P. 2002; A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids. Mol Cell 9:505–514
    [Google Scholar]
  37. Shi X., Elliott R. M. 2002; Golgi localization of Hantaan virus glycoproteins requires coexpression of G1 and G2. Virology 300:31–38
    [Google Scholar]
  38. Simons K., Toomre D. 2000; Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31–39
    [Google Scholar]
  39. Smallwood S., Cevik B., Moyer S. A. 2002; Intragenic complementation and oligomerization of the L subunit of the sendai virus RNA polymerase. Virology 304:235–245
    [Google Scholar]
  40. Taverna E., Francolini M., Jeromin A., Hilfiker S., Roder J., Rosa P. 2002; Neuronal calcium sensor 1 and phosphatidylinositol 4-OH kinase beta interact in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis. J Cell Sci 115:3909–3922
    [Google Scholar]
  41. Vapalahti O., Lundkvist Å., Kukkonen S. K. 10 other authors 1996; Isolation and characterization of Tula virus, a distinct serotype in the genus Hantavirus , family Bunyaviridae . J Gen Virol 77:3063–3067
    [Google Scholar]
  42. Vapalahti O., Lundkvist Å., Vaheri A. 2001; Human immune response, host genetics, and severity of disease. Curr Top Microbiol Immunol 256:153–169
    [Google Scholar]
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