1887

Abstract

Although inserting exogenous viral genome segments into rotavirus particles remains a hard challenge, this study describes the incorporation of a recombinant viral capsid protein (VP6) into newly assembled rotavirus particles. biotinylation technology was exploited to biotinylate a recombinant VP6 protein fused to a 15 aa biotin-acceptor peptide (BAP) by the bacterial biotin ligase BirA contextually co-expressed in mammalian cells. To avoid toxicity of VP6 overexpression, a stable HEK293 cell line was constructed with tetracycline-inducible expression of VP6–BAP and constitutive expression of BirA. Following tetracycline induction and rotavirus infection, VP6–BAP was biotinylated, recruited into viroplasms and incorporated into newly assembled virions. The biotin molecules in the capsid allowed the use of streptavidin-coated magnetic beads as a purification technique instead of CsCl gradient ultracentrifugation. Following transfection, double-layered particles attached to beads were able to induce viroplasm formation and to generate infective viral progeny.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.040089-0
2012-07-01
2019-10-20
Loading full text...

Full text loading...

/deliver/fulltext/jgv/93/7/1474.html?itemId=/content/journal/jgv/10.1099/vir.0.040089-0&mimeType=html&fmt=ahah

References

  1. Arnoldi F. , Campagna M. , Eichwald C. , Desselberger U. , Burrone O. R. . ( 2007; ). Interaction of rotavirus polymerase VP1 with nonstructural protein NSP5 is stronger than that with NSP2. . J Virol 81:, 2128–2137. [CrossRef] [PubMed]
    [Google Scholar]
  2. Beckett D. , Kovaleva E. , Schatz P. J. . ( 1999; ). A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. . Protein Sci 8:, 921–929. [CrossRef] [PubMed]
    [Google Scholar]
  3. Bican P. , Cohen J. , Charpilienne A. , Scherrer R. . ( 1982; ). Purification and characterization of bovine rotavirus cores. . J Virol 43:, 1113–1117.[PubMed]
    [Google Scholar]
  4. Campagna M. , Eichwald C. , Vascotto F. , Burrone O. R. . ( 2005; ). RNA interference of rotavirus segment 11 mRNA reveals the essential role of NSP5 in the virus replicative cycle. . J Gen Virol 86:, 1481–1487. [CrossRef] [PubMed]
    [Google Scholar]
  5. Charpilienne A. , Nejmeddine M. , Berois M. , Parez N. , Neumann E. , Hewat E. , Trugnan G. , Cohen J. . ( 2001; ). Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells. . J Biol Chem 276:, 29361–29367. [CrossRef] [PubMed]
    [Google Scholar]
  6. Charpilienne A. , Lepault J. , Rey F. , Cohen J. . ( 2002; ). Identification of rotavirus VP6 residues located at the interface with VP2 that are essential for capsid assembly and transcriptase activity. . J Virol 76:, 7822–7831. [CrossRef] [PubMed]
    [Google Scholar]
  7. Chen D. , Luongo C. L. , Nibert M. L. , Patton J. T. . ( 1999; ). Rotavirus open cores catalyze 5′-capping and methylation of exogenous RNA: evidence that VP3 is a methyltransferase. . Virology 265:, 120–130. [CrossRef] [PubMed]
    [Google Scholar]
  8. Chen J. Z. , Settembre E. C. , Aoki S. T. , Zhang X. , Bellamy A. R. , Dormitzer P. R. , Harrison S. C. , Grigorieff N. . ( 2009; ). Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM. . Proc Natl Acad Sci U S A 106:, 10644–10648. [CrossRef] [PubMed]
    [Google Scholar]
  9. Ciarlet M. , Crawford S. E. , Cheng E. , Blutt S. E. , Rice D. A. , Bergelson J. M. , Estes M. K. . ( 2002; ). VLA-2 (α2β1) integrin promotes rotavirus entry into cells but is not necessary for rotavirus attachment. . J Virol 76:, 1109–1123. [CrossRef] [PubMed]
    [Google Scholar]
  10. Contin R. , Arnoldi F. , Campagna M. , Burrone O. R. . ( 2010; ). Rotavirus NSP5 orchestrates recruitment of viroplasmic proteins. . J Gen Virol 91:, 1782–1793. [CrossRef] [PubMed]
    [Google Scholar]
  11. Delmas O. , Durand-Schneider A. M. , Cohen J. , Colard O. , Trugnan G. . ( 2004; ). Spike protein VP4 assembly with maturing rotavirus requires a postendoplasmic reticulum event in polarized Caco-2 cells. . J Virol 78:, 10987–10994. [CrossRef] [PubMed]
    [Google Scholar]
  12. Dennehy P. H. . ( 2008; ). Rotavirus vaccines: an overview. . Clin Microbiol Rev 21:, 198–208. [CrossRef] [PubMed]
    [Google Scholar]
  13. Eichwald C. , Vascotto F. , Fabbretti E. , Burrone O. R. . ( 2002; ). Rotavirus NSP5: mapping phosphorylation sites and kinase activation and viroplasm localization domains. . J Virol 76:, 3461–3470. [CrossRef] [PubMed]
    [Google Scholar]
  14. Eichwald C. , Rodriguez J. F. , Burrone O. R. . ( 2004; ). Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation. . J Gen Virol 85:, 625–634. [CrossRef] [PubMed]
    [Google Scholar]
  15. Estes M. . ( 2001; ). Rotaviruses and their replication. . In Fields Virology, , 4th edn., pp. 1747–1785. Edited by Knipe D. , Howley P. . . New York:: Lippincott Williams and Wilkins;.
    [Google Scholar]
  16. Estes M. K. , Kapikian A. . ( 2007; ). Rotaviruses. . In Fields Virology, , 5th edn., pp. 1917–1974. Edited by Howley P. M. , Knipe D. M. , Griffin D. A. , Lamb R. A. , Martin M. A. , Roizman B. , Straus S. E. . . Philadelphia:: Wolters Kluwer Health/Lippincott Williams & Wilkins;.
    [Google Scholar]
  17. Estes M. K. , Graham D. Y. , Smith E. M. , Gerba C. P. . ( 1979; ). Rotavirus stability and inactivation. . J Gen Virol 43:, 403–409. [CrossRef] [PubMed]
    [Google Scholar]
  18. Feng N. , Lawton J. A. , Gilbert J. , Kuklin N. , Vo P. , Prasad B. V. , Greenberg H. B. . ( 2002; ). Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb. . J Clin Invest 109:, 1203–1213.[PubMed] [CrossRef]
    [Google Scholar]
  19. Glatter T. , Wepf A. , Aebersold R. , Gstaiger M. . ( 2009; ). An integrated workflow for charting the human interaction proteome: insights into the PP2A system. . Mol Syst Biol 5:, 237. [CrossRef] [PubMed]
    [Google Scholar]
  20. González S. A. , Burrone O. R. . ( 1991; ). Rotavirus NS26 is modified by addition of single O-linked residues of N-acetylglucosamine. . Virology 182:, 8–16. [CrossRef] [PubMed]
    [Google Scholar]
  21. Humphrey W. , Dalke A. , Schulten K. . ( 1996; ). VMD: visual molecular dynamics. . J Mol Graph 14:, 33–38, 27–28. [CrossRef] [PubMed]
    [Google Scholar]
  22. Laemmli U. K. . ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. . Nature 227:, 680–685. [CrossRef] [PubMed]
    [Google Scholar]
  23. Lawton J. A. , Estes M. K. , Prasad B. V. . ( 1997a; ). Three-dimensional visualization of mRNA release from actively transcribing rotavirus particles. . Nat Struct Biol 4:, 118–121. [CrossRef] [PubMed]
    [Google Scholar]
  24. Lawton J. A. , Zeng C. Q. , Mukherjee S. K. , Cohen J. , Estes M. K. , Prasad B. V. . ( 1997b; ). Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal-deleted VP2: implications for the architecture of the VP2 capsid layer. . J Virol 71:, 7353–7360.[PubMed]
    [Google Scholar]
  25. Lawton J. A. , Estes M. K. , Prasad B. V. . ( 1999; ). Comparative structural analysis of transcriptionally competent and incompetent rotavirus–antibody complexes. . Proc Natl Acad Sci U S A 96:, 5428–5433. [CrossRef] [PubMed]
    [Google Scholar]
  26. Lepault J. , Petitpas I. , Erk I. , Navaza J. , Bigot D. , Dona M. , Vachette P. , Cohen J. , Rey F. A. . ( 2001; ). Structural polymorphism of the major capsid protein of rotavirus. . EMBO J 20:, 1498–1507. [CrossRef] [PubMed]
    [Google Scholar]
  27. Libersou S. , Siebert X. , Ouldali M. , Estrozi L. F. , Navaza J. , Charpilienne A. , Garnier P. , Poncet D. , Lepault J. . ( 2008; ). Geometric mismatches within the concentric layers of rotavirus particles: a potential regulatory switch of viral particle transcription activity. . J Virol 82:, 2844–2852. [CrossRef] [PubMed]
    [Google Scholar]
  28. Liu M. , Mattion N. M. , Estes M. K. . ( 1992; ). Rotavirus VP3 expressed in insect cells possesses guanylyltransferase activity. . Virology 188:, 77–84. [CrossRef] [PubMed]
    [Google Scholar]
  29. Mathieu M. , Petitpas I. , Navaza J. , Lepault J. , Kohli E. , Pothier P. , Prasad B. V. , Cohen J. , Rey F. A. . ( 2001; ). Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion. . EMBO J 20:, 1485–1497. [CrossRef] [PubMed]
    [Google Scholar]
  30. Meyer J. C. , Bergmann C. C. , Bellamy A. R. . ( 1989; ). Interaction of rotavirus cores with the nonstructural glycoprotein NS28. . Virology 171:, 98–107. [CrossRef] [PubMed]
    [Google Scholar]
  31. O’Gorman S. , Fox D. T. , Wahl G. M. . ( 1991; ). Recombinase-mediated gene activation and site-specific integration in mammalian cells. . Science 251:, 1351–1355. [CrossRef] [PubMed]
    [Google Scholar]
  32. Patton J. , Chizhikov V. , Taraporewala Z. , Chen D. Y. . ( 2000; ). Virus replication.. In: Rotaviruses. Methods and Protocols, pp. 33–66. Edited by Gray J. , Desselberger U. . . Totowa, NJ:: Humana Press;.
    [Google Scholar]
  33. Petris G. , Vecchi L. , Bestagno M. , Burrone O. R. . ( 2011; ). Efficient detection of proteins retro-translocated from the ER to the cytosol by in vivo biotinylation. . PLoS ONE 6:, e23712. [CrossRef] [PubMed]
    [Google Scholar]
  34. Pizarro J. L. , Sandino A. M. , Pizarro J. M. , Fernández J. , Spencer E. . ( 1991; ). Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3. . J Gen Virol 72:, 325–332. [CrossRef] [PubMed]
    [Google Scholar]
  35. Prasad B. V. , Rothnagel R. , Zeng C. Q.-Z. , Jakana J. , Lawton J. A. , Chiu W. , Estes M. K. . ( 1996; ). Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus. . Nature 382:, 471–473. [CrossRef] [PubMed]
    [Google Scholar]
  36. Predonzani A. , Arnoldi F. , López-Requena A. , Burrone O. R. . ( 2008; ). In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system. . BMC Biotechnol 8:, 41. [CrossRef] [PubMed]
    [Google Scholar]
  37. Settembre E. C. , Chen J. Z. , Dormitzer P. R. , Grigorieff N. , Harrison S. C. . ( 2011; ). Atomic model of an infectious rotavirus particle. . EMBO J 30:, 408–416. [CrossRef] [PubMed]
    [Google Scholar]
  38. Taylor J. A. , Meyer J. C. , Legge M. A. , O’Brien J. A. , Street J. E. , Lord V. J. , Bergmann C. C. , Bellamy A. R. . ( 1992; ). Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methionine residue is essential for ligand binding. . J Virol 66:, 3566–3572.[PubMed]
    [Google Scholar]
  39. Taylor J. A. , O’Brien J. A. , Lord V. J. , Meyer J. C. , Bellamy A. R. . ( 1993; ). The RER-localized rotavirus intracellular receptor: a truncated purified soluble form is multivalent and binds virus particles. . Virology 194:, 807–814. [CrossRef] [PubMed]
    [Google Scholar]
  40. Thouvenin E. , Schoehn G. , Rey F. , Petitpas I. , Mathieu M. , Vaney M. C. , Cohen J. , Kohli E. , Pothier P. , Hewat E. . ( 2001; ). Antibody inhibition of the transcriptase activity of the rotavirus DLP: a structural view. . J Mol Biol 307:, 161–172. [CrossRef] [PubMed]
    [Google Scholar]
  41. Towbin H. , Staehelin T. , Gordon J. . ( 1979; ). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. . Proc Natl Acad Sci U S A 76:, 4350–4354. [CrossRef] [PubMed]
    [Google Scholar]
  42. Valenzuela S. , Pizarro J. , Sandino A. M. , Vásquez M. , Fernández J. , Hernández O. , Patton J. , Spencer E. . ( 1991; ). Photoaffinity labeling of rotavirus VP1 with 8-azido-ATP: identification of the viral RNA polymerase. . J Virol 65:, 3964–3967.[PubMed]
    [Google Scholar]
  43. Viens A. , Mechold U. , Lehrmann H. , Harel-Bellan A. , Ogryzko V. . ( 2004; ). Use of protein biotinylation in vivo for chromatin immunoprecipitation. . Anal Biochem 325:, 68–76. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.040089-0
Loading
/content/journal/jgv/10.1099/vir.0.040089-0
Loading

Data & Media loading...

Most Cited This Month

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