Identification of the minimal replicase and the minimal promoter of (—)-strand synthesis, functional in rotavirus RNA replication in vitro

  • M. J. Wentz
  • C. Q.-Y. Zeng
  • J. T. Patton
  • M. K. Estes
  • R. F. Ramig
Conference paper
Part of the Archives of Virology book series (ARCHIVES SUPPL, volume 12)


An in vitro replication system supporting the initiation and synthesis of complete rotavirus (—)-strands on ( + )-strand template RNA (Chen et al., J Virol 68: 7030, 1994) was used to examine several parameters related to rotavirus RNA replication. Coexpression of VP1/2/3 in all possible combinations from baculovirus vectors revealed: [i] Virus-like particles (VLPs) were formed only if VP2 was present, and [ii] VP1/2 and VP1/2/3 VLPs had replicase activity in the in vitro system whereas VP2/3 and VP2 VLPs did not. Thus, the minimal replicase is composed of VP1 and VP2 and replicase activity is associated with VP1. In vitro replication reactions, using T7 transcripts of porcine rotavirus OSU genome segment 9 as reporter template, were performed to map cis-acting elements that regulate replication. Internal deletions and terminal truncations of the reporter RNA localized a replication signal, conferring full template activity, to the 5′-terminal 27 nucleotides (nt 1–27) and the 3′-terminal 26 nucleotides (nt 1037-1062). Further analysis showed that a minimal promoter of (—)-strand synthesis was contained in the 3′-terminal 7 nucleotides (nt 1056-1062); the sequence conserved at the 3′-terminus of all rotavirus genes. Hybrid constructs with this promoter had minimal, but detectable, template activity. This result indicated that upstream sequences between nucleotides 1037-1055 positively regulate the activity of the minimal promoter.


Minimal Promoter Strand Synthesis Replication System Internal Deletion Baculovirus Vector 
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  1. 1.
    Chen D, Ramig, RF (1992) Determinants of rotavirus stability and density during CsCl purification and storage. Virology 186: 228–237PubMedCrossRefGoogle Scholar
  2. 2.
    Chen D, Zeng Q-Y, Wentz M, Gorziglia M, Estes MK, Ramig RF (1994) Template-dependent, in vitro replication of rotavirus RNA. J Virol 68: 7030–7039PubMedGoogle Scholar
  3. 3.
    Cohen J, Charpilienne A, Shilmonczyk S, Estes MK (1989) Nucleotide sequence of bovine rotavirus gene 1 and expression of the gene product in baculovirus. Virology 171: 131–140PubMedCrossRefGoogle Scholar
  4. 4.
    Estes MK (1996) Rotaviruses and their replication. In: Fields BN, Knipe DM, Howley PM (eds) Fields Virology. Lippincott-Raven, Philadelphia, pp 1625–1655Google Scholar
  5. 5.
    Gorziglia M, Collins P (1992) Intracellular amplification and expression of a synthetic analog of rotavirus genomic RNA bearing a foreign marker gene: mapping cis-acting nucleotides in the 3′-noncoding region. Proc Natl Acad Sci USA 89: 5784–5788PubMedCrossRefGoogle Scholar
  6. 6.
    Imai M, Akatani K, Ikagami N, Furuichi Y (1983) Capped and conserved terminal structures in human rotavirus genome double-stranded RNA segments. J Virol 47: 125–136PubMedGoogle Scholar
  7. 7.
    Liu M, Mattion NM, Estes MK (1992) Rotavirus VP3 in insect cells possesses guanylyl-transferase activity. Virology 188: 77–84PubMedCrossRefGoogle Scholar
  8. 8.
    McCrae MA, McCorquodale JG (1983) Molecular biology of rotaviruses, V. Terminal structure of viral RNA species. Virology 126: 204–212PubMedCrossRefGoogle Scholar
  9. 9.
    Patton JT (1994) Rotavirus replication. In: Ramig RF (ed) Rotaviruses. Springer, Berlin, pp 107–127 Curr Top Microbiol Immunol vol 185)CrossRefGoogle Scholar
  10. 10.
    Pizarro JL, Sandino AM, Pizarro JM, Fernandez J, Spencer E (1991) Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3. J Gen Virol 72: 352–332CrossRefGoogle Scholar
  11. 11.
    Poch O, Saubaget I, Delarue M, Tordo N (1989) Indentification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J 8: 3867–3874PubMedGoogle Scholar
  12. 12.
    Ramig RF (1982) Isolation and genetic characterization of temperature-sensitive mutants of simian rotavirus SA11. Virology 120: 93–105PubMedCrossRefGoogle Scholar
  13. 13.
    Summers MD, Smith GE (1987) A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Bulletin No 1555. Texas A&M University, College StationGoogle Scholar
  14. 14.
    Valenzuela S, Pizzaro J, Sandino AM, Vasquez M, Fernandez J, Hernandez O, Patton J, Spencer E (1991) Photoaffinity labeling of rotavirus VP1 with 8-azido ATP: identification of the viral RNA polymerase. J Virol 65: 3964–3967PubMedGoogle Scholar
  15. 15.
    Zeng Q-Y, Wentz MJ, Cohen J, Estes MK, Ramig RF (1996) Characterization and replicase activity of double-and single-layered rotavirus-like particles expressed from baculovirus recombinants. J Virol 70 (in press)Google Scholar

Copyright information

© Springer-Verlag Wien 1996

Authors and Affiliations

  • M. J. Wentz
    • 1
  • C. Q.-Y. Zeng
    • 1
  • J. T. Patton
    • 2
  • M. K. Estes
    • 1
  • R. F. Ramig
    • 1
  1. 1.Division of Molecular VirologyBaylor College of MedicineHoustonUSA
  2. 2.Department of Microbiology and ImmunologyUniversity of Miami School of MedicineMiamiUSA

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