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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)

Summary

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.

Keywords

Minimal Promoter Strand Synthesis Replication System Internal Deletion Baculovirus Vector 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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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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