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Related Experiment Videos

Multiple pathways of reversion in viroids for conservation of structural elements

F Qu1, C Heinrich, P Loss

  • 1Heinrich-Heine-Universität Düsseldorf, Institut für Physikalische Biologie, Germany.

The EMBO Journal
|May 1, 1993
PubMed
Summary

Potato spindle tuber viroid (PSTVd) hairpin II (HP II) structure is essential for replication. While G:U pairs are tolerated, the wild-type sequence ensures optimal fitness and replicability.

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Area of Science:

  • Virology
  • Molecular Biology
  • Biochemistry

Background:

  • Previous studies indicated the critical role of hairpin II (HP II) in potato spindle tuber viroid (PSTVd) infectivity.
  • Site-directed mutagenesis suggested a thermodynamically metastable structure involving HP II is essential.

Purpose of the Study:

  • To distinguish between structural and sequence-specific effects in HP II.
  • To analyze the functional role of HP II during PSTVd replication and genetic stability.

Main Methods:

  • Site-directed double mutations were introduced into HP II of PSTVd without altering the overall rod-like structure.
  • Mutant viability, genetic stability, and reversion rates were assessed over generations.
  • Replicating sequence variants were separated using gel electrophoresis and their sequences/frequencies determined.

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Main Results:

  • Mutations in HP II were viable but showed reversion to wild-type in the second generation.
  • G:U pairs were transiently tolerated in the (-)strand HP II, with stability compensated by mutations outside HP II.
  • Reversions occurred spontaneously during (-)strand synthesis.

Conclusions:

  • HP II is a functional element in the (-)strand replication intermediate and essential for (+)strand template activity.
  • The double-stranded structure of HP II is crucial for short-term PSTVd replication.
  • The wild-type sequence of HP II provides superior fitness and replicability.