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Rotavirus RNA replication requires a single-stranded 3' end for efficient minus-strand synthesis
1Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Journal of Virology
|August 8, 1998
Summary
Rotavirus RNA replication requires a single-stranded 3' end on viral mRNA for efficient double-stranded RNA synthesis. Specific binding of RNA polymerase is necessary but not sufficient for this process.
Area of Science:
- Virology
- Molecular Biology
- RNA Replication
Background:
- Rotaviruses possess a segmented double-stranded (ds) RNA genome.
- Viral messenger RNA (mRNA) is the template for minus-strand RNA synthesis during replication.
- Conserved RNA termini contain cis-acting signals crucial for dsRNA synthesis.
Purpose of the Study:
- To investigate the role of secondary structure at rotavirus mRNA termini in minus-strand RNA synthesis.
- To determine the importance of the 3' single-stranded tail and 5' stem-loop motif in RNA replication.
Main Methods:
- Introduction of mutations into rotavirus gene 8 or 9 mRNAs affecting the 3' tail and 5' stem-loop.
- Analysis of mutant RNA replication using a cell-free system.
- Incubation of wild-type RNAs with complementary oligonucleotides.
- Assessment of viral RNA polymerase (VP1) binding to mutant RNAs.
Main Results:
- Mutations disrupting complementarity between the 5' end and 3' tail significantly inhibited minus-strand synthesis (>10-fold).
- Oligonucleotides complementary to the 3' tail also inhibited replication of wild-type RNAs.
- Mutant RNAs, despite being replication-defective, competitively interfered with wild-type replication and bound VP1 efficiently.
Conclusions:
- The single-stranded nature of the rotavirus mRNA 3' end is essential for efficient dsRNA synthesis.
- Specific binding of the viral RNA polymerase VP1 to the mRNA template is necessary but not sufficient for minus-strand synthesis.