Template role of double-stranded RNA in tombusvirus replication

Area of Science:

• Molecular Biology
• Virology
• Plant Pathology

Background:

• Positive-stranded RNA [(+)RNA] viruses replicate via complementary minus-strand RNA [(-)RNA] synthesis.
• The precise replicative form of the (-)RNA template in plant (+)RNA viruses remains unclear.

Purpose of the Study:

• To investigate the replicative form of the (-)RNA template during Tomato bushy stunt virus (TBSV) replication.
• To elucidate the mechanism of TBSV RNA replication and its implications for viral strategy.

Main Methods:

• Cell-free replication assay using yeast and plant extracts.
• RNase digestion, DNAzyme assays, and RNA mobility shift assays.
• Radioactive nucleotide chase experiments and time course analysis.

Main Results:

• TBSV replication generates double-stranded RNA (dsRNA) intermediates.
• Naked (-)RNA templates were not detected; dsRNA is the active template.
• dsRNA appears early and is involved in strand displacement synthesis of new (+)RNA.
• Replication involves host DEAD box helicases and viral p33 protein.

Conclusions:

• TBSV utilizes dsRNA as a template for (+)RNA synthesis through strand displacement.
• This mechanism allows for temporal separation of RNA synthesis and asymmetrical progeny production.
• The dsRNA template likely protects viral RNA and evades host immune responses.