Mapping RNA-capsid interactions and RNA secondary structure within virus particles using next-generation sequencing

Area of Science:

• Virology
• Molecular Biology
• Genomics

Background:

• Understanding RNA-capsid interactions is crucial for characterizing virus assembly and function.
• Existing methods lack the resolution to map these interactions genome-wide within intact viral particles.

Purpose of the Study:

• To develop and validate a novel platform for genome-wide characterization of RNA-capsid binding sites in mature virus particles.
• To identify specific RNA regions that interact with capsid proteins in Flock House virus (FHV).

Main Methods:

• Development of viral Photo-Activatable Ribonucleoside CrossLinking (vPAR-CL) using 4-thiouridine incorporation and UV-crosslinking.
• Next-Generation Sequencing (NGS) to detect crosslink-specific uridine to cytidine transitions.
• Combined vPAR-CL with dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) for high-resolution RNA structure analysis.

Main Results:

• vPAR-CL reliably identified significant RNA-capsid binding sites across the FHV genome.
• Predominant interaction sites were found in double-stranded RNA regions, confirmed by combining vPAR-CL and DMS-MaPseq.
• Disruption of RNA secondary structures at binding sites led to varied defects in virus replication, propagation, and packaging.

Conclusions:

• vPAR-CL is an effective tool for mapping RNA-capsid interactions within viral particles.
• Specific RNA-capsid binding sites are essential for multiple stages of the viral life cycle, indicating multifunctional roles.
• FHV packaging and replication are tightly coordinated and interdependent processes.