Detection of Specific mRNA Synthesis in Rotavirus using Single Molecule Hybridization

Rotavirus is the main cause of severe gastroenteritis in humans and animals. The capsid encloses 11 highly packed segments of double stranded RNA (dsRNA) and 12 transcription polymerases complexes arranged along 12 vertices of the icosahedral capsid. Inside the cell it becomes transcriptionally active and synthesises positive single stranded RNA (ssRNA). The synthesis occurs after a few seconds of setting transcription conditions. The newly ssRNAs are released simultaneously through twelve specialised channels, but the detection and assignment of specific segments to the channel (if any) is unknown with current standard microscopy techniques. A detailed description of this process is needed to understand the model of transcription of the virus, its genomic packaging and the assembly of the virus.Here, we use total internal reflection fluorescence combined with alternating laser excitation (TIRF-ALEX) microscopy and single molecule hybridization to identify, for the first time, the synthesis and release of specific single-stranded RNAs on the surface of a rotavirus capsid. A FRET pair consisting of a single stranded DNA labelled with Cy3B (FRET donor) or ATTO647N (FRET acceptor) was designed to hybridize with complementary RNA segment 11 with an expected value of approximately 35%. Biotinylated capsids were then immobilised on neutravidin-treated slides, incubated with ssDNA fragments that hybridize with the viral RNA only in presence of nucleotides; control experiments included parallel hybridization experiments in the absence of viral transcription. Our smFRET results show molecular populations with the expected FRET value of single hybridisation events on the viral surface. These findings support a transcription model in which one ssRNA segment is associated with a single transcription complex and individual dsRNA segment is packed with a single polymerase complex. Our results exclude alternative models in which an individual segment is transcribed by several polymerases.