The segmented nature of the influenza virus genome complicates the process of genome packaging because at least one complete set of eight viral RNA segments has to be packaged into a virus particle to produce infectious progeny. The mechanism by which influenza virus ensures correct packaging of its genome has been unclear. Two models have been proposed for the incorporation of influenza viral RNAs: (i) the random incorporation model; and (ii) the selective incorporation model.
This paper describes detection and quantification of the viral RNAs within influenza virions at single-virus resolution using single-molecule (sm)FISH technique. Most if not all influenza virus particles package heterogeneous viral RNAs and each viral RNA segment was packaged only once. These data provide direct evidence that the packaging mechanism in the influenza virus is very robust and the majority of virus particles package the unique eight viral RNAs.
One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis. PNAS USA 30 April 2012 doi: 10.1073/pnas.1206069109
Influenza A virus possesses a segmented genome of eight negative-sense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.