Abstract
Influenza A is a major cause of virus-related deaths worldwide. It has a segmented genome of eight single-stranded, negative-sense RNAs packed into ribonucleoproteins (vRNPs). This segmentation allows reassortment between different strains with the potential to create highly virulent, pandemic new strains. A packaging mechanism is supposed, ensuring the incorporation of one copy of each segment into budding virions. During their transport from the nucleus to budding at the plasma membrane, the vRNPs are thought to establish multi-segment complexes based on RNA-RNA interactions called packaging signals.This makes the assembly of viral RNA in infected cells a promising target of study. We have used RNA-FISH against all vRNA and mRNA segments of Influenza to study their location and colocalization during the time course of infection. This data allows us to learn more about the mechanisms and potential hierarchies within the packaging process. We also gained information about the abundance and inter-cell heterogeneity of vRNPs among large sets of infected cells. Further understanding of the vRNP packaging can open up paths to new antiviral medication.We used a new RNA-FISH method to overcome the spectral limits of multiplexing. Traditionally, RNA-FISH is limited to one target per spectral channel which prohibits the detection of all eight vRNP segments of IAV in the same cells. We extended this by removing the fluorescent oligos after each round of imaging. Other vRNA and mRNA targets were imaged using the same colors as before which results in highly multiplexed overlay images. We call this method MEGA-FISH which might well be of a more general interest, being compatible with DAPI staining and immunofluorescence. After initial experiments with human A/Panama/2007/1999 strain we are now applying the method to other viral strains and reassortant viruses.
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