Tracking the Switch of Influenza RNA Genesis by a Novel Multiplexed Fish Method in Single Cells

The genome of the Influenza A virus (IAV, Orthomyxoviridae) is divided into eight single ribonuclear protein segments (vRNPs) consisting of negative sense RNA (vRNA) covered with the IAV nucleoprotein. IAV infects cells via endocytosis and the vRNPs are released into the cytoplasm and are transported into the nucleus upon fusion of the viral envelope with the endosomal membrane.In the early infection, mRNA is transcribed from vRNPs. Simultaneously, vRNPs are used to produce complementary RNAs (cRNAs) in a stochastic manner as templates for vRNA-replication. Such cRNAs are transcribed to multiple short viral RNAs (svRNAs), which rapidly enhance vRNA-replication. Newly synthesized vRNPs are transported to the cell membrane for virus budding. To form an infectious virus, it is presumed that all eight different vRNPs have to assemble into a single viral envelope. Remarkably, the order of vRNP-complexing is not known.In this work, we study important steps of the Influenza RNA household - the switch of IAV RNA genesis and the vRNP assembly. Fluorescence in situ hybridization (FISH) with forced-intercalation probes targeting svRNAs was performed to visualize the reported switch in IAV-infected cells successfully. Since svRNAs are highly conserved among all IAV, our single nucleotide probe is suitable as a universal IAV infection marker in fixed and in living cells. Due to this, we detected IAV-infections independently of hemagglutinin and neuraminidase configurations (HxNx), the actual host, or the pathogenicity.To resolve the complete IAV RNA household at single cell level, we created a novel and simple FISH-technique enabling detection of more RNA targets than the number of separate detection channels available. So far, we established the system to detect nearly all IAV RNAs in single cells for different time points after infection with surprising results.