Orthohantaviruses are emerging zoonotic pathogens that can cause life-threatening diseases in humans. Their tripartite, negative-sense RNA genome is encapsidated by the viral nucleoprotein, but the subcellular localization and dynamics of these viral RNAs and proteins remain poorly characterized. Here, we present a comprehensive microscopy-based analysis of Puumala virus, the most prevalent orthohantavirus in northern and western Europe. Using fluorescence in situ hybridization (FISH) and Multiple Sequential FISH, we mapped the distribution of viral mRNAs, viral genomic RNAs (vRNAs), nucleoproteins and associated host cell factors, quantifying their intracellular abundance, co-localization and subcellular positioning. We observed distinct clustering of vRNAs with varying degrees of nucleoprotein association, a progressive increase in nucleoprotein expression levels during infection and a concomitant rise in the abundance of P-bodies. Moreover, we report a marked spatial reorganization of actin, microtubules and P-bodies, indicating substantial structural remodelling of host cells during orthohantavirus infections. Using a novel end-specific FISH assay, we observed a preferential 5'-end degradation of vRNAs in P-bodies, shedding new light on orthohantavirus RNA turnover within host RNA-processing compartments. Finally, co-localization analyses revealed the formation of potential 'viral factories' composed of nucleoprotein, vRNAs and viral mRNAs, indicating an intricate assembly hierarchy. Collectively, these findings improve our understanding of orthohantavirus replication and highlight the dynamic interplay between virus and host cell components.
Keywords: P-bodies; actin; cytoskeleton; fluorescence in situ hybridization (FISH); microscopy; orthohantaviruses.