P-BODY AND CYTOSKELETON REMODELING BY ORTHOHANTAVIRUSES.
Orthohantaviruses, are emerging zoonotic pathogens causing life-threatening diseases in humans. The orthohantavirus genome consists of three RNA segment (vRNAs) of negative polarity, which are encapsidated by the viral nucleoprotein (N). To date, the precise subcellular behavior of vRNAs and N has not been fully elucidated. Here, we present a comprehensive analysis of orthohantavirus infections using Fluorescence in situ Hybridization (FISH) and multiple sequential FISH (Mu-Seq FISH), which enables simultaneous detection of viral RNAs, viral mRNAs, N, and cellular factors. Our experiments revealed distinct patterns of viral RNA clustering with varying degrees of N association. Moreover, we found a significant spatial correlation of virus vRNAs and N with cellular processing (P)-bodies, underlining their key role in orthohantavirus replication. Throughout the course of an infection, we observed an increasing dominance of N expression, while concomitantly P-body numbers grew significantly. We also found indications for a preferential 5-end degradation of viral mRNAs in P-bodies. Furthermore, we report that orthohantavirus infection is accompanied by a significant redistribution of cellular components: while filamentous actin and microtubules become enriched in the perinuclear region, P-bodies move to the cell periphery. Finally, co-localization analyses suggest a formation of viral factories containing N, vRNAs, and viral mRNAs, indicating an intricate orthohantavirus assembly hierarchy. AUTHOR SUMMARYIn this study we used advanced imaging techniques to observe the dynamics of viral components and key cellular structures during orthohantavirus infections. Our experiments show that orthohantaviruses cause significant changes within the cell, particularly involving P-bodies and components of the cytoskeleton, such as actin and microtubules. We also provided comprehensive spatiotemporal maps of orthohantaviral components, including visualization of the viral nucleoprotein, genomic RNA and mRNAs. Finally, we found indications for a 5end degradation of virus mRNA in P-bodies, thus adding to our understanding of intracellular host-pathogen crosstalk. In summary, our work highlights the intricate relationship between viruses and host cells, emphasizing the dynamic changes that occur during orthohantaviral infections.