Tracking of Native Viral RNA of Enveloped Viruses in Living Host Cells

Abstract

Visualization of the temporal and spatial organization of viral RNA species in host cells is essential for understanding the viral replication cycle. This project aims at employing various techniques for imaging of viral RNA at single cell/virus level. For tracking in living cells, multiple short modified oligonucleotides carrying specific intercalating fluorophores whose brightness increases 5-20 fold via forced intercalation (FIT) only upon Watson-Crick hybridization with the complementary target sequence are used. Probes are delivered into living host cells via temporarily permeabilization by streptolysin O. Those probes are employed, for example, for in vivo tracking to investigate progress and localization of influenza virus RNA segments assembly during egress. Complementary, in fixed cells we established single molecule fluorescence in situ hybridization (single molecule FISH) for studying RNA segment organization, i.e., for hanta-virus infections, both for cell-free surface immobilized virions as well as for infected host cells. Parallel dual segment labeling via FISH allows measuring the degree of correlation of two segments in a single virion at a time. Employing a highly sensitive EMCCD camera with epifluorescence microscopy, bleaching steps can be recorded for each segment thus statistically determining the number of segments per virion and the degree of colocalization between them. This approach will resolve the packaging mechanism of hanta viruses.