Ulfert Rand, Helmholtz Centre for Infection Research, Braunschweig
A highly dynamic gene activation process begins upon entry of CMV genomes into the host cell nucleus, both on the viral and on the host-cell side. The timing and velocity of gene expression events follow distinct patterns, but are also subject to considerable cell-to-cell heterogeneity. Experimental models to assess the dynamics of these processes at singlecell levels – and thus understand the processes shaping viral gene expression during the lytic cycle – are limited.
We developed a novel approach to visualise and quantify both CMV gene expression and endothelial cell interferon (IFN)-β expression at the single-cell level with temporal resolution. Using live-cell imaging and single-cell quantification of reporter genes encoded by recombinant human and mouse CMVs and endothelial host cell line allows us to detect distinct temporal gene expression profiles.
Tracking the dynamic expression of essential immediate early (ie), early (e) and late (l) viral genes via fluorescence protein reporters in living infected cells, we observe that progression through the virus life cycle is remarkably robust. This observation even extended to a faction of cells in which the virus managed to initiate gene expression despite the presence of typeI IFN. Importantly, our method allows detecting subtle changes in gene expressiondynamics as well as phenotypes restricted to a fraction of the infected cell population. Here,we demonstrate the effects of inflammatory cytokines on CMV gene expression dynamicrevealing the potential of this technique to perform both powerful screening as well asproviding detailed insights into CMV biology.
