Applications for a Dual Fluorescent Human Cytomegalovirus in the Analysis of Viral Entry

The existence of cell type-specific entry pathways of human cytomegalovirus is an unresolved question as the course of viral entry in different cell types is still not fully understood. To gain more insight into these processes, we generated a dual fluorescent HCMV, where the capsid-associated tegument protein pp150 is labelled with EGFP and the envelope glycoprotein gM with mCherry. This dual labelled virus allows for the separate tracking of the viral envelope fusing with a cellular membrane and the viral capsid during its movement from the cellular membrane to the nucleus. We describe two applications for this virus in the analysis of viral entry: (a) Dynamic live-cell imaging allows for the visualization of viral de-envelopment and transport processes within the living cell. (b) Imaging of cell cultures fixed at different time points after infection enables a more comprehensive statistical analysis of the kinetics of viral entry events such as adsorption, fusion, and nuclear translocation. The techniques are described on the example of fibroblasts and endothelial cells, but can be adapted to other cell types as well. Furthermore, these protocols could provide suggestions for the establishment of live cell applications to other viruses.