Real-time visualization and quantification of human Cytomegalovirus replication in living cells using the ANCHOR DNA labeling technology.

Abstract

Human cytomegalovirus (HCMV) induces latent life-long infections in all human populations. Depending on geographic area and socio-economic conditions between 30 to nearly 100% of individuals are affected. The biology of this virus is difficult to explore due to its extreme sophistication and the lack of pertinent animal model. Here we present the first application of the ANCHOR DNA labeling system to a herpes virus, allowing real time imaging and direct monitoring of HCMV infection and replication in human living cells. The ANCHOR system is composed of a protein (OR) which specifically binds to a short, non-repetitive DNA target sequence (ANCH) and spreads onto neighboring sequences due to protein oligomerization. If OR protein is fused to GFP, this accumulation results in a site specific fluorescent focus. We have created a recombinant ANCHOR-HCMV harboring an ANCH target sequence and the gene encoding the cognate OR-GFP fusion protein. Infection of permissive cells with ANCHOR-HCMV enables visualization of the nearly complete viral cycle until cell fragmentation and death. Quantitative analysis of infection kinetics and of viral DNA replication revealed cell-type specific behavior of HCMV and sensitivity to inhibitors. Our results show that the ANCHOR technology is a very efficient tool for the study of complex DNA viruses and new highly promising biotechnology applications.\n\nIMPORTANCEThe ANCHOR technology is to date the most powerful tool to follow and quantify the replication of HCMV in living cells and to gain new insights into its biology. This technology is applicable to virtually any DNA virus or virus presenting a dsDNA phase, paving the way to infection imaging in various cell lines or even in animal models and opening fascinating fundamental and applied prospects. Associated to high content automated microscopy, this technology permitted rapid, robust and precise determination of Ganciclovir IC50 and IC90 on HCMV replication, with minimal hands-on investment. To search for new antiviral activities, the experiment is easy to up-grade towards efficient and cost-effective screening of large chemical libraries. The simple infection of permissive cells with ANCHOR-viruses in the presence of a compound of interest may even provide a first estimation about the stage of the viral cycle this molecule is acting upon.