(b) Same as panel a but treated to filter out the fluorescent background and then processed using the spot detector plug in for ImageJ (spot radius, 2; cutoff, 0; percentile, 7). composed of a protein (OR) that specifically binds to a short, nonrepetitive DNA target sequence (ANCH) and spreads onto neighboring sequences by protein oligomerization. When the OR protein is fused to green fluorescent protein (GFP), its accumulation results in a site-specific fluorescent focus. We created a recombinant ANCHOR-HCMV harboring an ANCH target sequence and Tobramycin sulfate the gene encoding the cognate OR-GFP fusion protein. Infection of permissive cells with ANCHOR-HCMV enables visualization of nearly the complete viral cycle until cell fragmentation and death. Quantitative analysis of PPP2R1B infection kinetics and of viral DNA replication revealed cell-type-specific HCMV behavior and sensitivity to inhibitors. Our results show that the ANCHOR technology provides an efficient tool for the study of complex DNA viruses and a new, highly promising system for the development of innovative biotechnology applications. IMPORTANCE The ANCHOR technology is currently the most powerful tool to follow and quantify the replication of HCMV in living cells and to gain new insights into its biology. The technology is applicable to virtually any DNA virus or viruses presenting a double-stranded DNA (dsDNA) phase, paving the way to imaging infection in various cell lines, or even in animal models, and opening fascinating fundamental and applied prospects. Associated with high-content automated microscopy, the technology permitted rapid, robust, and precise determination of ganciclovir 50% and 90% inhibitory concentrations (IC50 and IC90) on HCMV replication, with minimal hands-on time investment. To search for new antiviral activities, the experiment is easy to upgrade toward efficient and cost-effective screening of large chemical libraries. Simple infection of permissive cells with ANCHOR viruses in the presence of a compound of interest even provides a first estimation of the stage of the viral cycle the molecule is acting upon. family and, like all herpesviruses (HVs), is able to establish lifelong latency in infected individuals (1). HCMV is the largest HHV, with a double-stranded DNA (dsDNA) genome of about 240 kb. It is usually transmitted through body fluids, such as saliva, urine, or breast milk, but also through sexual contact (2). Primary infection is generally benign or silent in healthy individuals but may be much more serious and even life threatening in immunocompromised patients, especially those who have received hematopoietic Tobramycin sulfate cells or solid-organ transplants, or in AIDS patients. The virus is also able to cross the placental barrier, and primary HCMV infection during pregnancy, mainly during the first quarter, is the leading cause of birth defects, with an estimate of 1 1 million congenital HCMV infections worldwide per year (3, 4). Among those infected, possibly up to 25% of newborns suffer permanent sensorineural and intellectual deficits. infection is poorly understood but most likely initiates in mucosal tissue and then spreads through blood monocytes, which disseminate the virus. HCMV binds to heparan sulfate proteoglycan (5) and to numerous cell membrane structures, among which CD13 (6), annexin II (7), DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin) (8), EGFR (epidermal growth factor receptor) (9), and PDGFR- (platelet-derived growth factor receptor alpha) (10) are candidate receptors. This may Tobramycin sulfate in part explain the remarkably broad cell tropism of the virus, which is able to infect and replicate in many cell types, including epithelial, dendritic, fibroblastic, endothelial, and smooth muscle cells (11), and to establish latency in CD34+ hematopoietic progenitor cells (12). Extensive efforts have allowed partial deciphering of the biology of this highly sophisticated virus, but much remains to be learned about infection kinetics. Techniques to track real-time infections in live cells have been developed for RNA viruses (13,C15) and also for herpesviruses (16,C18). However, until now, fluorescent tracking of HVs relied on green fluorescent protein (GFP) expression alone or on fusion of the GFP gene with a viral structural gene. These engineered viruses have greatly contributed to some pioneering work but did not provide quantitative information about replication kinetics of the viral genome. Therefore, to gain a better understanding of the fundamental biology of HVs, we have introduced a new technology enabling real-time follow-up and counting of viral genomes during infection in live cells and also possibly in live-animal models. In this paper, we present the use of the patented ANCHOR DNA labeling technology (19) for tracking of HCMV in living cells. ANCHOR is a bipartite system derived from a bacterial ParABS chromosome segregation machinery. Under its natural form in bacteria, the ParABS system consists of a short, nonrepetitive target DNA sequence containing a limited number of nucleation parS sites to which ParB proteins bind and then spread onto adjacent DNA through a mechanism of protein-protein interaction. The third component of the system is an ATPase involved in the last steps of bacterial chromosome or plasmid segregation. Under its engineered form, called ANCHOR, OR.