Establishment of a Luciferase-Based Reporter System to Study Aspects of Human Cytomegalovirus Infection, Replication Characteristics, and Antiviral Drug Efficacy

• Published in: Pathogens (Basel) Vol. 13; no. 8; p. 645
• Main Authors: Tillmanns, Julia, Kicuntod, Jintawee, Ehring, Antonia, Elbasani, Endrit, Borst, Eva Maria, Obergfäll, Debora, Müller, Regina, Hahn, Friedrich, Marschall, Manfred
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 31.07.2024; MDPI
• Subjects: Antiviral activity; antiviral agents; Antiviral Agents - pharmacology; antiviral drug development; Antiviral drugs; Asymptomatic; Automation; Cell culture; Cytomegalovirus; Cytomegalovirus - drug effects; Cytomegalovirus - genetics; Cytomegalovirus - physiology; Cytomegalovirus infections; Cytomegalovirus Infections - drug therapy; Cytomegalovirus Infections - virology; Fibroblasts; firefly luciferase reporter (FLuc); Fluorescence; Genes, Reporter; Herpes viruses; host-pathogen relationships; Human betaherpesvirus 5; human cytomegalovirus (HCMV); Humans; Infections; L protein; luciferase; Luciferases - genetics; Luciferases - metabolism; Luciferases, Firefly - genetics; Luciferases, Firefly - metabolism; medically important viral pathogens; Molecular properties; Mutagenesis; Pathogenesis; pathogens; progeny; protein synthesis; Proteins; recombinant HCMV TB40-FLuc; Replication; Risk groups; Tropism; Vaccine development; Viral infections; viral replication characteristics and efficiency; virus replication; Virus Replication - drug effects; Viruses; St Louis Missouri; United Kingdom > UK; United States > US; Germany; medically important viral pathogens; antiviral drug development; future applications in molecular HCMV analysis; human cytomegalovirus (HCMV); infection of primary human cultured cells; viral replication characteristics and efficiency; recombinant HCMV TB40-FLuc; firefly luciferase reporter (FLuc); advantages of the reporter system
• ISSN: 2076-0817; 2076-0817
• DOI: 10.3390/pathogens13080645

Human cytomegalovirus (HCMV) represents a highly medically important pathogen which has constantly been the subject of both molecular and clinical investigations. HCMV infections, especially those in high-risk patients, still raise many unanswered questions, so current investigations are focused on viral pathogenesis, vaccine development, and options for antiviral drug targeting. To this end, the use of suitable viral strains as well as recombinant reporter constructs in cultured cells and model systems has specific significance. We previously reported on the application of various herpesviruses that express green, red, or related fluorescent proteins, especially in the fields of virus–host interaction and antiviral research. Here, we characterized a recombinant version of the clinically relevant and cell type-adaptable HCMV strain TB40, which expresses firefly luciferase as a quantitative reporter of viral replication (TB40-FLuc). The data provide evidence for five main conclusions. First, HCMV TB40-FLuc is employable in multiple settings in primary human cells. Second, viral reporter signals are easily quantifiable, even at early time points within viral replication. Third, the FLuc reporter reflects the kinetics of viral intracellular replication, cascade-like viral IE-E-L protein production, and progeny release. Fourth, as relates to specific applications of the TB40-FLuc system, we demonstrated the reliability of quantitative antiviral compound determination in multi-well formats and its independence from fluorescence-based measurements in the case of autofluorescent inhibitors. Finally, we illustrated increased reporter sensitivity in comparison to other recombinant HCMVs. In essence, recombinant HCMV TB40-FLuc combines several molecular properties that are considered beneficial in studies on viral host tropism, replication efficiency, and antiviral drug assessment.