Abstract
Human Cytomegalovirus (HCMV) infection may result in severe outcomes in immunocompromised individuals such as AIDS patients, transplant recipients, and neonates. To date, no vaccines are available and there are only few drugs for anti-HCMV therapy. Adverse effects and the continuous emergence of drug-resistance strains require the identification of new drug candidates in the near future. Identification and characterization of such compounds and biological factors requires sensitive and reliable detection techniques of HCMV infection, gene expression and spread. In this work, we present and validate a novel concept for multi-reporter herpesviruses, identified through iterative testing of minimally invasive mutations. We integrated up to three fluorescence reporter genes into replication-competent HCMV strains, generating reporter HCMVs that allow the visualization of replication cycle stages of HCMV, namely the immediate early (IE), early (E), and late (L) phase. Fluorescent proteins with clearly distinguishable emission spectra were linked by 2A peptides to essential viral genes, allowing bicistronic expression of the viral and the fluorescent protein without major effects on viral fitness. By using this triple color reporter HCMV, we monitored gene expression dynamics of the IE, E, and L genes by measuring the fluorescent signal of the viral gene-associated fluorophores within infected cell populations and at high temporal resolution. We demonstrate distinct inhibitory profiles of foscarnet, fomivirsen, phosphonoacetic acid, ganciclovir, and letermovir reflecting their mode-of-action. In conclusion, our data argues that this experimental approach allows the identification and characterization of new drug candidates in a single step.
Highlights
Cytomegalovirus (CMV) is a common opportunistic infection in immunocompromised hosts or in congenitally infected children (Cannon et al, 2013), where virus replication and cytopathic effects result in tissue damage and disease (Boppana and WJ, 2013)
Contemporary advances in transcriptome analysis have exposed a surprising complexity of CMV gene expression over time (Marcinowski et al, 2012; Stern-Ginossar et al, 2012; Weekes et al, 2014; Shnayder et al, 2018; Erhard et al, 2019)
Any such system would require that the virus replication matches the real-life conditions and that the reporter genes are driven by endogenous promoters driving the expression of essential viral genes
Summary
Cytomegalovirus (CMV) is a common opportunistic infection in immunocompromised hosts or in congenitally infected children (Cannon et al, 2013), where virus replication and cytopathic effects result in tissue damage and disease (Boppana and WJ, 2013). Several antivirals thwarting virus replication have been approved for clinical use in adult patients (Ljungman et al, 2019). The pharmacotherapy of CMV infection relies on compounds that target viral replication during the lytic phase, but do not affect cellular processes. They include substances targeting the virus DNA polymerization reaction, such as ganciclovir (Crumpacker, 1996) or foscarnet (Chrisp and Clissold, 1991). Ganciclovir is a guanosine analogue that requires phosphorylation by the CMV-encoded kinase UL97 prior to its incorporation in the DNA. Foscarnet blocks the pyrophosphate binding to the viral DNA polymerase and is closely related to the phosphonoacetic acid (PAA) which is not approved for clinical use. The antiviral activity of fomivirsen might be partially independent of its antisense binding, its mode of action remains unclear
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