Dataset Information

A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms.

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.

SUBMITTER: Rand U

PROVIDER: S-EPMC7820782 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms.

Rand Ulfert U Kubsch Tobias T Kasmapour Bahram B Cicin-Sain Luka L

Frontiers in cellular and infection microbiology 20210108

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 ...[more]

PMID: 33489928

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Project description:Human cytomegalovirus (HCMV) is the leading cause of congenital infection and is associated with a wide range of neurodevelopmental disabilities and intrauterine growth restriction. Yet our current understanding of the mechanisms modulating transplacental HCMV transmission is poor. The placenta, given its critical function in protecting the fetus, has evolved effective yet largely uncharacterized innate immune barriers against invading pathogens. Here we show that the intrinsic cellular restriction factor apolipoprotein B editing catalytic subunit-like 3A (APOBEC3A [A3A]) is profoundly upregulated following ex vivo HCMV infection in human decidual tissues-constituting the maternal aspect of the placenta. We directly demonstrated that A3A severely restricted HCMV replication upon controlled overexpression in epithelial cells, acting by a cytidine deamination mechanism to introduce hypermutations into the viral genome. Importantly, we further found that A3 editing of HCMV DNA occurs both ex vivo in HCMV-infected decidual organ cultures and in vivo in amniotic fluid samples obtained during natural congenital infection. Our results reveal a previously unexplored role for A3A as an innate anti-HCMV effector, activated by HCMV infection in the maternal-fetal interface. These findings pave the way to new insights into the potential impact of APOBEC proteins on HCMV pathogenesis.IMPORTANCE In view of the grave outcomes associated with congenital HCMV infection, there is an urgent need to better understand the innate mechanisms acting to limit transplacental viral transmission. Toward this goal, our findings reveal the role of the intrinsic cellular restriction factor A3A (which has never before been studied in the context of HCMV infection and vertical viral transmission) as a potent anti-HCMV innate barrier, activated by HCMV infection in the authentic tissues of the maternal-fetal interface. The detection of naturally occurring hypermutations in clinical amniotic fluid samples of congenitally infected fetuses further supports the idea of the occurrence of A3 editing of the viral genome in the setting of congenital HCMV infection. Given the widely differential tissue distribution characteristics and biological functions of the members of the A3 protein family, our findings should pave the way to future studies examining the potential impact of A3A as well as of other A3s on HCMV pathogenesis.

Dataset Information

A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms.

Publications

A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms.

Similar Datasets

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