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. 2021 Jan 8:10:536150.
doi: 10.3389/fcimb.2020.536150. eCollection 2020.

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

Ulfert Rand  1 , Tobias Kubsch  1 , Bahram Kasmapour  1   2 , Luka Cicin-Sain  1   2   3   4
Affiliations

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

Ulfert Rand et al. Front Cell Infect Microbiol. .

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.

Keywords: Human Cytomegalovirus; antivirals; herpesvirus; in vitro drug testing; letermovir; live-cell imaging; reporter assay; screening.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Design and function of a trifluorescent reporter HCMV. (A) Novel strain TB40/BAC4 HCMV3F was generated by minimally invasive genetic integrations. Cassettes encoding a fluorescent protein and a 2A peptide were positioned replacing the START codons of UL122/123 (ie1/2), UL112/113 (e1), and SCP (UL48A). (B) Expression of mNeonGreen (green), mTagBFP2 (blue), and mCherry (magenta) during the course of primary infection of MRC-5 fibroblasts in live-cell microscopy. (C) In vitro growth curves of TB40/BAC4 HCMV3F and the parental TB40/BAC4WT virus in MRC-5 fibroblasts. (D) Virus growth area measurement. MRC-5 cells were infected with HCMV3F on 96-well plates and mean areas per well showing mNeonGreen-ie1/2 fluorescence were determined microscopically at 5 dpi. (mean values +/− SD; n = 3; PFU, plaque-forming units; DL, detection limit; dpi, days postinfection; D: ****p < 0.0001 with one-way ANOVA and Dunnett’s multiple comparisons test).
Figure 2
Figure 2
Immediate early, early, and late gene expression dynamics of HCMV. RPE-1 or MRC-5 cells were infected with TB40/BAC4 HCMV3F at MOI 0.5 or MOI 5, respectively, and subjected to live-cell confocal microscopy acquiring images at 20 min intervals. (A) Fluorescence profiles of single cells for mNeonGreen-ie1/2, mTagBFP2-e1, and SCP-mCherry [curves were generated by smoothing raw data using the Savitzky-Golay algorithm (10-neighbor), n = 19]. (B) RNA-FISH was performed on RPE-1 cells infected with TB40/BAC4 wild type HCMV-infected cells at indicated times postinfection (0 h = not infected). Fluorescence intensity of the fluorescently labeled probe was integrated over whole cell bodies. Each dot represents one cell (n ≥ 20; error bars represent SD). (C) Averaged and normalized signals (mean: thick line; SD: dotted line) of fluorescence reporter signals in A (green: mNeonGreen-ie1/2; blue: mTagBFP2-e1; magenta: mCherry-SCP) are compared to averaged and normalized RNA-FISH signals from (B). Error bars and error lines represent SD.
Figure 3
Figure 3
Reporter gene expression dynamics characterize antiviral action of different drugs. Fluorescence signals of RPE-1 or MRC-5 cells infected with HCMV3F at MOI 0.5 or MOI 5, respectively. Cells were either left untreated (solid lines) or treated at the time of infection (except for fomivirsen which was given 1 h before infection) with antiherpesviral drugs (dashed lines) followed by live-cell imaging. Graphs represent mean fluorescence intensity of infected cells within fields of view. Treatment of cells was done with 50 µM ganciclovir (A), 200 µM foscarnet (B), 1.8 mM PAA (C), 5 µM fomivirsen (D), or 10 nM letermovir (E), or 10 nM letermovir. Data represents mean values (n ≥ 3).
Figure 4
Figure 4
Lytic phase frequency gating of HCMV3F-infected cells. MRC-5 or RPE-1 cells were infected with HCMV3F using centrifugal enhancement to achieve 10–20% initially infected cells, respectively. Cells were then either left untreated or treated at the time of infection (except for fomivirsen which was given 1 h before infection) with antiherpesviral drugs and analyzed by flow cytometry at 24 h intervals for 5 days. (A) Gating strategy to classify stages of lytic infection in MRC-5 cells. (B) Infected MRC-5 and RPE-1 cells were gated into four mutually exclusive lytic stage phases: mNeonGreen-ie1/2+, mTagBFP2-e1+, SCP-mCherryint, and SCP-mCherryhi. (B) Frequency distribution of lytic stage phases. Representative data from one out of three independent experiments are shown.
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