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. 2013 Jun 17:10:197.
doi: 10.1186/1743-422X-10-197.

A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells

Franziska Dag, Adrien Weingärtner, Milada Butueva, Ianina Conte, Julia Holzki, Tobias May, Barbara Adler, Dagmar Wirth, Luka Cicin-Sain

A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells

Franziska Dag et al. Virol J. .

Abstract

Background: The MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie1, ie2 and ie3. The regulation of their expression is intensively studied, but still incompletely understood.

Methods: We constructed a reporter MCMV, (MCMV-MIEPr) expressing YFP and tdTomato under the control of the MIEP as proxies of ie1 and ie2, respectively. Moreover, we generated a liver sinusoidal endothelial cell line (LSEC-uniLT) where cycling is dependent on doxycycline. We used these novel tools to study the kinetics of MIEP-driven gene expression in the context of infection and at the single cell level by flow cytometry and by live imaging of proliferating and G0-arrested cells.

Results: MCMV replicated to higher titers in G0-arrested LSEC, and cycling cells showed less cytopathic effect or YFP and tdTomato expression at 5 days post infection. In the first 24 h post infection, however, there was no difference in MIEP activity in cycling or G0-arrested cells, although we could observe different profiles of MIEP gene expression in different cell types, like LSECs, fibroblasts or macrophages. We monitored infected LSEC-uniLT in G0 by time lapse microscopy over five days and noticed that most cells survived infection for at least 96 h, arguing that quick lysis of infected cells could not account for the spread of the virus. Interestingly, we noticed a strong correlation between the ratio of median YFP and tdTomato expression and length of survival of infected cells.

Conclusion: By means of our newly developed genetic tools, we showed that the expression pattern of MCMV IE1 and IE2 genes differs between macrophages, endothelial cells and fibroblasts. Substantial and cell-cycle independent differences in the ie1 and ie2 transcription could also be observed within individual cells of the same population, and marked ie2 gene expression was associated with longer survival of the infected cells.

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Figures

Figure 1
Figure 1
Growth and susceptibility to MCMV infection of the conditionally immortalized liver sinusoidal endothelial cell line LSEC-uniLT (LSEC). (A) 103 LSEC-uniLT were cultured for 10 days in 10 cm Petri dishes in the presence of 2 μg/ml doxycycline (+DOX) or in its absence (-DOX). Representative crystal violet stains visualizing cell proliferation are shown. (B) Representative brightfield microscopy picture of confluent LSEC-uniLT is shown. (C) LSEC-uniLT were infected at 0.1 MOI of MCMV in presence or absence of doxycycline and virus growth was monitored by plaque assay of supernatants at indicated time-points post infection. The average PFU/ml +/- SD of three experiments is shown. The limit of detection (DL) is marked by the dashed line.
Figure 2
Figure 2
Generation and characterization of the reporter MCMV MIEPr. (A) Graphic representation of the MIEPr reporter construct and its integration into the MCMV genome. The bidirectional major immediate early promoter enhancer (MIEP) was flanked by the yellow fluorescent protein (YFP) and tdTomato (Tom) and inserted ectopically into the MCMV genome, replacing the viral genomic region between the genes m07 and m17. (B) LSEC-uniLT were infected with 1 MOI of MIEPr and representative pictures were visualized by epifluorescence microscopy at indicated hours post infection (hpi) are shown. (C) NIH-3 T3 cells were infected with MIEPr or MCMV wild type (WT) at an MOI of 0.1 and virus growth was monitored by plaque assay of cell supernatants at indicated days post infection (dpi). Averages (+/- SD) from three independent experiments are shown. The dashed line represents the limit of detection (DL). (D) Endogenous (ie1 and ie2) and reporter (YFP and Tom) transcripts were measured by qRT-PCR. The cDNA was synthesized from RNA obtained from LSEC-uniLT infected at an MOI of 10 for the indicated hours post infection (hpi). Copy numbers were normalized to GAPDH and are represented as averages (+/- SD) from three independent experiments.
Figure 3
Figure 3
In vivo growth of MCMV MIEPr. Growth of MCMV WT (black line) and MIEPr (grey line) in organ homogenates of (A) spleen, (B) liver and (C) salivary glands of C57BL/6 mice on day 2, 4, 7, 10 and 14 post infection. Each data point depicts the mean obtained from 5 mice and error bars indicate SEM.
Figure 4
Figure 4
Cell proliferation has no effect on the early reporter gene expression of the MIEPr. (A) LSEC-uniLT (LSEC) were cultured in the presence (+ DOX) or absence (- DOX) of doxycycline. Cells were infected with an MOI of 10 and analyzed at 0, 4, 6, 12 and 24 hpi by flow cytometry. Representative dot blots of tdTomato and YFP expression are shown. (B) LSEC-uniLT were cultured in the presence (+DOX) or absence (-DOX) of doxycycline and infected with an MOI of 1 or left uninfected (control). At day 5 post infection confocal images were acquired to visualize cells by differential interference contrast (DIC) and the fluorescent reporters (YFP, tdTomato) by fluorescence imaging.
Figure 5
Figure 5
Comparison of fluorescence profiles in different cell lines infected with the MIEPr. NIH/3T3, LSEC-uniLT (LSEC), IC-21, and Ana-1 cells were infected with MIEPr at an MOI of 10 and analysed by flow cytometry 0, 4, 6, 12 and 24 hpi. (A) Representative dot blots of tdTomato and YFP expression are shown. (B) Mean fluorescence intensities of YFP and tdTomato (+ SEM) for the indicated cell lines and times post infection are shown as histograms.
Figure 6
Figure 6
Live cell imaging and single cell analysis of LSEC-uniLT infected with the MIEPr. Cells were infected at an MOI of 10 and monitored by fluorescence microscopy in a CO2 regulated chamber for 5 days. (A) Representative time courses of fluorescence intensities for YFP and tdTomato (Tom) in individual LSEC-uniLT. The signal intensity is represented on the ordinate, whereas the time post infection is shown on the abscissa. (B) The median fluorescence intensity ratio of tdTomato/YFP was established for individual infected cells and plotted against the survival time of the infected cell. (C) LSEC-uniLT shown in panel B were subdivided into terciles according to their median tdTomato/YFP-ratio. Symbols show survival times of individual cells in the lower (1), centre (2) and upper tercile (3), while horizontal lines show group average (+/- SEM) values. * and ** denote p values below 0.05 and 0.0035, respectively, according to Kruskal-Wallis followed by Dunns post-analysis test.
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