Abstract
BackgroundThe 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.MethodsWe 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.ResultsMCMV 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.ConclusionBy 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.
Highlights
The murine cytomegalovirus (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
Analysis of different cell types revealed that ie2 accumulates predominantly in macrophages, whereas ie1/3 expression is more pronounced in fibroblasts or endothelial cells
In the cell line Liver sinusoidal endothelial cells (LSEC)-uniLT, proliferation was completely abrogated in the absence of doxycycline for at least 10 days while the presence of doxycycline allowed rapid cell proliferation (Figure 1A)
Summary
The MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie, ie2 and ie. HCMV shares many similarities with the murine cytomegalovirus (MCMV) [1,2] and MCMV has been used as a model for HCMV in numerous studies Upon infection, both the HCMV and the MCMV express viral genes controlled by the major immediate early promoter/enhancer (MIEP) at high levels [1,3], and their transcripts are detected as early as one hour post infection [4]. In follow-up studies the same group has shown that the major immediate early enhancer (MIE) may act as a genetic switch by preferentially enhancing the transcription of ie or ie, but not of both genes at the same time [20] All these studies were performed by PCR based testing of viral mRNA in lungs of latently infected mice, and it remained unclear if the MIEP acts as a genetic switch at the single-cell level and during lytic infection. Analysis of different cell types revealed that ie accumulates predominantly in macrophages, whereas ie1/3 expression is more pronounced in fibroblasts or endothelial cells
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