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  3. Medizin - Open Access LMU - Teil 20/22 Podcast
  4. A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells
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 G(0)-arrested cells.
Results: MCMV replicated to higher titers in G(0)-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 G(0)-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 G(0) 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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„A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells“

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