Interferon alpha signaling in viral hepatitis

Summarizing all our results, we can draw the following picture (Fig.6). The expression of
HCV proteins induces an ER stress response. The consequence of prolonged ER stress is
the release of Ca2+ from the ER into the cytosol. Cytosolic Ca2+-transients activate Ca2+
dependent kinases, which phosphorylate the transcription factor CREB. Phosphorylated
CREB binds to the CRE-element in the PP2Ac promoter and induces transcription.
Elevated PP2Ac level inhibits the enzymatic activity of PRMT1. Two consequences of
reduced PRMT1 activity are the hypomethylation of STAT 1 and the hypomethylation of
NS3 helicase. Hypomethylated STAT1 shows increased association with PIAS1 and
decreased binding to IFNa-target genes upon IFNa treatment. The consequence of the
inhibited IFNa-signaling is a reduced antiviral response, which contributes to the
development of chronic liver infection, which later on can progress to liver cirrhosis and
HCC. On the other hand, the unwinding activity of hypomethylated NS3 is increased
compared to methylated NS3. The higher unwinding activty of NS3 causes an increase in
viral replication. Therefore the upregulation of PP2Ac has two advantages for the virus:
reduced cellular antiviral response and increased viral replication.
Perspectives:
The promising results of the improved IFNa signaling in UHCV 57.3 cells (express HCV
proteins in the absence of tetracycline) after AdoMet/Betaine treatment and the fact that
the expression of HBV proteins in Huh7.93 cells induces obviously the same IFNa
inhibiting mechanism (increased PP2Ac levels, hypomethylated STAT1 and decreased
induction of IFNa target genes), it opens the interesting perspective of correcting the
HBV induced defect in IFNa signaling also by treating cells with AdoMet and betaine.
We would expect, that the AdoMet/betaine treatment would at least partially restore the
IFNa signaling in Huh7.93 cells and would lead to an increased induction of antiviral
genes compared to Huh7.93 cells not treated with AdoMet/Betaine.
AdoMet and Betaine are two compounds, which are already on the market as over the
counter drugs, and which are used to treat liver diseases. Now that we could show
improved IFNa signaling in UHCV57.3 cell pretreated with AdoMet and betaine, we
plan to apply these drugs to HCV patients in a clinical study. The plan is to retreat nonresponders
with a combination of AdoMet, Betaine, IFNa and rabavirin.
To increase the activity of PRMT1 by manipulating the methionine/AdoMet cycle is one
possibility to correct the inhibition of the IFNa signaling by HCV. Another possibility
would be to target the first event, induction of ER stress by HCV. Without the induction
of ER stress during expression of HCV proteins, Ca2+ would not leak out of the ER and
the phosphorylation of CREB would not occur, so the level of PP2A would not rise and
therefore there would be no inhibition of PRMT1. One possibility to avoid ER stress is
the treatment of cells with chemical chaperones. Chemical chaperones are a group of
low-molecular-weight componds, which can reverse the mislocalization and/or
aggregation of proteins(138). Polyos such as glycerol, trimethylamines such as
trimethlyamine N-oxide (TMAO), amino acid derivatives and other compounds like 4-
phenylbutyric acid (PBA) and membrane-permeable forms of enzyme antagonists belong
to the chemical chaperones. The mechanisms by which chemical chaperones function are
not fully understood but are thought to include stabilization of improperly folded
proteins, reduction of aggregation, prevention of nonproductive interactions with other
resident proteins and alteration of the activity of endogenous chaperones in such a way
that the affected proteins are more efficiently transported to the appropriate intracellular
or extracellular destination.
An interesting experiment would be the expression of viral proteins in UHCV57.3 cells
for 24h in the presence of chemical chaperones, and check afterwards for the level of
PP2Ac and for the induction of ER stress markers like BiP or spliced XBP1. We would
expect, that the increased level of chaperones in the ER prevent the induction of ERstress
and the Ca2+ -transients in the cytosol. Therefore the transcription factor CREB
does not get phosphorylated, and the PP2Ac level stays constant even in the presence of
viral proteins.