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244
Prenylation Inhibition of the Large Hepatitis
Delta Virus Antigen Leads to Intracellular
Accumulation of Viral RNA and Enhanced
Innate Immune Responses
Florian Lempp1,2, Franziska Schlund1, Lisa Rieble1, Lea
Nussbaum1, Corinna Link1, Yi Ni1,2 and Stephan Urban1,2,
(1)Department of Infectious Diseases, Molecular Virology,
Heidelberg University Hospital, (2)Heidelberg Partner Site,
German Center for Infection Research (DZIF)
Background: Chronic Hepatitis Delta Virus (HDV) infection
represents the most severe form of viral hepatitis. Current
treatment options for chronic HDV are limited to interferonalpha
(IFNα), but sustained response rates are rare. Two
novel drugs with distinct antiviral mechanisms are currently
approaching registration trials: Myrcludex B inhibits viral entry
by blocking the HBV/HDV specific receptor NTCP. Lonafarnib
inhibits viral secretion by interfering with prenylation of
the large Hepatitis Delta antigen (L-HDAg). Since the
identification of NTCP, different cell culture models that allow
viral entry and replication have been developed, however,
these models do not support assembly and secretion of
progeny virus. We developed a novel cell line that allows
both entry & secretion of progeny virus and used this cell
line to study the antiviral mechanisms of IFNα and the new
drugs. Methods: Using recombinant lentiviruses, we stably
transduced HepG2 cells with NTCP and the HBV envelope
proteins to create HepNB2.7 cells. The cells were treated
with the three drugs at increasing concentrations and primary
infection as well as viral progeny secretion were evaluated
by in-cell ELISA. Intracellular accumulation of viral RNAs
and proteins was measured by RT-qPCR, FISH and western
blot. Results: After infection with HDV, HepNB2.7 cells stably
secreted infectious progeny virions for more than 30 days.
Myrcludex B inhibited de novo infection with an IC50 of 1 nM,
IFNα inhibited primary infection (IC50: 20 IU/ml) but only to
a level of 20-30% of an uncompeted infection. Lonafarnib
inhibited HDV progeny secretion with an IC50 of 35 nM but
resulted in a substantial intracellular accumulation of HDAg
(mostly L-HDAg) and viral RNAs in primary infection. This
was accompanied by an increased induction of the antiviral
innate immunity, as observed by enhanced induction of
interferons and interferon-stimulated genes. Conclusion: We
have established the first hepatic cell line that recapitulates
the complete replication cycle of HDV, thus allowing host
factor screening, drug screening and drug evaluation. While
Myrcludex B completely blocked de novo infection but had no
effect on virus release, inhibition of prenylation resulted in a
block of HDV release but at the same time an accumulation of
replicating HDV RNA and an enhanced induction of innate
immune responses. Whether both drugs act synergistically in
a combination treatment needs further investigations |
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发表于 2018-10-8 16:46
