Hepatitis delta virus (HDV) is a single-stranded RNA virus that encodes two viral
nucleocapsid proteins named small and large form hepatitis delta antigen (SHDAg
and LHDAg). The SHDAg is essential for viral RNA replication while the LHDAg is
required for viral assembly. We had identified HDAg as an acetylated protein and
Lys-72 of SHDAg as one of the acetylation sites. Substitution of Lys-72 to Arg
modulated HDAg subcellular localization and might participate in viral RNA
nucleocytoplasmic shuttling and replication. In the following study, overexpression of
SIRT, the type III histone deacetylase, translocated HDAg from mainly nucleolar
distribution to nucleoplasmic or even cytoplasmic distribution. To elucidate the
correlation between acetylation and nucleolar location of HDAg, putative acetylation
motif (K/R)XKK of nuclear receptor and the nucleolar localization motif
(R/K)(R/K)X(R/K) was found between 38KKKLKK43 of SHDAg. SHDAg mutant
with Lys-to-Arg substitutions of 38KKKLKK43 (SHDAg-(K38-43R)) localized
outside the nucleoli and could not facilitate the replication of HDV RNA. We added a
heterologous nucleolar localization sequence to this mutant to restore its nucleolar
localization. This will disclose the role of nucleolar localization for HDV replication.
The functions and mechanisms of nucleolus-localization of SHDAg on HDV RNA
replication are under investigation. In another part of our study, we also analyzed the
cellular machinery for HDV RNA replication. By ultracentrifugation, nuclear extracts
from HDV cDNA transfected cells were fractionated and the distribution of HDV
RNA and HDAg were analyzed. RNA smearing in fraction 8-11 was observed,
suggesting replication intermediates were produced during HDV replication cycle.
These fractions were perforned in vitro transcription with 32P labeling and the
products then hybridized with cold HDV genomic or antigenomic RNA probe in slot
blot. Unfortunately, the signal in slot blot was not strong enough to show the
replication activity in these fractions. Optimization of the system is proceeding.
Keywords: HDV, nucleolus, NoLS, acetylation, in vitro transcription
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