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The first part of this thesis is to study roles of HDAgs in
packaging the viral genome during viral assembly. In
addition, functions of HDAgs involved in supporting and
inhibiting the replication of HDV were study. To approach the
problems, culture cells were transfected with various mutant
constructs of large and small HDAgs in the presence of
HDV cDNA and a plasmid encoding the small form of HBAgs.
Viral particles were collected four to six days
posttransfection and the presence of HDV RNA and HDAgs were
analyzed. Results are summarized as following: (1) Both HDAgs
can package the HDV genome and the RNA-binding region in the
middle domain is essential for this function. (2)The short
leucine zipper-like motifs of small HDAg is not essential for
the assembly of HDV RNA. (3) Small and large HDAgs, with a
proper ratio, can package viral genome cooperatively during
assembly.(4) In the presence of small HDAg, the viral particles
contain mainly the full-length RNA genome. (5) Interactions
between the N- termini of small and large HDAgs may not be
the only mechanism of large HDAg in inhibiting the viral
replication. (6) The C-ter- minal Pro/Gly-rich region of
large HDAg may be important for the inhibition of HDV
replication. The second part of this study focuses on the
expression of HDAgs as the first step towards
understanding the structure difference between two HDAgs. The
pET expression system which is under the control of T7 RNA
polymerases was used. Both HDAgs could be expressed as
soluble proteins after induction with IPTG for 2 hr at 37℃.
From immunoprecipitation assay, antigenicities of the
recombinant HDAgs may be similar to those of HDAgs ex-
pressed in transfected cells.
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