ABSTRACT
Instead
of displaying the wild-type selective export of virions containing
mature genomes, human hepatitis B virus (HBV) mutant I97L, changing
from an isoleucine to a leucine at amino acid 97 of HBV core antigen
(HBcAg), lost the high stringency of selectivity in genome maturity
during virion export. To understand the structural basis of this
so-called “immature secretion” phenomenon, we compared
the stability and morphology of self-assembled capsid particles from
the wild-type and mutant I97L HBV, in either full-length (HBcAg1-183)
or truncated core protein contexts (HBcAg1-149 and HBcAg1-140). Using
negative staining and electron microscopy, full-length particles appear
as “thick-walled” spherical particles with little
interior space, whereas truncated particles appear as“
thin-walled” spherical particles with a much larger
inner space. We found no significant differences in capsid stability
between wild-type and mutant I97L particles under denaturing pH and
temperature in either full-length or truncated core protein contexts.
In general, HBV capsid particles (HBcAg1-183, HBcAg1-149, and
HBcAg1-140) are very robust but will dissociate at pH 2 or 14, at
temperatures higher than 75°C, or in 0.1% sodium dodecyl
sulfate (SDS). An unexpected upshift banding pattern of the SDS-treated
full-length particles during agarose gel electrophoresis is most likely
caused by disulfide bonding of the last cysteine of HBcAg. HBV capsids
are known to exist in natural infection as dimorphic T=3 or
T=4 icosahedral particles. No difference in the ratio between
T=3 (78%) and T=4 particles (20.3%) are
found between wild-type HBV and mutant I97L in the context of
HBcAg1-140. In addition, we found no difference in capsid stability
between T=3 and T=4 particles successfully separated by
using a novel agarose gel electrophoresis
procedure.
Joining of simian virus 40 DNA molecules at endonuclease R Eco RI sites by polynucleotide ligase and analysis of the products by agarose gel electrophoresis
