Biology Reference
In-Depth Information
RNA Replication Model
RNA replication of most RNA viruses involves certain intracellular
membrane structures, including the ER,
70-72
Golgi,
73
endosomes and
lysosomes.
74
Although NS5B protein has RdRp activity
in vitro
, its
recombinant product alone is presumably short of the strict template
specificity and fidelity, which are essential for the viral RNA synthesis.
It is highly likely that other viral and/or host factors are important for
conferring proper RNA replication and that the replication complexes
(RCs) that are composed of NS5B and additional components
required for modulating polymerase activity are involved in catalyzing
HCV RNA synthesis during the replication process.
Several coprecipitation and immunostaining studies revealed that
the newly synthesized HCV RNA was localized to distinct speckle-like
structures, where all of the viral nonstructural proteins coexisted.
48
These distinct structures may be equivalent to the membranous webs,
as reported by Gosert
et al
.
45
and described above. Expression of all
structural and nonstructural proteins in the context of the entire
HCV polyprotein induced similar membrane changes.
46
It is of inter-
est that morphologically similar structures, termed sponge-like inclu-
sions,
75
have been found by electron microscopy in liver cells of
HCV-infected chimpanzees. Thus, the membranous web may com-
prise the HCV RC in infected cells.
Recently, several groups have succeeded in showing the
in vitro
replication activities of the HCV RCs in crude membrane fractions of
cells harboring the subgenomic replicons.
76-80
These cell-free systems
provide a valuable complement to the
in vitro
RdRp assays for bio-
chemical dissection of HCV RNA replication and a useful source for
isolation of viral RCs. The replication activity in the crude membrane
fraction, which contains HCV ribonucleoprotein complexes associ-
ated with cellular membranes, is measured by incorporation of radio-
labeled nucleotides into newly synthesized RNA
in vitro
, and the
products can be resolved from replicative intermediates by native or
denaturing gel electrophoresis. The RNA synthesis can be initiated in
the absence of added negative-strand template RNA, suggesting that
preinitiated template RNA copurifies with the RC.
77-79
Although the
