Biology Reference
In-Depth Information
newly synthesized single-strand RNA can be used as a template for a
further round of double-strand RNA synthesis, no exogenous RNA
can be served as a template for HCV RC preparation.
79
The added
RNA templates might not access the active site of the HCV RCs due
to sequestration by membranes. The HCV RCs contain both minus-
and plus-strand RNAs,
77,78
with the plus-strand RNA being approx-
imately 10-fold more abundant than the minus strand.
76
This is
consistent with the replication process for other positive-strand RNA
viruses, which produce a negative-sense replication intermediate in
smaller amounts than that of the genomic RNA. It has also been
reported that the cell-free replication activity increases at tempera-
tures ranging from 25
C, and divalent cations (Mn
2+
and
Mg
2+
) can be used in the reaction.
78,79
Shi
et al
.
48
have demonstrated that newly synthesized HCV RNA
and the nonstructural proteins colocalized on distinct speckle-like struc-
tures in the cytoplasm of the replicon-containing cells. Membrane
flotation analysis and replication assay have shown that the viral RNA
and proteins were present in detergent-resistant membrane (DRM)
structures, most likely a lipid-raft structure, and that RNA replication
activity was detected even after treatment with detergent.
76,81
Lipid
rafts are cholesterol- and sphingolipid-rich microdomains and are
characterized by their detergent insolubility.
82-84
The structures are
known to play a critical role in a number of biological processes such
as regulators and organizing centers of signal transduction and mem-
brane traffic pathways, including virus entry and assembly of, for
example, influenza virus,
85-87
human immunodeficiency virus type-1
(HIV-1),
88-90
Ebola, Marburg virus,
91
enterovirus,
92
avian sarcoma and
leukosis virus,
93
Coxsackie B virus, adenovirus,
94
measles virus,
1
and
respiratory syncytial virus.
95
However, HCV may be the first example
of the association of lipid rafts with viral RNA replication.
On the other hand, it has been widely thought that most of the
HCV life cycle, including the protein processing and genome replica-
tion, takes place at the ER, where cholesterol-sphingolipid rafts are not
assembled.
46,96-98
Several studies using the replicon system have indi-
cated that the nonstructural proteins were associated with the ER.
97,99
Nevertheless, it is still possible that HCV nonstructural proteins that
°
C to 40
°
