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frequency, followed by the identification of replicons harboring cell
culture-adaptive mutations yielding higher replication efficiency. 14-16
Some of the most adaptive mutations are located at highly conserved
serine residues within NS5A upstream of the region putatively involved
in IFN sensitivity. A combination of adaptive mutation in NS3 and
NS5A resulted in the highest level of replication of particular HCV
genotype 1b isolate. 16 Later work, however, has indicated that adap-
tive mutations can arise in most of the viral nonstructural proteins. 17,18
The mechanisms by which adaptive mutations increase RNA replica-
tion efficiency are not well understood.
Huh-7 cells have been permissive for adapted HCV replicons,
although variability in the permissiveness for replicons has been observed
for these cells. Recently, some studies showed that HCV subgenomic
RNAs could replicate in human hepatic HepG2 and IMY-N9 cells,
human cervical carcinoma HeLa cells, human embryonic kidney 293
cells, 19,20 a human T cell line MT-2C 21 and mouse hepatoma cells. 22
These findings suggest that host factors required for HCV RNA repli-
cation are not hepatocyte specific and are not restricted to cells of
human origin.
Finally, taking advantage of cell culture-adaptive mutations, full-
length dicistrinic HCV genomes that efficiently and stably replicate in
Huh-7 cells have been developed. By immuno-electron microscopy, the
core protein expressed in the cells harboring the genomic HCV RNA
was localized mainly to the boundary of cytoplasmic lipid storage vesi-
cles and was also found at the endoplastic recticulum (ER). 23 In the cells,
viral envelope proteins E1 and E2 formed heterodimers and existed at
the ER and cis-Golgi compartments. Cell culture systems based on the
selectable subgenomic and genome-length dicistronic HCV RNAs,
which produce abundant viral RNA and nonstructural proteins, open
avenues of biochemical and genetic studies for HCV replication.
Polyprotein Translation and Processing
-NTR, an open reading frame (ORF)
that encodes a polyprotein with a length of
The HCV genome carries a 5
3010 amino acids, and
a 3
-NTR. The precursor polyprotein is co-translationally or post-
processed by both viral and host proteases into at least ten viral products.
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