Article

Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 10021, USA.
Journal of Virology (Impact Factor: 4.44). 04/2003; 77(5):3181-90. DOI: 10.1128/JVI.77.5.3181-3190.2003
Source: PubMed

ABSTRACT

Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease
worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from
genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication
of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid
substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting,
and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs
and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive
mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons
should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development
of adapted replicons for the remaining HCV genotypes.

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    • "The H56A and D79A mutations in HPgV NS3/4AB-HA were made using site-directed mutagenesis. The coding regions for HCV NS3/4A (nt 3419–5473) were amplified from the HCV 1a strain H77 clone (kind gift of Dr. Charles Rice) (Blight et al., 2003) using HiFi DNA polymerase (Roche) and inserted into pCDNA3.1/Zeoþ expression vector (Invitrogen). "
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    • "This system only depends on the ability to replicate in cells. HCV-RMT was the first genotype 1a clone that could be established in authentic replicon cells without artificially introduced adaptive mutations that are required by H77 [30], [31], although the three spontaneously occurring mutations (E1056V, E1202G, and A2199T) are not novel [11], [31]. Among the mutants with single mutations or a combination of these three adaptive mutations, the amounts of HCV genome and viral proteins did not reflect the colony-forming abilities (Figure 1D, E). "
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