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


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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    • "In DDX3 knockdown cells, lower luciferase activities were observed for all of the time points in both peaks (Fig. 7B), indicating that viral protein translation and viral RNA replication were all hampered by the knockdown of DDX3. Because JEV RNA replication depends on viral proteins, we next confirmed the roles of DDX3 on viral translation using a replication-deficient replicon, which contains a GDD-AAG mutation in NS5 polymerase (Blight et al., 2003). The results showed that luciferase activity derived from deficient-replicon RNA was lower in DDX3 knockdown cells when compared with the control cells (Fig. 7C). "
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    ABSTRACT: Japanese encephalitis virus is one of the most common causes for epidemic viral encephalitis in humans and animals. Herein we demonstrated that cellular helicase DDX3 is involved in JEV replication. DDX3 knockdown inhibits JEV replication. The helicase activity of DDX3 is crucial for JEV replication. GST-pulldown and co-immunoprecipitation experiments demonstrated that DDX3 could interact with JEV non-structural proteins 3 and 5. Co-immunoprecipitation and confocal microscopy analysis confirmed that DDX3 interacts and colocalizes with these viral proteins and viral RNA during the infection. We determined that DDX3 binds to JEV 5′ and 3′ un-translated regions. We used a JEV-replicon system to demonstrate that DDX3 positively regulates viral RNA translation, which might affect viral RNA replication at the late stage of virus infection. Collectively, we identified that DDX3 is necessary for JEV infection, suggesting that DDX3 might be a novel target to design new antiviral agents against JEV or other flavivirus infections.
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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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