Article

Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations.

Institute for Virology, Johannes-Gutenberg University Mainz, 55131 Mainz, Germany.
Journal of Virology (impact factor: 5.4). 06/2001; 75(10):4614-24. DOI:10.1128/JVI.75.10.4614-4624.2001 pp.4614-24
Source: PubMed

ABSTRACT Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.

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Keywords

adaptive mutations
 
amino acid substitutions
 
cell culture-adaptive mutations
 
clear correlation
 
cultured cells
 
gene encoding firefly luciferase
 
HCV RNA replication
 
HCV structural proteins
 
highest replication level
 
naive Huh-7 cells
 
neomycin phosphotransferase gene
 
nonadapted RNA replicated
 
region encoding
 
replicate autonomously
 
replication efficiencies
 
RNA replication
 
selectable marker gene
 
subgenomic selectable RNAs
 
support high-level replication
 
transfected cells
 

N Krieger