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PLoS One. 2011;6(8):e22808. doi: 10.1371/journal.pone.0022808. Epub 2011 Aug 4.

Generation of a cell culture-adapted hepatitis C virus with longer half life at physiological temperature.

Author information

1
Molecular Virology Laboratory, POSTECH Biotech Center, Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk, Republic of Korea.

Abstract

BACKGROUND:

We previously reported infectious HCV clones that contain the convenient reporters, green fluorescent protein (GFP) and Renilla luciferase (Rluc), in the NS5a-coding sequence. Although these viruses were useful in monitoring viral proliferation and screening of anti-HCV drugs, the infectivity and yield of the viruses were low.

METHODOLOGY/PRINCIPAL FINDINGS:

In order to obtain a highly efficient HCV cultivation system, we transfected Huh7.5.1 cells [1] with JFH 5a-GFP RNA and then cultivated cells for 20 days. We found a highly infectious HCV clone containing two cell culture-adapted mutations. Two cell culture-adapted mutations which were responsible for the increased viral infectivity were located in E2 and p7 protein coding regions. The viral titer of the variant was ∼100-fold higher than that of the parental virus. The mutation in the E2 protein increased the viability of virus at 37°C by acquiring prolonged interaction capability with a HCV receptor CD81. The wild-type and p7-mutated virus had a half-life of ∼2.5 to 3 hours at 37°C. In contrast, the half-life of viruses, which contained E2 mutation singly and combination with the p7 mutation, was 5 to 6 hours at 37°C. The mutation in the p7 protein, either singly or in combination with the E2 mutation, enhanced infectious virus production about 10-50-fold by facilitating an early step of virion production.

CONCLUSION/SIGNIFICANCE:

The mutation in the E2 protein generated by the culture system increases virion viability at 37°C. The adaptive mutation in the p7 protein facilitates an earlier stage of virus production, such as virus assembly and/or morphogenesis. These reporter-containing HCV viruses harboring adaptive mutations are useful in investigations of the viral life cycle and for developing anti-viral agents against HCV.

PMID:
21829654
PMCID:
PMC3150383
DOI:
10.1371/journal.pone.0022808
[Indexed for MEDLINE]
Free PMC Article

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