Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication


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Article: article from journal or magazin.
Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication
Polyak  S. J., Paschal  D. M., McArdle  S., Gale, M. J., Jr. , Moradpour  D., Gretch  D. R.
0270-9139 (Print)
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Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S. --- Old month value: Apr
The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. Biochemical studies have demonstrated that NS5A interacts in vitro with and inhibits the IFN-induced, RNA-dependent protein kinase, PKR, and that NS5A interacts with at least one other cellular kinase. The present study describes the establishment and characterization of various stable NS5A-expressing human cell lines, and the development of a cell culture-based assay for determining the inherent IFN resistance of clinical NS5A isolates. Human epithelioid (Hela) and osteosarcoma (U2-OS) cell lines were generated that express NS5A under tight regulation by the tetracycline-dependent promoter. Maximal expression of NS5A occurred at 48 hours following the removal of tetracycline from the culture medium. The half-life of NS5A in these cell lines was between 4 to 6 hours. NS5A protein expression was localized cytoplasmically, with a staining pattern consistent with the location of the Golgi apparatus and endoplasmic reticulum. In the majority of cell lines, no obvious phenotypic changes were observed. However, three genotype 1b NS5A-expressing osteosarcoma cell lines exhibited cytopathic effect and severely reduced proliferation as a result of high-level NS5A expression. Full-length NS5A protein isolated from a genotype 1b IFN-nonresponsive patient (NS5A-1b) was capable of rescuing encephalomyocardititis virus replication during IFN challenge up to 40-fold, whereas a full-length NS5A-1a and an interferon sensitivity determining region (ISDR) deletion mutant (NS5A-1a-triangle upISDR) isolated from a genotype 1a IFN-nonresponsive patient showed no rescue activity. The NS5A-1b and NS5A-1a proteins also rescued vesicular stomatitis virus replication during IFN treatment by two- to threefold. These data cummulatively suggest that NS5A expression alone can render cells partially resistant to the effects of IFN against IFN-sensitive viruses, and that in some systems, these effects may be independent of the putative ISDR. A scenario is discussed in which the NS5A protein may employ multiple strategies contributing to IFN resistance during HCV infection.
Amino Acid Sequence Blotting, Western Cell Division Drug Resistance, Microbial Encephalomyocarditis virus/drug effects/physiology Female Fluorescent Antibody Technique Gene Expression Regulation, Viral/drug effects Hela Cells Humans Interferons/*pharmacology Molecular Sequence Data Plaque Assay/methods RNA Replicase/*immunology/*metabolism Tetracycline/pharmacology Transfection Tumor Cells, Cultured Vesicular stomatitis-Indiana virus/drug effects/physiology Viral Nonstructural Proteins/genetics/immunology/*metabolism Virus Replication/*drug effects
Web of science
Open Access
Create date
25/01/2008 16:05
Last modification date
20/08/2019 12:38
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