Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication
Sj. Polyak et al., Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication, HEPATOLOGY, 29(4), 1999, pp. 1262-1271
The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been implic
ated in the inherent resistance of HCV to interferon (IFN) antiviral therap
y in clinical studies. Biochemical studies have demonstrated that NS5A inte
racts in vitro with and inhibits the IFN-induced, RNA-dependent protein kin
ase, PKR, and that NS5A interacts with at least one other cellular kinase,
The present study describes the establishment and characterization of vario
us stable NS5A-expressing human cell lines, and the development of a cell c
ulture-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 tetracyclin
e-dependent promoter. Maximal expression of NS5A occurred at 48 hours follo
wing 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 Ib NS5A-expressing osteosarcoma cell lines exhibited cytopathic ef
fect and severely reduced proliferation as a result of high-level NS5A expr
ession. Full-length NS5A protein isolated from a genotype Ib IFN-nonrespons
ive patient (NS5A-1b) was capable of rescuing encephalomyo-cardititis 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-Delta ISDR) isolated from a genotype la IFN-nonresponsive patient
showed no rescue activity The NS5A-1b and NS5A-1a proteins also rescued ve
sicular stomatitis virus replication during IFN treatment by two- to threef
old. These data cummulatively suggest that NS5A expression alone can render
cells partially resistant to the effects of IFN against IFN-sensitive viru
ses, and that in some systems, these effects may be independent of the puta
tive ISDR. A scenario is discussed in which the NS5A protein may employ mul
tiple strategies contributing to IFN resistance during HCV infection.