Abstract
Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.
| Original language | English |
|---|---|
| Pages (from-to) | 13306-13314 |
| Number of pages | 9 |
| Journal | Journal of Virology |
| Volume | 78 |
| Issue number | 23 |
| DOIs | |
| Publication status | Published - Dec 2004 |
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ASJC Scopus subject areas
- Immunology
Cite this
Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture. / Neddermann, Petra; Quintavalle, Manuela; Di Pietro, Chiara; Clementi, Angelica; Cerretani, Mauro; Altamura, Sergio; Bartholomew, Linda; De Francesco, Raffaele.
In: Journal of Virology, Vol. 78, No. 23, 12.2004, p. 13306-13314.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture
AU - Neddermann, Petra
AU - Quintavalle, Manuela
AU - Di Pietro, Chiara
AU - Clementi, Angelica
AU - Cerretani, Mauro
AU - Altamura, Sergio
AU - Bartholomew, Linda
AU - De Francesco, Raffaele
PY - 2004/12
Y1 - 2004/12
N2 - Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.
AB - Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.
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U2 - 10.1128/JVI.78.23.13306-13314.2004
DO - 10.1128/JVI.78.23.13306-13314.2004
M3 - Article
VL - 78
SP - 13306
EP - 13314
JO - Journal of Virology
JF - Journal of Virology
SN - 0022-538X
IS - 23
ER -