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Tesi di dottorato in scienze matematiche e fisiche >
Please use this identifier to cite or link to this item:
http://hdl.handle.net/2108/595
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Full metadata record
| DC Field | Value | Language |
|---|
| contributor.advisor | Cesareni, Gianni | - |
| contributor.advisor | Santoro, Gabriella | - |
| contributor.author | Quintavalle, Manuela | - |
| date.accessioned | 2008-08-29T09:17:38Z | - |
| date.available | 2008-08-29T09:17:38Z | - |
| date.issued | 2008-08-29T09:17:38Z | - |
| identifier.uri | http://hdl.handle.net/2108/595 | - |
| description | 19. ciclo | en |
| description.abstract | Hepatitis C virus (HCV) has been the subject of intensive studies for nearly
two decades. Nevertheless, some aspect of the virus life cycle are still a
mystery. The HCV Nonstructural protein 5A (NS5A) has been shown to be a
modulator of cellular processes possibly required for the establishment of
viral persistence. NS5A is heavily phosphorylated, and a switch between a
basally phosphorylated form of NS5A (p56) and a hyperphosphorylated
form of NS5A (p58) seems to play a pivotal role in regulating HCV
replication.
Efficient replication of HCV subgenomic RNA in cell culture requires the
introduction of adaptive mutations. Some of the most effective adaptive
mutations occur at the serine residues that have been shown to be implicated
in NS5A hyperphosphorylation and adaptive mutations at these sites result in
a significant reduction of NS5A hyperphosphorylation (p58).
After screening of a panel of kinase inhibitors, we selected three compounds
which inhibited NS5A phosphorylation in vitro, as well as the formation of
NS5A p58 in cell culture. Cells transfected with the HCV wild type replicon
sequence supported HCV RNA replication upon addition of any of the three
compounds.
Thus, reduction of the formation of p58 below a certain threshold either by
adaptive mutations or by inhibition of the NS5A–specific kinase(s) would
enable HCV replication in cell culture.
Although large amounts of NS5A-p58 appear to inhibit HCV RNA
replication, the complete inhibition of NS5A hyperphosphotylation by the
kinase inhibitors we identified abolishes HCV replication of already adapted
replicons indicating that a small quantity of p58 is required for replication.
Using kinase inhibitors that specifically inhibit the formation of NS5A-p58
in cells, we identified CK1 kinase family as a target.
NS5A-p58 increased upon overexpression of CK1alpha, CK1δ and CK1ε,
whereas the RNA interference of only CK1alpha reduced NS5A
hyperphosphorylation. Rescue of inhibition of NS5A-p58 was achieved by
CK1 alpha overexpression, and we demonstrated that the CK1 alpha isoform is
targeted by NS5A hyperphosphorylation inhibitors in living cells and that the
down-regulation of NS5A attenuates HCV RNA replication.
Finally, we demonstrate here that NS5A is a direct substrate of CKI- alpha and
phosphorylation of NS5A in vitro by CKI- alpha resulted in the production of two
phosphorylated forms that resemble those products produced in cells.
In vitro kinase reactions performed with NS5A peptides show that S2204 is
a preferred substrate residue for CKI- alpha after pre-phosphorylation of S2201 | en |
| format.extent | 3287567 bytes | - |
| format.mimetype | application/pdf | - |
| language.iso | en | en |
| subject.classification | BIO/11 Biologia molecolare | en |
| title | Identification of cellular kinases responsible for Hepatitis C Virus NS5A hyperphosphorylation | en |
| type | Doctoral thesis | en |
| degree.name | Dottorato in biologia cellulare e molecolare | en |
| degree.level | Dottorato | en |
| degree.discipline | Facoltà di Scienze Matematiche Fisiche e Naturali | en |
| degree.grantor | Università degli studi di Roma Tor Vergata | en |
| date.dateofdefense | A.A. 2006/2007 | en |
| Appears in Collections: | Tesi di dottorato in scienze matematiche e fisiche
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Files in This Item:
| File |
Description |
Size | Format |
|---|
| Tesi Dottorato Biologia Molecolare e Cellulare XIX ciclo-Man.pdf | | 3210Kb | Adobe PDF | View/Open |
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