Hepatitis C virus NS5A is a direct substrate of casein kinase I-alpha, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors.
The hepatitis C virus encodes a single polyprotein that is processed by host and viral proteases to yield at least 10 mature viral proteins. The nonstructural (NS) protein 5A is a phosphoprotein, and experimental data indicate that the phosphorylation state of NS5A is important for the outcome of viral RNA replication. We were able to identify kinase inhibitors that specifically inhibit the formation of the hyperphosphorylated form of NS5A (p58) in cells. These kinase inhibitors were used for inhibitor affinity chromatography in order to identify the cellular targets of these compounds. The kinases casein kinase I (CKI), p38 MAPK, CIT (Citron Rho-interacting kinase), GAK, JNK2, PKA, RSK1/2, and RIPK2 were identified in the high affinity binding fractions of two NS5A hyperphosphorylation inhibitors (NS5A-p58-i). Even though these kinases are targets of the NS5A-p58-i, the only kinase showing an effect on NS5A hyperphosphorylation was confirmed to be CKI-alpha. Although this finding does not exclude the possibility that other kinase(s) might be involved in basal or regulatory phosphorylation of NS5A, we show here that NS5A is a direct substrate of CKI-alpha. Moreover, in vitro phosphorylation of NS5A by CKI-alpha resulted for the first time in the production of basal and hyperphosphorylated forms resembling those produced in cells. In vitro kinase reactions performed with NS5A peptides show that Ser-2204 is a preferred substrate residue for CKI-alpha after pre-phosphorylation of Ser-2201.
Quintavalle
Manuela
M
Istituto di Ricerche di Biologia Molecolare "P. Angeletti," Via Pontina Km 30.6, 00040 Pomezia, Rome, Italy.
Sambucini
Sonia
S
Summa
Vincenzo
V
Orsatti
Laura
L
Talamo
Fabio
F
De Francesco
Raffaele
R
Neddermann
Petra
P
D000818:Animals; D048128:Casein Kinase Ialpha / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity; D016174:Hepacivirus / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology*; D012324:RNA-Dependent RNA Polymerase
D048128:Casein Kinase Ialpha; D047428:Protein Kinase Inhibitors; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase