Identification of human kinases involved in hepatitis C virus replication by small interference RNA library screening.
The propagation of the hepatitis C virus (HCV) is a complex process that requires both host and viral proteins. To facilitate identification of host cell factors that are required for HCV replication, we screened a panel of small interference RNAs that preferentially target human protein kinases using an HCV replicon expressing the firefly luciferase gene as a genetic reporter. Small interference RNAs specific for three human kinases, Csk, Jak1, and Vrk1, were identified that reproducibly reduce viral RNA and viral protein levels in HCV replicon-bearing cells. Treatment of replicon cells with a small molecule inhibitor of Csk also resulted in a significant reduction in HCV RNA and proteins, further supporting a role for Csk in HCV replication. The effects of siRNAs targeting eight kinases known to be negatively regulated by Csk were then examined; knock down of one of these kinases, Fyn, resulted in up-regulation of the HCV replicon, suggesting that Csk mediates its effect on HCV replication through Fyn. This conclusion was further corroborated by demonstration that replicon cells treated with Csk inhibitor contained lower levels of the phosphorylated form of Fyn than control cells.
Supekova
Lubica
L
Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037.
Supek
Frantisek
F
Department of Drug Discovery, Genomics Institute of the Novartis Research Foundation, San Diego, California 92121.
Lee
Jongkook
J
Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037.
Chen
Shawn
S
Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037.
Gray
Nathanael
N
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Dana Farber Cancer Institute, Boston, Massachusetts 02115.
Pezacki
John P
JP
Steacie Institute for Molecular Sciences Ottawa, Ontario K1A 0R6, Canada, and.
Schlapbach
Achim
A
Novartis Institute for Biomedical Research, Basel CH-4002, Switzerland.
Schultz
Peter G
PG
Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037; Department of Drug Discovery, Genomics Institute of the Novartis Research Foundation, San Diego, California 92121. Electronic address: schultz@scripps.edu.
D000081247:CSK Tyrosine-Protein Kinase; D045744:Cell Line, Tumor; D005839:Gentamicins / Q000494:pharmacology; D016174:Hepacivirus / Q000187:drug effects / Q000254:growth & development*; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D053613:Janus Kinase 1 / Q000235:genetics / Q000378:metabolism; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D012093:Replicon / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014779:Virus Replication / Q000187:drug effects*; D019061:src-Family Kinases
D005839:Gentamicins; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D012367:RNA, Viral; C010680:antibiotic G 418; D049409:Luciferases, Firefly; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; C507904:JAK1 protein, human; D053613:Janus Kinase 1; D019061:src-Family Kinases; C000631346:CSK protein, human; D017346:Protein Serine-Threonine Kinases; C109890:VRK1 protein, human