Internal de novo initiation sites of the HCV NS5B polymerase and use thereof

An isolated de novo initiation site for an RNA-dependent RNA polymerase (RdRp), said site selected from the group consisting of:

• U13CU3CCUUCU3 (SEQ ID. NO. 21)
• U11GU29CU30CCU4CCU10AUAUUCCUUCU (SEQ ID. NO. 22);
• U14GGU47CCU5CCU10AUAUUCCUUCU (SEQ ID. NO. 23);
• and
• U13GU58CCU13AUAUUCCUUCU (SEQ ID. NO. 24).

A method of identifying a compound that inhibits primer-independent de novo RNA synthesis catalyzed by a flavivirus RNA-dependent RNA polymerase, comprising:

• a) contacting a synthetic heteropolymeric RNA template comprising a cluster of cytidylate nucleotides or a mixed cluster of cytidylate and uridylate nucleotides with said polymerase in the absence of a primer and in the absence of said compound under conditions permitting RNA synthesis, and determining the amount of RNA product thus formed;
• b) contacting an RNA template as in a) with said polymerase in the absence of a primer and in the presence of said compound under the same conditions as in a), and determining the amount of RNA product thus formed;
• and
• c) comparing the amount of RNA product formed in b) with that in a),
• wherein any reduction in the amount of RNA product formed in b) compared with that formed in a) indicates a compound that is an inhibitor of primer-independent de novo RNA synthesis catalyzed by said polymerase.