HCV replicase complexes
View Patent ↗This invention provides efficient HCV replicase complexes comprising novel RNA template and primer pair. Assay systems are also provided, which use such complexes, for detecting replicase activity, quantitatively studying the kinetics and mechanism of HCV NS5B-catalyzed nucleotide incorporation, and identifying inhibitors of HCV replicase. The assay systems use small and well-defined synthetic RNAs which allow efficient assembly of all catalytic components in the quaternary complex for HCV NS5B-directed RNA replication. Specific template-primer requirements for efficient RNA synthesis by HCV NS5B replicase are provided for use in assay systems.
1. An assay system for detecting HCV replicase activity comprising
an enzymatically active amount of HCV NS5B protein;
RNA template which comprises at least five nucleotides;
an RNA primer which is complementary to the 3′ terminus of the template and comprises two or three nucleotides, wherein the template and primer do not form stable duplex solution in the absence of the NS5B protein,
ATP, GTP, CTP, and UTP nucleotlde triphosphates (NTPs), wherein only one of the NTPs or the primer is radiolabeled; and
an assay buffer that permits replication activity of the NS5B protein.
2. The assay system according to claim 1 , wherein the base of the first nucleotide of the primer is a guanine.
3. The assay system according to claim 1 , wherein the NS5B protein is in contact with an inhibitory compound of the NS5B protein.
4. The assay system according to claim 1 , wherein the NS5B protein is a soluble enzymatically active NSB5B protein expressed in Escherichia coli.
5. The assay system according to claim 1 , wherein the RNA template lacks any stable secondary structure at the 3′ terminus.
6. The assay system according to claim 1 , wherein the radiolabeled NTP or primer comprises a phosphate isotope.
7. The assay system according to claim 6 , wherein the radiolabeled NTP is an α- 33 P-NTP which hydrogen bonds to a nucleotide of the template.
8. The assay system according to claim 1 , wherein
the assay buffer comprises 50 mM HEPES (pH 7.3), 10 mM β-mercaptoethanol, 50 mM NaCl, and 5 mM MgCl 2 ;
the template RNA is 5 μM;
the primer is 10 μM;
the HCV NS5B protein is 3 to 5 μM; and
the NTP substrate is 100 μM.
9. A method for detecting HCV replicase activity, comprising
(a) providing an assay system comprising
an enzymatically active amount of HCV NS5B protein;
an RNA template which comprises at least five nucleotides;
an RNA primer which is complementary to the 3′ terminus of the template and comprises two or three nucleotides, wherein the template and primer do not form stable duplex in solution in the absence of the NS5B protein,
ATP, GTP, CTP, and UTP nucleotide triphosphates (NTPs), wherein one of the NTPs or the primer is radiolabeled; and
an assay buffer that permits replication activity of the NS5B protein;
(b) incubating said assay system under conditions that permit NS5B polymerase activity; and
detecting a nucleic acid synthesized by the HCV NS5B protein.
10. The method according to claim 9 , wherein said detecting comprises evaluating an autoradiograph of reaction products separated by gel electrophoresis.
11. The method according to claim 9 , wherein the NS5B protein is a soluble NS5B protein expressed in Escherichia coli.
12. The method according to claim 9 , wherein the base of the first nucleotide of the primer is a guanine.
13. The method according to claim 9 , wherein the radiolabeled NTP or primer comprises a phosphate isotope.
14. The method according to claim 13 , wherein the radiolabeled NTP is an α- 33 P-NTP which hydrogen bonds to a nucleotide of the template.
15. The method according to claim 9 , wherein
the assay buffer comprises 50 mM HEPES (pH 7.3), 10 mM β-mecaptoethanol, 50 mM NaCl, and 5 mM MgCl 2 ;
the template RNA is 5 μM;
the primer is 10 μM;
the HCV NS5B protein is 3-5 μM;
the NTP is 100 μM; and
the assay is performed at 30° C.
16. The method according to claim 9 , wherein said assay system further comprises a candidate inhibitory compound.
17. The method according to claim 16 , wherein said method is a high throughput screen.
18. The method according to claim 16 , wherein said candidate inhibitory compound directly interferes with the replicase activity of the NS5B protein.
19. The assay system according to claim 1 , wherein said HCV NS5B protein, RNA template and RNA primer form a replicase complex.