Inhibition of picornavirus genome replication by interference with VPg-nucleotidylylation and elongation
AssigneeThe Research Foundation of State University of New York
InventorsAniko V. Paul, Eckard Wimmer, Elizabeth Rieder
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Abstract
The present invention provides methods of inhibiting picornavirus genome replication in a subject. In particular, methods for interfering with VPg uridylylation and elongation are provided. The methods comprise administering to a subject an effective amount of at least one of VPg, VPg analog, VPg homology or biologically active fragment thereof as well as oligonucleotides, divalent cations, ribonucleotide or deoxyribonucleotide. Also provided are methods of identifying an inhibitor of picornaviral replication which comprise adding a potential inhibitor of picornaviral replication to an in vitro assay and analyzing levels of VPg uridylylation reaction products.
Claims13
A method of identifying an inhibitor of picornaviral replication which comprises the steps of:
- (a) incubating for a time and under conditions sufficient to detect uridylylation of picornaviral VPg a first reaction mixture comprising a buffer with an approximate pH of 7.0-8.0 and a buffer concentration of approximately 10-50 mM;
- a divalent metal in the form of at least one of approximately 0.1-0.5 mM manganous acetate, approximately 1-7 mM magnesium acetate, approximately 0.1-0.5 mM cobaltous acetate, or approximately 0.1-0.5 mM zinc acetate;
- approximately 2-10% glycerol;
- approximately 0.1-1.0 μg of poly(A);
- approximately 0.5-2.0 μg of purified picornaviral RNA polymerase;
- labeled UTP sufficient to identify labeled uridylylation reaction products;
- approximately 0.1-100 μM UTP;
- and approximately 0.5-5 μg of picornaviral VPg;
- (b) incubating a second reaction mixture comprising the same reactants and under the same conditions as enumerated in step (a) with a potential inhibitor of picornaviral replication;
- (c) comparing the level of uridylylation reaction products obtained in the first and second reaction mixtures;
- and
- (d) correlating a decrease in the level of uridylylation reaction products obtained in the second reaction mixture with the identification of an inhibitor of picornaviral RNA replication.
A method of identifying an inhibitor of picornaviral replication which comprises the steps of:
- (a) incubating for a time and under conditions sufficient to detect uridylylation of picornaviral VPg a first reaction mixture comprising a buffer with an approximate pH of 7.0-8.0 and a buffer concentration of approximately 10-50 mM;
- a divalent metal in the form of at least one of approximately 0.1-0.5 mM manganous acetate, approximately 1-7 mM magnesium acetate, approximately 0.1-0.5 mM cobaltous acetate, or approximately 0.1-0.5 mM zinc acetate;
- approximately 2-10% glycerol;
- approximately 0.1-1.0 μg of purified picornaviral transcript corresponding to a an RNA hairpin structure located within the coding sequence of the picornavirus, approximately 0.5-2.0 μg of purified picornavirus RNA polymerase;
- approximately 0.2-1.2 μg of purified picornavirus protein 3CDpro;
- labeled UTP in an amount sufficient to detect uridylylated reaction products;
- 0.1-100 μM UTP and approximately 0.5-5.0 μg of picornaviral VPg;
- (b) incubating a second reaction mixture comprising the same reactants and under the same conditions as enumerated in step (a) with a potential inhibitor of picornaviral replication;
- (c) comparing the level of uridylylation reaction products obtained in the first and second reaction mixtures;
- and
- (e) correlating a decrease in the level of uridylylation reaction products obtained in the second reaction mixture with the identification of an inhibitor of picornaviral RNA replication.
Dependencies are inferred from the claim text.