Induction of viral mutation by incorporation of miscoding ribonucleoside analogs into viral RNA
AssigneeUniversity of Washington
InventorsLawrence A Loeb, James I Mullins
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Abstract
The present invention is directed to the identification and use of ribonucleoside analogs to induce the mutation of an RNA virus, including BVDV, HIV and HCV, or a virus which otherwise replicates through an RNA intermediate. The increase in the mutation rate of the virus results in reduced viability of progeny generations of the virus, thereby inhibiting viral replication. In addition to these methods and related compositions, the invention provides methods and combinatorial chemistry libraries for screening ribonucleoside analogs for mutagenic potential.
Claims67
A method of increasing the mutation rate of a virus, comprising administering an RNA nucleoside analog to a virally infected cell, wherein the analog is incorporated by a polymerase into an RNA copy of a genomic nucleic acid encoding the virus, said analog replacing a first natural occurring nucleotide having a first complementary nucleotide wherein said analog complements a second nucleotide which is other than the first nucleotide, thereby inducing the virus to mutate.
A viral particle comprising viral genomic RNA, wherein the viral genomic RNA comprises an RNA nucleoside analog.
A population of cells comprising a highly variable population of replicated homologous viral nucleic acids.
A cell comprising a viral genomic nucleic acid, an RNA analog, a cellular mRNA analog and a viral genomic RNA analog.
A method of detecting the mutagenic potential of a ribonucleoside analog comprising integrating the ribonucleoside analog into a viral RNA synthesized by a polymerase, and determining whether the incorporation causes a mutation in a progeny virus.
A method of screening for a ribonucleoside analog which is incorporated by a cellular polymerase, comprising incubating the cellular polymerase with the ribonucleoside analog in the presence of a nucleic acid template and detecting whether the ribonucleoside analog is polymerized.
A pharmaceutical composition comprising a therapeutically effective dose of an RNA nucleoside analog, wherein the analog is one that in a infected cell with a virus of interest is incorporated by a polymerase into an RNA copy of a genomic nucleic acid encoding the virus, said analog replacing a first natural occurring nucleotide having a first complementary nucleotide wherein said analog complements a second nucleotide which is other than the first nucleotide together with a pharmaceutically acceptable carrier.
A method of identifying a mutagenic ribonucleoside analog, comprising:
- providing a plurality of ribonucleoside analogs;
- incorporating a portion of the ribonucleoside analogs into a ribonucleoside polymer using a RNA polymerase;
- isolating the ribonucleoside polymer;
- and,
- determining the chemical composition of a ribonucleoside analogs which is incorporated into the ribonucleoside polymer.
A method of making a mutagenic ribonucleoside analog comprising:
- chemically modifying a nucleotide analog selected from the group consisting of uridine, cytidine, adenosine, guanosine, N4-aminocytidine, N1-methyl-N4-aminocytidine, 3,N4-ethenocytidine, 3-methylcytidine, 5-hydroxycytidine, N4-dimethylcytidine, 5-(2-hydroxyethyl)cytidine, 5-chlorocytidine, 5-bromocytidine, N4-methyl-N4-aminocytidine, 5-aminocytidine, 5-nitrosocytidine, 5-(hydroxyalkyl)-cytidine, 5-(thioalkyl)-cytidine and cytidine glycol, 5-hydroxyuridine, 3-hydroxyethyluridine, 3-methyluridine, O2-methyluridine, O2-ethyluridine, 5-aminouridine, O4-methyluridine, O4-ethyluridine, O4-isobutyluridine, O4-alkyluridine, 5-nitrosouridine, 5-(hydroxyalkyl)-uridine, and 5-(thioalkyl)-uridine, 1,N6-ethenoadenosine, 3-methyladenosine, and N6-methyladenosine, 8-hydroxyguanosine, O6-methylguanosine, O6-ethylguanosine, O6-isopropylguanosine, 3,N2-ethenoguanosine, 06-alkylguanosine, 8-oxo-guanosine, 2,N3-ethenoguanosine, and 8-aminoguanosine to yield a chemically modified ribonucleoside analog;
- determining whether the chemically modified analog is incorporated by an RNA polymerase into a polyribonucleotide molecule;
- and,
- measuring the mutagenic potential of the chemically modified analog.
A kit comprising a container and one or more of the following components:
- a control mutagenic RNA analog, a test mutagenic RNA analog, an RNA polymerase, reagents for detecting incorporation of the RNA analog by the RNA polymerase, and instructions in the use of the kit components for detecting the mutagenic potential of the test mutagenic analog as compared to the control mutagenic RNA analog.
A method of increasing the mutation rate of a virus in an animal comprising administering to the animal (a) a therapeutically effective dose of a ribonucleoside analog to a virally infected cell, wherein the analog is incorporated by a polymerase into an RNA copy of a genomic nucleic acid encoding the virus, said analog replacing a first natural occurring nucleotide having a first complementary nucleotide wherein said analog complements a second nucleotide which is other than the first nucleotide, in combination with (b) a drug that reduces the concentration of the first natural occurring nucleotide.
A method of increasing the mutation rate of a virus, comprising administering a free base selected from the group comprising adenine, cytosine, guanine, uracil and thymine to a virally infected cell, wherein the base is incorporated by a polymerase into an RNA or DNA copy of a genomic nucleic acid encoding the virus, said base replacing a first natural occurring nucleotide having a first complementary nucleotide wherein said base complements a second nucleotide which is other than the first nucleotide, thereby inducing the virus to mutate.
Dependencies are inferred from the claim text.