Induction of viral mutation by incorporation of miscoding ribonucleoside analogs into viral RNA

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.

Description

RELATED APPLICATIONS [0001] This application claims priority from U.S. Ser. No. 60 / 029,404, filed Oct. 28, 1996, and U.S. Ser. No. 60 / 040,535, filed Feb. 27, 1997, and is a continuation-in-part of U.S. Ser. No. 08 / 958,065, filed Oct. 27, 1997, which are herein incorporated by reference in their entirety for all purposes. BACKGROUND OF THE INVENTION [0002] The present invention is directed to the identification and use of ribonucleoside analogs to induce the mutation of an RNA virus, including HIV and HCV, or a virus which otherwise replicates through an RNA intermediate. [0003] RNA viral diseases are responsible for the vast majority of viral morbidity and mortality of viral diseases of mankind, including AIDS, hepatitis, rhinovirus infections of the respiratory tract, flu, measles, polio and others. [0004] Acquired Immune Deficiency Syndrome (“AIDS”) is a fatal human disease that has recently grown in epidemic proportions. Current estimate is that there are approximately one millio...

AI Technical Summary

Benefits of technology

[0013] Thus, in one class of embodiments, the invention provides methods of increasing the mutation rate of a virus by administering an RNA nucleoside analog to a virally infected cell. The cell can be in culture, or present in an animal such as a human patient. The analog is incorporated by an RNA polymerase into an RNA copy of a genomic nucleic acid encoding the virus, thereby inducing the virus to mutate.

[0019] The invention also provides a population of cells containing a highly variable population of replicated viral nucleic acids. This population of highly variable virus results from administering mutagenic nucleoside analogs to virally infected cells and increasing the mutation rate of the virus population. Thus, the highly variable population of viruses is an indicator that the mutation rate of the virus was increased by the administration of the nucleoside analogs. Measuring the variability of the population provides an assessment of the viability of the viral population. In turn, the viability of the viral population is a prognostic indicator for the health of the cell population. For example, low viability for an HIV population in a human patient corresponds to an improved outlook for the patient. In addition, the viral nucleic acids can also be used as probes to detect corresponding naturally occurring nucleic acids, e.g., in a Southern or northern blot, which is a useful diagnostic indicator for the presence of the wild-type virus.

[0020] In one embodiment, the invention provides a cell comprising a viral genomic nucleic acid, an RNA analog, a cellular mRNA analog and a viral genomic RNA analog. These are, e.g., cells which are being treated for a viral infection. These cells are useful for producing mutant viruses, which serve, e.g., as molecular decoys in subsequent cellular infections by the mutant viruses. The mutant viruses are also useful as diagnostic reagents for the detection of antisera against wild-type viruses, e.g., in an ELISA or western assay, or a Southern or northern blot. Because the mutant viruses have reduced viability, they are less pathogenic than wild-type viruses.

[0029] Also included within the invention is 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. The base replaces a first natural occurring nucleotide having a first complementary nucleotide, but the base also complements a second nucleotide which is other than the first nucleotide, thereby inducing the virus to mutate.

Problems solved by technology

Subsequent copying of the incorporated analog will result in mutations in the viral genome.

This results in variability in template-dependent copies of the nucleic acid.

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