RNA silencing in animals as an antiviral defense

Abstract

The present invention provides recombinant DNA constructs for inactivation of viral or endogenous genes in a cell, wherein the construct comprises viral sequence sufficient to activate RNA silencing. In another aspect, the invention provides methods for identifying RNA silencing suppressors by sequence analysis and functional tests. In yet another aspect, the invention provides a method for identifying inhibitors of RNA silencing suppressors. In still other aspects, the invention comprises methods for identifying genes in the antiviral RNA silencing pathway, enhancers of the antiviral pathway, and methods of treating or preventing viral infections using enhancers of the pathway.

Description

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0001] This work was made with Government support under Grant No.2002-35319-11537. awarded by the U.S. Department of Agriculture. The government has certain rights in this invention.CROSS-REFERENCES TO RELATED APPLICATIONS [0002] NOT APPLICABLE REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK [0003] NOT APPLICABLE BACKGROUND OF THE INVENTION [0004] RNA interference (RNAi) is a process where introduction of dsRNA into a cell causes destruction of RNA in a sequence-specific manner (see, D. Baulcombe, Curr. Biol., 12:R83 (2002); Hutvagner et al., Curr. Opin. Genet. Dev., 12:225 (2002)) RNAi has been observed in plants, Neurospora, flies, protozoans, and mice. Available data show that double-stranded (ds) RNA serves as the initial trigger of RNA interference and upon recognition, is processed by the Dicer RNAse into short fragments of...

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Benefits of technology

[0011] In yet another aspect, the invention provides methods for identifying an inhibitor of a RNA silencing suppressor. The methods typically comprise (a) infecting an animal cell with FHV; (b) contacting the cell with a candidate inhibitor of a RNA silencing suppressor; and (c) testing for a rate or extent of FHV RNA accumulation less than that for a cell not contacted with the candidate inhibitor. In certain embodiments, the candidate inhibitor is a small molecule. Typically, RNA accumulation is measured by visual assays for expressed reporter molecules and Northern blots.

[0012] In still yet another aspect, the invention provides methods for identifying a gene in the antiviral RNA silencing pathway of an animal. Typically, the methods comprise: (a) providing an animal cell expressing a polynucleotide encoding FHV-ΔB2; (b) inhibiting the expression of a candidate gene in the antiviral RNA silencing pathway; and (c) testing for a rate or extent of FHV-ΔB2 RNA accumulation greater than that for a cell where the expression of the candidate gene in the antiviral RNA silencing pathway has not been inhibited. In some embodiments,

Problems solved by technology

However, co-transfection with dsRNAs targeting mRNAs of the two Drosophila Dicer genes (Bernstein et al., Nature, 411:494 (2001)), was not effective (lane 17) under the same conditions.

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