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.

What is claimed is:  
     
         1 . A recombinant DNA construct for inactivation of a gene in a cell, said construct comprising: 
 (a) polynucleotide sequence of a virus sufficient to activate RNA silencing and    (b) the polynucleotide sequence of said gene.    
     
     
         2 . The recombinant DNA construct of  claim 1 , wherein said construct is a viral vector capable of infecting said cell.  
     
     
         3 . The recombinant DNA construct of  claim 1 , wherein said construct is a recombinant vector.  
     
     
         4 . The recombinant DNA construct of  claim 1 , wherein said virus is FHV.  
     
     
         5 . The recombinant DNA construct of  claim 4 , wherein said construct further comprises polynucleotide sequences enabling introduction into said cell.  
     
     
         6 . The recombinant DNA construct of  claim 1 , wherein said cell is in an animal.  
     
     
         7 . The recombinant DNA construct of  claim 1 , wherein said gene is a polynucleotide sequence from a heterologous virus.  
     
     
         8 . The recombinant DNA construct of  claim 1 , wherein said gene is a polynucleotide sequence from an endogenous gene.  
     
     
         9 . The recombinant DNA construct of  claim 8 , wherein expression or overexpression of said endogenous gene induces a disease or a medical condition.  
     
     
         10 . The recombinant DNA construct of  claim 8 , wherein said endogenous gene is an oncogene.  
     
     
         11 . A method for identifying a RNA silencing suppressor, said method comprising: 
 (a) introducing a polynucleotide sequence encoding FHV-AB2 into an animal cell;    (b) introducing a polynucleotide sequence encoding a candidate RNA silencing suppressor into said cell; and    (c) testing for a rate or extent of FHV-AB2 RNA accumulation greater than that for a cell not contacted with said candidate suppressor.    
     
     
         12 . The method of  claim 11 , wherein said RNA silencing suppressor is from a virus.  
     
     
         13 . The method of  claim 11 , wherein said RNA silencing suppressor is an endogenous gene.  
     
     
         14 . A method for identifying a viral RNA silencing suppressor, said method comprising identifying a polynucleotide sequence that is an overlapping gene.  
     
     
         15 . The method of  claim 14 , wherein said gene overlaps a RNA polymerase gene.  
     
     
         16 . A method for identifying an inhibitor of a RNA silencing suppressor, said method comprising: 
 (a) infecting an animal cell with FHV;    (b) contacting said 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 said candidate inhibitor of a RNA silencing suppressor.    
     
     
         17 . The method of  claim 16 , wherein said candidate inhibitor is a small molecule.  
     
     
         18 . The method of  claim 16 , wherein said testing for accumulation of FHV RNA comprises a method selected from the group consisting of: visual assays for expressed reporter molecules and Northern blots.  
     
     
         19 . A method for identifying a gene in the antiviral RNA silencing pathway of an animal, said method comprising: 
 (a) providing an animal cell expressing a polynucleotide encoding FHV-AB2;    (b) inhibiting the expression of a candidate gene in the antiviral RNA silencing pathway; and    (c) testing for a rate or extent of FHV-AB2 RNA accumulation greater than that for a cell where the expression of said candidate gene in the antiviral RNA silencing pathway has not been inhibited.    
     
     
         20 . The method of  claim 19 , wherein said polynucleotide encoding FHV-ΔB2 further comprises a reporter molecule.  
     
     
         21 . The method of  claim 20 , wherein said reporter molecule is GFP.  
     
     
         22 . The method of  claim 19 , wherein the expression of said candidate gene is inhibited by RNA interference, antisense DNA, or a ribozyme.  
     
     
         23 . A method for identifying an enhancer of the antiviral RNA silencing pathway in an animal, said method comprising: 
 (a) providing an animal cell infected with FHV;    (b) contacting said cell with a candidate enhancer of the antiviral RNA silencing pathway; and    (c) testing for a rate or extent of FHV RNA accumulation which is less than that for a cell which is not contacted with said candidate enhancer.    
     
     
         24 . A method for treating or preventing a viral infection in an animal, said method comprising enhancing the antiviral RNA silencing pathway by contacting said animal with a compound that modulates a gene identified by the method of  claim 19 .  
     
     
         25 . The method of  claim 24 , wherein said gene is AGO2 or an AGO2 homologue in said animal.