Methods of screening for antiviral compounds

Abstract

The present invention demonstrates that the M2-1 protein of respiratory syncytial virus has a conserved Cys 3 —His 1 motif known to bind zinc ions in other proteins and that mutations of the predicted zinc coordinating residues in the Cys 3 —His 1 motif affect the transcriptional antitermination activity of M2-1, its ability to interact with nucleocapsid protein, and the phosphorylation state of M2-1. This invention clearly demonstrates the requirement for conservation of the Cys 3 —His 1 motif in order to maintain the functional integrity of the M2-1 protein. Therefore, the present invention provides for methods of designing and screening compounds for antiviral activity towards respiratory syncytial virus based upon the loss of function of the M2-1 protein.

What is claimed is:  
     
         1 . A method of screening for antiviral compounds directed towards respiratory syncytial virus, comprising the steps of: 
 a) treating a sample of respiratory syncytial virus with a compound, thereby producing a treated sample and a n untreated sample;    b) producing respiratory syncytial virus RNA transcripts in the presence of said treated sample or in the presence of said untreated sample; and    c) comparing said transcripts produced in the presence of said treated sample with said transcripts produced in the presence of said untreated sample, wherein less readthrough transcripts due to termination at gene end signal produced in the presence of said treated sample is indicative of an antiviral compound directed towards respiratory syncytial virus.    
     
     
         2 . The method of  claim 1 , wherein said sample of respiratory syncytial virus is selected from the group consisting of a purified respiratory syncytial virus M2-1 protein, M2-1 protein produced in cell from an expression vector, an isolated respiratory syncytial virus, a respiratory syncytial virus-infected cell and a respiratory syncytial virus-infected animal.  
     
     
         3 . A method of screening for antiviral compounds directed towards respiratory syncytial virus, comprising the steps of: 
 a) treating a sample of respiratory syncytial virus with a compound, thereby producing a treated sample and an untreated sample; and    b) comparing said treated sample with said untreated sample, wherein an inhibitory effect on said treated sample of a characteristic selected from the group consisting of transcriptional antitermination by M2-1 protein, zinc binding of M2-1 protein, phosphorylation of M2-1 protein, M2-1 protein binding to respiratory syncytial virus N protein, viral transcription, viral replication and generation of progeny virus particles is indicative of an antiviral compound directed towards respiratory syncytial virus.    
     
     
         4 . The method of  claim 3 , wherein said sample of respiratory syncytial virus is selected from the group consisting of a purified respiratory syncytial virus M2-1 protein, M2-1 protein produced in cell from an expression vector, an isolated respiratory syncytial virus, a respiratory syncytial virus-infected cell and a respiratory syncytial virus-infected animal.  
     
     
         5 . A method of screening for antiviral compounds directed towards respiratory syncytial virus, comprising the steps of: 
 a) treating a sample of respiratory syncytial virus with a chelator or a compound that inhibits zinc binding, thereby producing a treated sample and an untreated sample; and    b) comparing said treated sample with said untreated sample, wherein an inhibitory effect on said treated sample of a characteristic selected from the group consisting of transcriptional antitermination by M2-1 protein, zinc binding of M2-1 protein, phosphorylation of M2-1 protein, M2-1 protein binding to respiratory syncytial virus N protein, viral transcription, viral replication and generation of progeny virus particles is indicative of an antiviral compound directed towards respiratory syncytial virus.    
     
     
         6 . The method of  claim 5 , wherein said inhibition of zinc binding is selected from the group consisting of competing with zinc for binding to the Cys 3 —His 1  motif within the M2-1 protein, preventing zinc from binding, destroying the formation of the Cys 3 —His 1  motif, and interfering with the interaction of the properly formed Cys 3 —His 1  motif with its target.  
     
     
         7 . The method of  claim 5 , wherein said sample of respiratory syncytial virus is selected from the group consisting of a purified respiratory syncytial virus M2-1 protein, M2-1 protein produced in cell from an expression vector, an isolated respiratory syncytial virus, a respiratory syncytial virus-infected cell and a respiratory syncytial virus-infected animal.  
     
     
         8 . A method of screening for antiviral compounds directed towards respiratory syncytial virus, comprising the steps of: 
 a) treating a sample of respiratory syncytial virus with a compound, thereby producing a treated sample and a n untreated sample; and    b) producing respiratory syncytial virus RNA transcripts in the presence of said treated sample or in the presence of said untreated sample, wherein an inhibition of virus RNA transcription or production of progeny virus particles in the presence of said treated sample is indicative of an antiviral compound directed towards respiratory syncytial virus.    
     
     
         9 . The method of  claim 8 , wherein said sample of respiratory syncytial virus is selected from the group consisting of core polymerase protein, nucleocapsid protein, phosphoprotein, an isolated respiratory syncytial virus, a respiratory syncytial virus-infected cell and a respiratory syncytial virus-infected animal.  
     
     
         10 . A method of designing antiviral compounds directed towards respiratory syncytial virus, comprising the steps of: 
 a) designing a compound that inhibits zinc binding to a Cys 3 —His 1  motif of respiratory syncytial virus M2-1 protein;    b) treating a sample of respiratory syncytial virus with said compound, thereby producing a treated sample and an untreated sample; and    c) comparing said treated sample with said untreated sample, wherein an inhibitory effect on said treated sample of a characteristic selected from the group consisting of transcriptional antitermination by M2-1 protein, zinc binding of M2-1 protein, phosphorylation of M2-1 protein, M2-1 protein binding to respiratory syncytial virus N protein, viral transcription, viral replication and generation of progeny virus particles is indicative of an antiviral compound directed towards respiratory syncytial virus.    
     
     
         11 . The method of  claim 10 , wherein said inhibition of zinc binding is selected from the group consisting of competing with zinc for binding to the Cys 3 —His 1  motif, preventing zinc from binding, destroying the formation of the Cys 3 —His 1  motif, and interfering with the interaction of the properly formed Cys 3 —His 1  motif with its interacting target.  
     
     
         12 . The method of  claim 10 , wherein said sample of respiratory syncytial virus is selected from the group consisting of a purified respiratory syncytial virus M2-1 protein, M2-1 protein produced in cell from an expression vector, an isolated respiratory syncytial virus, a respiratory syncytial virus-infected cell and a respiratory syncytial virus-infected animal.  
     
     
         13 . The method of  claim 10 , wherein said designing is done by computer modeling.