Use of MDA-5 as an antiviral and antiproliferative agent

Abstract

The present invention relates to mda-5 nucleic acids, MDA-5 proteins, the mda-5 promoter, and related molecules, and the use of each of these elements in protecting against or limiting viral infection, controlling cell proliferation, and promoting apoptosis. It is based upon the discovery that, contrary to earlier hypotheses based on the amino acid sequence of MDA-5, the protein is not a defective ATPase but rather exhibits ATPase activity. This supports the role of MDA-5 as a RNA helicase protein and consequently its use in promoting RNA degradation, for example in the context of antiviral defense, limitation of cell proliferation, or induction of apoptosis.

What is claimed is:  
     
         1 . A method of protecting a subject against contracting a viral infection comprising administering, to the subject, an effective amount of an agent which increases the level of MDA-5 protein activity in the subject.  
     
     
         2 . The method of  claim 1 , where the viral infection is caused by a virus which produces a viral helicase.  
     
     
         3 . The method of  claim 1 , where the viral infection is caused by a virus which does not produce a viral helicase.  
     
     
         4 . The method of  claim 1 , wherein the agent is a MDA-5 protein.  
     
     
         5 . The method of  claim 4 , where the MDA-5 protein comprises a protein having an amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         6 . The method of  claim 2 , wherein the agent is a MDA-5 protein.  
     
     
         7 . The method of  claim 6 , where the MDA-5 protein comprises a protein having an amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         8 . The method of  claim 3 , wherein the agent is a MDA-5 protein.  
     
     
         9 . The method of  claim 8 , where the MDA-5 protein comprises a protein having an amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         10 . The method of  claim 1 , wherein the agent is an mda-5 nucleic acid, in expressible form.  
     
     
         11 . The method of  claim 10 , wherein the mda-5 nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         12 . The method of  claim 10 , wherein the mda-5 nucleic acid comprises a nucleic acid which is capable of hybridizing, under stringent conditions, to a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         13 . The method of  claim 2 , wherein the agent is an mda-5 nucleic acid, in expressible form.  
     
     
         14 . The method of  claim 13 , wherein the mda-5 nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         15 . The method of  claim 13 , wherein the mda-5 nucleic acid comprises a nucleic acid which is capable of hybridizing, under stringent conditions, to a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         16 . The method of  claim 3 , wherein the agent is an mda-5 nucleic acid, in expressible form.  
     
     
         17 . The method of  claim 16 , wherein the mda-5 nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         18 . The method of  claim 16 , wherein the mda-5 nucleic acid comprises a nucleic acid which is capable of hybridizing, under stringent conditions, to a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         19 . The method of  claim 1 , wherein the agent increases the activity of an mda-5 promoter and is not an interferon.  
     
     
         20 . The method of  claim 19 , wherein the agent is tumor necrosis factor alpha.  
     
     
         21 . The method of  claim 19 , wherein the agent is poly(I)(C).  
     
     
         22 . The method of  claim 2 , wherein the agent increases the activity of an mda-5 promoter and is not an interferon.  
     
     
         23 . The method of  claim 22 , wherein the agent is tumor necrosis factor alpha.  
     
     
         24 . The method of  claim 22 , wherein the agent is poly(I)(C).  
     
     
         25 . The method of  claim 3 , wherein the agent increases the activity of an mda-5 promoter.  
     
     
         26 . The method of  claim 25 , wherein the agent is an interferon.  
     
     
         27 . The method of  claim 25 , wherein the agent is beta interferon.  
     
     
         28 . The method of  claim 1 , wherein the agent increases the RNA degradation activity of MDA-5 protein.  
     
     
         29 . The method of  claim 1 , wherein the agent increases the ATPase activity of MDA-5 protein.  
     
     
         30 . The method of  claim 1 , further comprising administering, to the subject, an interferon in an amount such that the combined effects of interferon and agent confer protection against viral infection.  
     
     
         31 . The method of  claim 1 , further comprising administering to the subject an antiviral agent in an amount such that the combined effects of the antiviral agent and the agent confer protection against viral infection.  
     
     
         32 . A method of limiting a viral infection in a subject comprising administering, to the subject, an effective amount of an agent which increases the level of MDA-5 protein activity in the subject.  
     
     
         33 . The method of  claim 32 , where the viral infection is caused by a virus which produces a viral helicase.  
     
     
         34 . The method of  claim 32 , where the viral infection is caused by a virus which does not produce a viral helicase.  
     
     
         35 . The method of  claim 32 , wherein the agent is a MDA-5 protein.  
     
     
         36 . The method of  claim 35 , where the MDA-5 protein comprises a protein having an amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         37 . The method of  claim 33 , wherein the agent is a MDA-5 protein.  
     
     
         38 . The method of  claim 37 , where the MDA-5 protein comprises a protein having an amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         39 . The method of  claim 34 , wherein the agent is a MDA-5 protein.  
     
     
         40 . The method of  claim 39 , where the MDA-5 protein comprises a protein having an amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         41 . The method of  claim 32 , wherein the agent is an mda-5 nucleic acid, in expressible form.  
     
     
         42 . The method of  claim 41 , wherein the mda-5 nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         43 . The method of  claim 41 , wherein the mda-5 nucleic acid comprises a nucleic acid which is capable of hybridizing, under stringent conditions, to a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         44 . The method of  claim 33 , wherein the agent is an mda-5 nucleic acid, in expressible form.  
     
     
         45 . The method of  claim 44 , wherein the mda-5 nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         46 . The method of  claim 44 , wherein the mda-5 nucleic acid comprises a nucleic acid which is capable of hybridizing, under stringent conditions, to a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         47 . The method of  claim 34 , wherein the agent is an mda-5 nucleic acid, in expressible form.  
     
     
         48 . The method of  claim 47 , wherein the mda-5 nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         49 . The method of  claim 47 , wherein the mda-5 nucleic acid comprises a nucleic acid which is capable of hybridizing, under stringent conditions, to a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element active in a cell of the subject.  
     
     
         50 . The method of  claim 32 , wherein the agent increases the activity of an mda-5 promoter and is not an interferon.  
     
     
         51 . The method of  claim 50 , wherein the agent is tumor necrosis factor alpha.  
     
     
         52 . The method of  claim 50 , wherein the agent is poly(I)(C).  
     
     
         53 . The method of  claim 33 , wherein the agent increases the activity of an mda-5 promoter and is not an interferon.  
     
     
         54 . The method of  claim 53 , wherein the agent is tumor necrosis factor alpha.  
     
     
         55 . The method of  claim 53 , wherein the agent is poly(I)(C).  
     
     
         56 . The method of  claim 34 , wherein the agent increases the activity of an mda-5 promoter.  
     
     
         57 . The method of  claim 56 , wherein the agent is an interferon.  
     
     
         58 . The method of  claim 56 , wherein the agent is beta interferon.  
     
     
         59 . The method of  claim 32 , wherein the agent increases the RNA degradation activity of MDA-5 protein.  
     
     
         60 . The method of  claim 32 , wherein the agent increases the ATPase activity of MDA-5 protein.  
     
     
         61 . A method of inhibiting cell proliferation in a cell population comprising administering, to the cell population, an effective amount of an MDA-5 protein.  
     
     
         62 . The method of  claim 61 , wherein the cell population comprises cancer cells.  
     
     
         63 . The method of  claim 61 , wherein the MDA-5 protein comprises a protein having the sequence set forth as SEQ ID NO:2.  
     
     
         64 . The method of  claim 62 , wherein the MDA-5 protein comprises a protein having the sequence set forth as SEQ ID NO:2.  
     
     
         65 . The method of  claim 61 , further comprising administering, to the cell population, an effective amount of an interferon.  
     
     
         66 . The method of  claim 65 , further comprising administering, to the cell population, an effective amount of mezerein.  
     
     
         67 . A method of inhibiting cell proliferation in a cell population comprising administering, to the cell population, an effective amount of an mda-5 nucleic acid in expressible form.  
     
     
         68 . The method of  claim 67 , wherein the cell population comprises cancer cells.  
     
     
         69 . The method of  claim 67 , wherein the mda-5 nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element.  
     
     
         70 . The method of  claim 67 , wherein the mda-5 nucleic acid comprises a nucleic acid which hybridizes to a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element.  
     
     
         71 . The method of  claim 68 , wherein the mda-5 nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element.  
     
     
         72 . The method of  claim 68 , wherein the mda-5 nucleic acid comprises a nucleic acid which hybridizes to a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element.  
     
     
         73 . The method of  claim 67 , further comprising administering, to the cell population, an effective amount of an interferon.  
     
     
         74 . The method of  claim 67 , further comprising administering, to the cell population, an effective amount of mezerein.  
     
     
         75 . The method of  claim 68 , further comprising administering, to the cell population, an effective amount of an interferon.  
     
     
         76 . The method of  claim 68 , further comprising administering, to the cell population, an effective amount of mezerein.  
     
     
         77 . A method of inhibiting tumor growth in a subject comprising administering, to the subject, an effective amount of an MDA-5 protein.  
     
     
         78 . The method of  claim 77 , wherein the cell population comprises cancer cells.  
     
     
         79 . The method of  claim 77 , wherein the MDA-5 protein comprises a protein having the sequence set forth as SEQ ID NO:2.  
     
     
         80 . The method of  claim 78 , wherein the MDA-5 protein comprises a protein having the sequence set forth as SEQ ID NO:2.  
     
     
         81 . The method of  claim 77 , further comprising administering, to the subject, an effective amount of an interferon.  
     
     
         82 . The method of  claim 77 , further comprising administering, to the subject, an effective amount of mezerein.  
     
     
         83 . A method of inhibiting tumor growth in a subject comprising administering, to the subject, an effective amount of an mda-5 nucleic acid in expressible form.  
     
     
         84 . The method of  claim 83 , wherein the cell population comprises cancer cells.  
     
     
         85 . The method of  claim 83 , wherein the mda-5 nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element.  
     
     
         86 . The method of  claim 83 , wherein the mda-5 nucleic acid comprises a nucleic acid which hybridizes to a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element.  
     
     
         87 . The method of  claim 84 , wherein the mda-5 nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element.  
     
     
         88 . The method of  claim 84 , wherein the mda-5 nucleic acid comprises a nucleic acid which hybridizes to a nucleic acid having a sequence as set forth in SEQ ID NO:1, and is operably linked to a promoter element.  
     
     
         89 . The method of  claim 83 , further comprising administering, to the cell population, an effective amount of an interferon.  
     
     
         90 . The method of  claim 83 , further comprising administering, to the cell population, an effective amount of mezerein.  
     
     
         91 . The method of  claim 84 , further comprising administering, to the cell population, an effective amount of an interferon.  
     
     
         92 . The method of  claim 84 , further comprising administering, to the cell population, an effective amount of mezerein.  
     
     
         93 . An isolated mda-5 nucleic acid having a sequence as set forth in FIG. 9 (SEQ ID NO:4).  
     
     
         94 . A method of sensitizing a cell to a growth-inhibitory effect of a protein kinase C inhibitor, comprising increasing the level of MDA-5 protein activity in the cell and administering, to the cell, a protein kinase C inhibitor.  
     
     
         95 . A viral vector comprising a first cancer-inhibitory gene operably linked to a promoter selectively active in cancer cells, and further comprising a second cancer-inhibitory gene operably linked to a mda-5 promoter.  
     
     
         96 . An isolated protein comprising a protein having an amino acid sequence as set forth in FIG. 10 (SEQ ID NO:7).  
     
     
         97 . An isolated nucleic acid comprising a nucleic acid having a nucleic acid sequence as set forth in FIG. 11 (SEQ ID NO:8).  
     
     
         98 . An isolated protein comprising a protein having the amino acid sequence as set forth in FIG. 12 (SEQ ID NO:9).