Pro-apoptotic fragments of the dengue virus envelope glycoproteins

Abstract

The present invention relates to pro-apoptotic fragments of the Dengue virus pRM and E glycoproteins, methods of screening for molecules capable of inducing apoptosis and methods of inducing apoptosis in a cell.

1 . An isolated polypeptide of the sequence in SEQ ID NO:1.  
     
     
         2  The isolated polypeptide of  claim 1 , wherein said peptide induces apoptosis in a cell.  
     
     
         3 . An isolated polynucleotide which encodes the polypeptide of  claim 1 .  
     
     
         4 . A vector comprising the polynucleotide of  claim 3 .  
     
     
         5 . A prokaryotic cell comprising the polynucleotide of  claim 3 .  
     
     
         6 . A eukaryotic cell comprising the polynucleotide of  claim 3 .  
     
     
         7 . A composition comprising the polynucleotide of  claim 3  and a physiologically acceptable carrier.  
     
     
         8 . A composition comprising the peptide of  claim 1  and a physiological acceptable carrier.  
     
     
         9 . An isolated polypeptide of the sequence in SEQ ID NO:2.  
     
     
         10 . An isolated polynucleotide which encodes the polypeptide of  claim 9 .  
     
     
         11 . A vector comprising the polynucleotide of  claim 10 .  
     
     
         12 . A prokaryotic cell comprising the polynucleotide of  claim 10 .  
     
     
         13 . A eukaryotic cell comprising the polynucleotide of  claim 10 .  
     
     
         14 . A composition comprising the polynucleotide of  claim 10  and a physiologically acceptable carrier.  
     
     
         15 . A composition comprising the peptide of  claim 9  and a physiological acceptable carrier.  
     
     
         16 . An isolated polypeptide of the sequence SEQ ID NO:1 operably linked to the sequence SEQ ID NO:2.  
     
     
         17 . An isolated polynucleotide which encodes the polypeptide of  claim 16 .  
     
     
         18 . A vector comprising the polynucleotide of  claim 17 .  
     
     
         19 . The vector of  claim 18  which is the plasmid p[95-114]EGFP[206-245] deposited in the CNCM on Jan. 21, 2000 under the accession number I-2380.  
     
     
         20 . The vector of  claim 18  which is the plasmid p[95-114][211-245] deposited in the CNCM on May 10, 2000 under the accession number I-2475.  
     
     
         21 . A prokaryotic cell comprising the polynucleotide of  claim 17 .  
     
     
         22 . A eukaryotic cell comprising the polynucleotide of  claim 17 .  
     
     
         23 . A composition comprising the polynucleotide of  claim 17  and a physiological acceptable carrier.  
     
     
         24 . A composition comprising the polypeptide of  claim 16  and a physiological acceptable carrier.  
     
     
         25 . A method of inducing apoptosis in a cell comprising administering an effective amount of the polypeptide of  claim 16  to the cell to induce apoptosis.  
     
     
         26 . The method of  claim 25 , wherein said cell is in a human patient.  
     
     
         27 . The method of  claim 25 , wherein said patient is suffering from cancer.  
     
     
         28 . The method of  claim 25 , wherein said patient is infected with a Flavivirus  
     
     
         29 . A method of inducing apoptosis in a cell comprising delivering the polypeptide of  claim 16  in an amount sufficient to induce apoptosis.  
     
     
         30 . The method of  claim 29 , wherein said delivering comprises delivering a polynucleotide encoding said polypeptide to the cell, wherein said polynucleotide is in an expression vector suitable to express said polypeptide in the cell.  
     
     
         31 . The method of  claim 29 , wherein said cell is in a human patient.  
     
     
         32 . The method of  claim 31 , wherein said patient is suffering from cancer.  
     
     
         33 . The method of  claim 31 , wherein said patient is infected with a Flavivirus.  
     
     
         34 . A method of screening for peptides capable of inducing apoptosis comprising 
 introducing a recombinant protein into a cell, wherein said recombinant protein comprises the peptide to be screened operably linked SEQ ID NO:2; and    detecting apoptosis in the cell.    
     
     
         35 . The method of  claim 34 , wherein said introducing step comprises introducing an expression vector comprising a polynucleotide which encodes said recombinant protein.  
     
     
         36 . The method of  claim 34 , wherein said recombinant protein further comprises a green fluorescent protein.  
     
     
         37 . A method of screening for molecules which inhibit apoptosis induced by the polypeptide of the sequence SEQ ID NO:1 comprising 
 introducing said polypeptide into a cell;    contacting said cell containing said polypeptide, with the molecule to be screened; and    detecting the presence or absence of apoptosis in the cell.    
     
     
         38 . The method of  claim 37 , wherein said polypeptide is operably linked to the polypeptide of the sequence SEQ ID NO:2.  
     
     
         39 . The method of  claim 37 , wherein said polypeptide is operably linked to a green fluorescent protein.  
     
     
         40 . The method of  claim 37 , wherein said polypeptide is not linked to a green fluorescent protein.  
     
     
         41 . The method of  claim 37 , wherein said introducing comprises introducing a polynucleotide which encodes said polypeptide, wherein said polynucleotide is an expression vector capable of expressing the polypeptide in a cell.  
     
     
         42 . An isolated polypeptide of the sequence in SEQ ID NO:3.  
     
     
         43 . The isolated polypeptide of  claim 42 , wherein said peptide induces apoptosis in a cell.  
     
     
         44 . An isolated polynucleotide which encodes the polypeptide of  claim 42 .  
     
     
         45 . A vector comprising the polynucleotide of  claim 44 .  
     
     
         46 . The vector of  claim 45  which is the plasmid p[95-114]EGFP[206-245]DEN-2 deposited in the CNCM on Jan. 29, 2001 under the accession number I-2620.  
     
     
         47 . A prokaryotic cell comprising the polynucleotide of  claim 44 .  
     
     
         48 . A eukaryotic cell comprising the polynucleotide of  claim 44 .  
     
     
         49 . A composition comprising the polynucleotide of  claim 44  and a physiologically acceptable carrier.  
     
     
         50 . A composition comprising the peptide of  claim 42  and a physiological acceptable carrier.  
     
     
         51 . A method of inducing apoptosis in a cell comprising administering an effective amount of the polypeptide of  claim 42  to the cell to induce apoptosis.  
     
     
         52 . The method of  claim 51 , wherein said cell is in a human patient.  
     
     
         53 . The method of  claim 51 , wherein said patient is suffering from cancer.  
     
     
         54 . The method of  claim 51 , wherein said patient is infected with a Flavivirus.  
     
     
         55 . A method of screening for molecules which inhibit apoptosis induced by the polypeptide of the sequence SEQ ID NO:3 comprising 
 introducing said polypeptide into a cell;    contacting said cell containing said polypeptide, with the molecule to be screened; and    detecting the presence or absence of apoptosis in the cell.    
     
     
         56 . The method of  claim 55 , wherein said polypeptide is operably linked to the polypeptide of the sequence SEQ ID NO:3.  
     
     
         57 . The method of  claim 55 , wherein said polypeptide is operably linked to a green fluorescent protein.  
     
     
         58 . The method of  claim 55 , wherein said polypeptide is not linked to a green fluorescent protein.  
     
     
         59 . The method of  claim 55 , wherein said introducing comprises introducing a polynucleotide which encodes said polypeptide, wherein said polynucleotide is an expression vector capable of expressing the polypeptide in a cell.  
     
     
         60 . Monoclonal antibodies raised against DEN-1 viral M protein.  
     
     
         61 . Monoclonal antibodies raised against DEN-2 viral M protein.  
     
     
         62 . The plasmid [95-114]EGFP[M10-M40]DEN-2 deposited at the CNCM under the accession number I-2684.  
     
     
         63 . The plasmid pTripΔU3[95-114]EGFP[206-245]DEN-2 deposited at the CNCM under the accession number I-2686.  
     
     
         64 . The plasmid pTripΔU3[95-114]EGFP[206-245]DEN-1 deposited at the CNCM under the accession number I-2685.