Membrane fusion proteins derived from reovirus

Abstract

In accordance with the present invention, a family of membrane fusion protein and polynucleotides encoding the proteins have been identified. The proteins and nucleotides are derived from the family Reoviridae. Two membrane fusion proteins have been isolated from reoviruses isolated from poikilothermic hosts: the p14 protein from reptilian reovirus (RRV) isolated from python, and the p16 protein from aquareovirus (AQV) isolated from salmon. The genes encoding these proteins have been cloned and sequenced. Analysis of the amino acid sequences of these proteins show that both lack the typical fusion peptide motif found in other membrane fusion proteins. Expression of these proteins in cells results in cell-cell fusion.

1 . A method for facilitating membrane fusion, comprising contacting the membranes to be fused with an effective amount of a peptide comprising a hydrophobic amino acid sequence from the N-terminal domain of a Reovirus membrane fusion protein whose amino acid sequence is free of fusion peptide motif I and II, wherein the fusion peptide motif I comprises an amino acid sequence about 17 to about 28 residues long having a hydrophobicity of about 0.6 to about 0.7 and an alanine plus glycine content of about 29 to about 43% and fusion peptide motif II comprises an amino acid sequence about 16 to about 20 residues long having a hydrophobicity of about 0.3 to about 0.4, an alanine plus glycine content of about 29 to about 43% and a heptad repeat. 
     
     
         2 . The method of  claim 1 , wherein the membranes are individually selected from cell membranes, liposome membranes, proteoliposome membranes and cationic lipids. 
     
     
         3 . The method of  claim 1 , wherein one membrane is from an immortalized cell and the other membrane is from an antibody-synthesizing cell. 
     
     
         4 . The method of  claim 3 , wherein the immortalized cell is selected from human or mouse B cell myeloma cells, and T cell myelomas. 
     
     
         5 . The method of  claim 3 , wherein the antibody-synthesizing cell is a purified spleen cell from an immunized mammal. 
     
     
         6 . The method of  claim 2 , wherein the one membrane is the cell membrane and the other membrane is the cationic lipid. 
     
     
         7 . The method of  claim 1 , wherein the Reovirus membrane fusion protein is isolated from  Orthoreovirus  genus or  Aquareovirus  genus. 
     
     
         8 . The method of  claim 7 , wherein the Reovirus membrane fusion protein is isolated from avian reovirus, mammalian reovirus, Nelson Bay virus, Baboon reovirus, or reptilian reovirus. 
     
     
         9 . The method of  claim 1 , wherein the hydrophobic amino acid sequence comprises an N-terminal myristylation consensus sequence defined by SEQ ID NO:9 after the initiation methionine wherein X 2 , X 3 , X 4 , X 5  and X 6  of SEQ ID NO:9 are small uncharged residues and X 6  is not proline. 
     
     
         10 . The method of  claim 1 , wherein the peptide further comprises a transmembrane domain. 
     
     
         11 . The method of  claim 1 , wherein the peptide further comprises at least one positive cluster comprising at least three positively charged amino acid residues within a contiguous sequence of about 25 residues, wherein the contiguous sequence is within about 100 residues flanking the transmembrane domain at the C-terminal side and at least one polyproline motif comprising at least 3 contiguous proline residues within the residues flanking the transmembrane domain at the C-terminal side. 
     
     
         12 . A method for facilitating membrane fusion, comprising contacting the membranes to be fused with an effective amount of a peptide comprising an amino acid sequence which is at least 50% similar to the sequence defined by SEQ ID NO:2 or a fragment thereof. 
     
     
         13 . The method of  claim 12 , wherein the membranes are individually selected from cell membranes, liposome membranes, proteoliposome membranes and cationic lipids. 
     
     
         14 . The method of  claim 12 , wherein the fragment comprises up to 25 contiguous amino acids of the N-terminal region sequence of SEQ ID NO:2. 
     
     
         15 . A method for facilitating membrane fusion, comprising contacting the membranes to be fused with an effective amount of a membrane fusion protein comprising an amino acid sequence which is at least 50% similar to the sequence defined by SEQ ID NO:2, wherein said amino acid sequence comprises an N-terminal myristylation consensus sequence defined by SEQ ID NO:9 after the initiation methionine, wherein X 2 , X 3 , X 4 , X 5  and X 6  of SEQ ID NO:9 are small uncharged residues and X 6  is not proline; a transmembrane domain; at least one positive cluster comprising at least three positively charged amino acid residues within a contiguous sequence of about 25 residues, wherein the contiguous sequence is within about 100 residues flanking the transmembrane domain at the C-terminal side and at least one polyproline motif comprising at least 3 contiguous proline residues within the residues flanking the transmembrane domain at the C-terminal side, or a fragment thereof. 
     
     
         16 . The method of  claim 15 , wherein the membranes are individually selected from cell membranes, liposome membranes, proteoliposome membranes and cationic lipids. 
     
     
         17 . The method of  claim 15 , wherein one membrane is from an immortalized cell and the other membrane is from an antibody-synthesizing cell. 
     
     
         18 . The method of  claim 17 , wherein the immortalized cell is selected from human or mouse B cell myeloma cells, and T cell myelomas. 
     
     
         19 . The method of  claim 17 , wherein the antibody-synthesizing cell is a purified spleen cell from an immunized mammal. 
     
     
         20 . A transfection agent comprising the peptide defined by  claim 1 . 
     
     
         21 . The transfection agent of  claim 20 , further comprising a nucleic acid molecule.