Membrane fusion proteins derived from reovirus

Abstract

In accordance with the present invention, a family of membrane fusion proteins and polynucleotides encoding the proteins have been identified. The proteins and polynucleotides 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 . An isolated protein: 
 (a) which is encoded by the genome of the family Reoviridae;    (b) which is a membrane fusion protein;    (c) which comprises at least one transmembrane domain;    (d) whose amino acid sequence is free of fusion peptide motif I; wherein the fusion peptide motif I: 
 (i) is an amino acid sequence 17 to 28 residues long;  
 (ii) has a hydrophobicity value from 0.6 to 0.7; and  
 (iii) has a content of alanine plus glycine from 29 to 43%; and  
   (e) whose amino acid sequence is free of fusion peptide motif II; wherein the fusion peptide motif II: 
 (i) is an amino acid sequence 16 to 28 residues long;  
 (ii) has a hydrophobicity value from 0.3 to 0.4;  
 (iii) has a content of alanine plus glycine from 29 to 43%; and  
 (iv) contains a heptad repeat.  
   
     
     
         2 . A protein: 
 (a) which is a membrane fusion protein;    (b) whose amino acid sequence is free of fusion peptide motif I; wherein the fusion peptide motif I: 
 (i) is an amino acid sequence 17 to 28 residues long;  
 (ii) has a hydrophobicity value from 0.6 to 0.7; and  
 (iii) has a content of alanine plus glycine from 29 to 43%;  
   (c) whose amino acid sequence is free of fusion peptide motif II; wherein the fusion peptide motif II: 
 (i) is an amino acid sequence 16 to 28 residues long;  
 (ii) has a hydrophobicity value from 0.3 to 0.4;  
 (iii) has a content of alanine plus glycine from 29 to 43%; and  
 (iv) contains a heptad repeat;  
   (d) which comprises an amino acid sequence which has at least 33% identity overall to the protein of  claim 1;  and    (e) which comprises a transmembrane domain whose amino acid sequence has at least 60% amino acid sequence identity to the transmembrane domain of the protein of  claim 1 .    
     
     
         3 . The protein of  claim 1  which is encoded by the genome of a reovirus selected from the genus consisting of Orthoreovirus and Aquareovirus.  
     
     
         4 . The protein of  claim 1  which is encoded by the genome of a reovirus which naturally infects a poikilothermic host.  
     
     
         5 . The protein of any one of  claims 1  to  4  which has an isoelectric point equal to or greater than 7.  
     
     
         6 . The protein of any one of  claims 1  to  5  further comprising a positive cluster; wherein the positive cluster consists of at least three positively charged amino acid residues within a contiguous sequence of at most 25 residues, wherein the contiguous sequence is within at most 100 residues flanking the transmembrane domain at the C-terminal side.  
     
     
         7 . The protein of any one of  claims 1  to  6  further comprising a non-transmembrane domain which comprises a polyproline motif comprising at least 3 contiguous proline residues.  
     
     
         8 . The protein of any one of  claims 1  to  7  further comprising a fatty acylation sequence.  
     
     
         9 . An isolated protein encoded by the genome of the family Reoviridae, said isolated protein being a membrane fusion protein and having at least one transmembrane domain with the proviso that said isolated protein is free of fusion peptide motif I and fusion peptide motif II, said isolated protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 6.  
     
     
         10 . An isolated protein comprising an amino acid sequence of SEQ ID NO: 2.  
     
     
         11 . An isolated protein comprising an amino acid sequence of SEQ ID NO: 6.  
     
     
         12 . A protein: 
 (a) which is a membrane fusion protein;    (b) whose amino acid sequence is free of fusion peptide motif I; wherein the fusion peptide motif I: 
 (i) is an amino acid sequence 17 to 28 residues long;  
 (ii) has a hydrophobicity value from 0.6 to 0.7; and  
 (iii) has a content of alanine plus glycine from 29 to 43%;  
   (c) whose amino acid sequence is free of fusion peptide motif II; wherein the fusion peptide motif II: 
 (i) is an amino acid sequence 16 to 28 residues long;  
 (ii) has a hydrophobicity value from 0.3 to 0.4;  
 (iii) has a content of alanine plus glycine from 29 to 43%; and  
 (iv) contains a heptad repeat;  
   (d) which comprises an amino acid sequence which has at least 33% identity overall to SEQ ID NO: 2; and    (e) which comprises a transmembrane domain whose amino acid sequence has at least 60% amino acid sequence identity to the sequence from amino acid 39 to 57 of SEQ ID NO: 2.    
     
     
         13 . A protein: 
 (a) which is a membrane fusion protein;    (b) whose amino acid sequence is free of fusion peptide motif I; wherein the fusion peptide motif I: 
 (i) is an amino acid sequence 17 to 28 residues long;  
 (ii) has a hydrophobicity value from 0.6 to 0.7; and  
 (iii) has a content of alanine plus glycine from 29 to 43%;  
   (c) whose amino acid sequence is free of fusion peptide motif II; wherein the fusion peptide motif II: 
 (i) is an amino acid sequence 16 to 28 residues long;  
 (ii) has a hydrophobicity value from 0.3 to 0.4;  
 (iii) has a content of alanine plus glycine from 29 to 43%; and  
 (iv) contains a heptad repeat;  
   (d) which comprises an amino acid sequence which has at least 33% identity overall to SEQ ID NO: 6; and    (e) which comprises a transmembrane domain whose amino acid sequence has at least 60% amino acid sequence identity to the sequence 5′ WAIPPLAICCCCCICCTGGLYLV 3′ of SEQ ID NO: 6, or has at least 60% amino acid sequence identity to the sequence 5′ YIALASVILVTLVISLIWNCLGTGLIL 3′ of SEQ ID NO: 6.    
     
     
         14 . The protein of  claim 12  or  13  further comprising a positive cluster; wherein the positive cluster consists of at least three positively charged amino acid residues within a contiguous sequence of at most 25 residues, wherein the contiguous sequence is within at most 100 residues flanking the transmembrane domain at the C-terminal side.  
     
     
         15 . The protein of  claim 12  further comprising a non-transmembrane domain which comprises a polyproline motif comprising at least 3 contiguous proline residues.  
     
     
         16 . The protein of  claim 12  or  15  further comprising a fatty acylation sequence.  
     
     
         17 . The protein of  claim 16  wherein the fatty acylation sequence is the myristylation consensus sequence, wherein the consensus sequence is: 
 (initiator Met removed) Gly1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-; where: 
 AA2,AA3,AA4,AA5,AA6 are small uncharged residues; and  
 AA6 is not proline.  
 
 
     
     
         18 . The protein of  claim 17  wherein AA5 is serine or threonine.  
     
     
         19 . An isolated polynucleotide encoding the protein according to any one of  claims 1  to  15 .  
     
     
         20 . An isolated polynucleotide comprising SEQ ID NO: 1 or SEQ ID NO: 5, or degenerate variations thereof which encode the same amino acid sequences, or splice variant nucleotide sequences thereof.  
     
     
         21 . An isolated polynucleotide according to  claim 19  operatively associated with a promoter.  
     
     
         22 . The polynucleotide of  claim 21  wherein the promoter is inducible.  
     
     
         23 . A cell containing the polynucleotide according to any one of  claims 19  to  22 .  
     
     
         24 . The cell of  claim 23  wherein the protein of any one of  claims 1  to  15  is expressed from the isolated polynucleotide.  
     
     
         25 . A liposome containing the protein according to any one of  claims 1  to  15 .  
     
     
         26 . A liposome containing the polynucleotide according to any one of  claims 19  to  22 .  
     
     
         27 . An antibody against the protein according to any one of  claims 1  to  15 .  
     
     
         28 . A method to promote fusion between two or more membranes, comprising contacting the membranes to be fused with an effective amount of the protein according to any one of  claims 1  to  15 .  
     
     
         29 . The method according to  claim 28  wherein the membranes are selected from the group consisting of cell membranes, liposome membranes and proteoliposome membranes.  
     
     
         30 . The method according to  claim 28  wherein the membranes are membranes of cells, and wherein heterokaryons are produced.  
     
     
         31 . The method according to  claim 28  wherein the membranes are of an immortalized cell and a primary B cell or T cell, for producing a hybridoma cell.  
     
     
         32 . A method according to  claim 31  wherein the hybridoma cell produces a substance selected from: monoclonal antibodies, cytokines and immune modulators.  
     
     
         33 . The method according to  claim 32  wherein the hybridoma cells produce monoclonal antibodies, and wherein the membranes are of an immortalized cell and an antibody-synthesizing cell.  
     
     
         34 . A method according to  claim 33  wherein said immortalized cell is a human or mouse B cell myeloma cell or a T cell myeloma cell, and wherein said antibody-synthesizing cell is an isolated spleen cell from an immunized mammal.  
     
     
         35 . A method according to  claim 28 , wherein the membranes are selected from the group consisting of: liposome membranes, liposome membrane and cell membrane, and proteoliposome membrane and cell membrane.  
     
     
         36 . The method according to  claim 35  wherein the liposome or proteoliposome contains a bioactive drug.