Integrase cofactor

Abstract

In a study of HIV-1 integrase (IN) complexes derived from nuclei of human cells stably expressing the viral protein from a synthetic gene it was demonstrated that in the nuclear extracts IN exists as part of a large distinct complex with apparent Stokes radius of 61 Å, which dissociates upon dilution yielding a core molecule of 41 Å. The IN complexes were isolated from cells expressing FLAG-tagged IN. By present invention it was demonstrated that the 41 Å core is tetramer of IN, whereas 61 Å molecules are composed of IN tetramers associated with a cellular protein with an apparent molecular weight of 76 kDa. This integrase interacting protein (Inip76) was found to be identical to LEDGF/DFS70/p75 a protein implicated in regulation of gene expression and cellular stress-response. HIV-1 IN and Inip76 co-localized in the nuclei of human cells stably expressing IN. Furthermore, it has been demonstrated by present invention that recombinant Inip76 strongly promoted strand-transfer activity of HIV-1 IN in vitro. Our findings reveal that the minimal IN molecule in human cells is a tetramer and clearly demonstrates that Inip76 is plays a role in retroviral integration. Therefore the present invention provides integrase interacting proteins and more particularly cofactors which promote strand transfer activity of viral integrase, more particularly HIV integrase, and methods and uses relating thereto. The present invention relates to a cellular protein that associates with integrase (integrase interacting protein-Inip), to molecules interacting with Inip and their use as an antiviral. The present invention also relates to antibodies, RNA interference, antigen therapy, gene silencing or antisense inhibition of said integrase interacting protein. The novel integrase interaction protein is a target for HIV replication prevention or inhibition.

1 . A complex formed between an LEDGF polypeptide, a variant, analogue or fragment thereof and an HIV-1 integrase polypeptide (IN), a variant, analogue or fragment thereof.  
     
     
         2 . The complex of  claim 1 , wherein said LEDGF polypeptide is Inip76.  
     
     
         3 . A complex between two nucleotides encoding the polypeptide of  claim 1 .  
     
     
         4 . A recombinant host cell containing a polynucleotide encoding an LEGDF polypeptide, a variant, analogue or fragment thereof according to  claim 1 , wherein said polynucleotide is expressed.  
     
     
         5 . The recombinant host cell of  claim 4 , wherein said polynucleotide encodes Inip76.  
     
     
         6 . A polynucleotide encoding a polypeptide selected from the group consisting essentially of Inip76, a variant, analogue or fragment thereof and a polynucleotide which codes for at least a portion of the HIV integrase.  
     
     
         7 . A vector comprising the polynucleotide according to  claim 6 .  
     
     
         8 . A recombinant host cell containing the vector of  claim 7 .  
     
     
         9 . A composition comprising an LEDGF polypeptide, a variant, analogue or fragment thereof and a carrier.  
     
     
         10 . A composition comprising the recombinant host cell of  claim 4  and a carrier.  
     
     
         11 . An antibody that specifically binds the complex of  claim 1  or a functional fragment thereof.  
     
     
         12 . A method for screening molecules for their ability to modulate the activity of a integrase-interacting protein: 
 (a) exposing said molecule to a polypeptide of the hepatoma-derived growth factor family or to a polynucleotide encoding said polypeptide; and    (b) determining whether said molecule specifically binds to a polypeptide of the hepatoma-derived growth factor family or hybridises to a polynucleotide encoding said polypeptide and suppresses the binding of said polypeptide of the hepatoma-derived growth factor family to an HIV-1 integrase.    
     
     
         13 . The method of  claim 12 , wherein said polypeptide of the hepatoma-derived growth factor family is Inip76.  
     
     
         14 . The method according to  claim 12 , wherein said screening occurs in a cell culture.  
     
     
         15 . The method according to  claim 14 , which comprises introducing a vector comprising said polynucleotide encoding Inip76 into said cell culture.  
     
     
         16 . The method according to  claim 14 , wherein said cell culture is a culture of mammalian cells or yeast cells.  
     
     
         17 . The method according to  claim 12 , wherein said binding is detected by fluorescence or radioactive techniques.  
     
     
         18 . The method of  claim 12 , which further comprises monitoring the prevention or suppression of retroviral replication or integration by said compound.