Structure of adenovirus bound to cellular receptor car

Abstract

The present invention is based on the solving of the crystal structure of adenovirus fiber protein knob domain bound to domain 1 of the coxsackie-adenovirus receptor. One aspect of the present invention relates to a mutant adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain, the viral particle encoded by the genome being characterized by a significantly weakened binding affinity for CARD1 relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Another aspect of the present invention is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type., from an adenovirus which binds CARD1. In the method, residues of the adenovirus fiber protein knob domain which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus, and whether or not the mutant produced exhibits altered adenovirus-CAR binding properties is determined. Mutants produced by this method include those which under physiological conditions, have significantly weakened binding affinity for CARD1 relative to wild type adenovirus and those which bind a receptor other than CARD1, including an engineered receptor. Introduced mutations may produce an amino acid insertion, deletion or substitution in the encoded viral particle, and may serve to alter the conformation of one or more residues of knob which participate directly in D1 binding. Such residues include residues of the AB loop, the CD loop, the DE loop, the FG loop, the E strand and the F strand. Alternatively, the mutation may be directly introduced in a codon encoding the residue of knob which participates directly in D1 binding. Specific residues in the AB loop, the CD loop, the FG loop, the E strand, the F strand, and the DE loop which participate directly in binding are identified. Another aspect of the present invention is a method for identifying an inhibitor of adenovirus binding to CAR. In the method, a three-dimensional structure derived by X-ray diffraction from a crystal of adenovirus knob trimer bound to CARD1 is provided and then employed to design or select a potential inhibitor. The potential inhibitor is synthesized and then whether or not the potential inhibitor inhibits adenovirus binding to CAR is determined. Preferred crystal structures and space group symmetry is listed. A set of atomic coordinates which define the three dimensional structure is provided. The potential inhibitor may be designed to interact non-covalently with one or more residues of the adenovirus fiber knob protein domain. Alternatively, the potential inhibitor is designed to interact non-covalently with one or more residues of CARD1. Specific residues for covalent and non-covalent interaction are listed. The potential inhibitor may also be designed to interact non-covalently with residues which line a cavity formed during adenovirus knob trimer/CARD1 binding. The potential inhibitor can be designed by identifying chemical entities or fragments capable of associating with the adenovirus knob trimer, and assembling the identified chemical entities or fragments into a single molecule to provide the structure of said potential inhibitor. Such an inhibitor may be designed de novo or from a known inhibitor. Methods of inhibition include competitive inhibition, non-competitive inhibition and uncompetitive inhibition.

1 . A mutant adenovirus having a genome comprising one or more mutations in sequences which encode the AB loop of the fiber protein knob domain, the viral particle encoded by the genome being characterized by a significantly weakened binding affinity for CARD1 relative to wild-type adenovirus.  
     
     
         2 . The mutant adenovirus of  claim 1  which is adenovirus serotype 2.  
     
     
         3 . The mutant adenovirus of  claim 1  which is adenovirus serotype 5.  
     
     
         4 . The mutant adenovirus of  claim 1  wherein the mutation results in an amino acid substitution at one or both positions which correspond to residue 417 and 418 of Adenovirus serotype 12 in the encoded viral particle.  
     
     
         5 . The mutant adenovirus of  claim 4  wherein the mutation results in amino acid substitutions which correspond to P417E and P418A of Adenovirus serotype 12 in the encoded viral particle.  
     
     
         6 . The mutant adenovirus of  claim 1  wherein the mutation results in an insertion of threonine and isoleucine between residues which correspond to residue 421 and 422 of Adenovirus serotype 12 in the encoded viral particle.  
     
     
         7 . The mutant adenovirus of  claim 1  wherein the mutation results in a deletion of residues which correspond to E425 and L426 of Adenovirus serotype 12 in the encoded viral particle.  
     
     
         8 . A mutant adenovirus having a genome comprising one or more mutations in sequences which encode the HI loop of the fiber protein knob domain, the viral particle encoded by the genome being characterized by a significantly weakened binding affinity for CARD1.  
     
     
         9 . The mutant adenovirus of  claim 8  which is Adenovirus Serotype 2.  
     
     
         10 . The mutant adenovirus of  claim 8  which is Adenovirus Serotype 5.  
     
     
         11 . The mutant adenovirus of  claim 8  wherein the mutation results in a deletion of amino acids which correspond to amino acids G550 and I551 of Adenovirus serotype 12 of the encoded viral particle.  
     
     
         12 . A method for generating a mutant adenovirus, the mutant being characterized by a receptor binding affinity or specificity which differs substantially from wild type, comprising: 
 a) providing an adenovirus which binds CARD1;    b) identifying residues of the adenovirus fiber protein knob domain which when mutated are predicted to alter D1 binding from the crystal structure coordinates of the AD12knob:CAR-D1 complex;    c) introducing a mutation into the genome of the adenovirus which alters one or more of the residues identified in step b); and    d) determining that the mutant produced exhibits altered adenovirus-CAR binding properties.    
     
     
         13 . The method of  claim 12  wherein the mutant adenovirus generated has significantly weakened binding affinity for CARD1 relative to wild type adenovirus under physiological conditions.  
     
     
         14 . The method of  claim 12  wherein the mutant adenovirus generated binds a receptor other than CARD1.  
     
     
         15 . The method of  claim 14  wherein the receptor to which the mutant binds is an engineered receptor.  
     
     
         16 . The method of  claim 12  wherein the introduced mutation results in an amino acid substitution, an amino acid deletion, or an amino acid insertion in the encoded viral particle.  
     
     
         17 . The method of  claim 16  wherein the introduced mutation serves to alter the conformation of one or more residues of knob which participate directly in D1 binding.  
     
     
         18 . The method of  claim 17  wherein the residue which participates directly in D1 binding is located in a region of knob selected from the group consisting of the AB loop, the CD loop, the DE loop, the FG loop, the E strand and the F strand.  
     
     
         19 . The method of  claim 16  wherein the mutation is introduced in a codon encoding the residue of knob which participates directly in D1 binding.  
     
     
         20 . The method of  claim 19  wherein the residue of knob which directly participates in D1 binding is in the AB loop.  
     
     
         21 . The method of  claim 20  wherein the mutation is introduced at the codon for the residue which corresponds to the Ad12 residue selected from the group consisting of 409, 415, 417, 418, 419, 426, and 429.  
     
     
         22 . The method of  claim 19  wherein the residue which directly participates in D1 binding is in the CD loop.  
     
     
         23 . The method of  claim 22  wherein the mutation is introduced at the codon for the residue which corresponds to the Ad12 residue selected from the group consisting of 450 and 451.  
     
     
         24 . The method of  claim 19  wherein the residue which directly participates in D1 binding is in the FG loop of knob.  
     
     
         25 . The method of  claim 24  wherein the mutation is introduced at the codon for the residue which corresponds to the Ad12 residue selected from the group consisting of 517, 519, 520 and 523.  
     
     
         26 . The method of  claim 19  wherein the residue which directly participates in D1 binding is in the E strand of knob.  
     
     
         27 . The method of  claim 26  wherein the mutation is introduced at the codon which encodes the residue corresponding to residue 494 of Adenovirus serotype 12.  
     
     
         28 . The method of  claim 19  wherein the residue which directly participates in D1 binding is in the F strand of knob.  
     
     
         29 . The method of  claim 28  wherein the mutation is introduced at the codon which encodes a residue corresponding to residues 497 and 498 of Adenovirus serotype 12.  
     
     
         30 . The method of  claim 19  wherein the residue which directly participates in D1 binding is in the DE loop of knob.  
     
     
         31 . The method of  claim 30  wherein the mutation is introduced at the codon encoding the residue corresponding to residue 487 of Adenovirus serotype 12.  
     
     
         32 . A method for identifying an inhibitor of adenovirus binding to CAR, comprising: 
 a) providing a three-dimensional structure derived by X-ray diffraction from a crystal of adenovirus knob trimer bound to CARD1;    b) employing the three-dimensional structure to design or select a potential inhibitor;    c) synthesizing the potential inhibitor; and    d) determining whether the potential inhibitor inhibits adenovirus binding to CAR.    
     
     
         33 . The method of  claim 32  wherein the crystal of the Ad12knob:CARD1 complex has P4 3 32 space group symmetry with a cubic unit cell with 167.85 angstroms per side.  
     
     
         34 . The method of  claim 33  wherein the three dimensional structure is defined by atomic coordinates listed in the Protein Data Bank under code 1KAC.  
     
     
         35 . The method of  claim 32  wherein the atomic coordinates of the Ad12knob:CARD1 complex are obtained by means of computational analysis.  
     
     
         36 . The method of  claim 32  wherein the potential inhibitor is designed to interact non-covalently with one or more residues of the adenovirus fiber knob protein domain.  
     
     
         37 . The method of  claim 36  wherein the adenovirus fiber knob protein domain residues correspond to Ad12 residues selected from the group consisting of D415, P417, P418, I426, V450, K451, Q487, Q494, S497, V498, P517, P519, N520, and E523.  
     
     
         38 . The method of  claim 32  wherein the potential inhibitor is designed to interact non-covalently with one or more residues of CARD1.  
     
     
         39 . The method of  claim 38  wherein the residues of CARD1 are selected from the group consisting of P33, D37, L39, V48, D49, V51, L54, S56, Y61, E62, E63, Y64, K102, K104, A106 and P107.  
     
     
         40 . The method of  claim 32  wherein the potential inhibitor is designed to interact non-covalently with residues which line a cavity formed during adenovirus knob trimer/CARD1 binding.  
     
     
         41 . The method of  claim 32  wherein the potential inhibitor is designed by identifying chemical entities or fragments capable of associating with the adenovirus knob trimer, and assembling the identified chemical entities or fragments into a single molecule to provide the structure of said potential inhibitor.  
     
     
         42 . The method of  claim 41  wherein the potential inhibitor is designed de novo.  
     
     
         43 . The method of  claim 41  wherein the potential inhibitor is designed from a known inhibitor.  
     
     
         44 . The method of  claim 32  wherein the potential inhibitor is a competitive inhibitor of adenovirus-CAR binding.  
     
     
         45 . The method of  claim 32  wherein the potential inhibitor is a non-competitive or uncompetitive inhibitor of adenovirus-CAR binding.