Vif as a target for hiv inhibition

Abstract

The present invention relates, e.g., to a method for inhibiting infectivity of a lentivirus (e.g., a lentivirus which expresses a Viral infectivity factor (Vif) protein), such as, e.g., SIV, SHIV and/or HIV, comprising contacting a cell which is producing the virus with an antiviral-effective amount of a membrane-permeable Zinc (Zn) chelator, wherein the antiviral-effective amount of the Zn chelator does not substantially inhibit proteins in the cell which contain Zn-binding motifs other than lentivirus Vif. Kits and pharmaceutical compositions are also disclosed, as is a method for identifying inhibitors of lentiviruses that target a specific zinc-binding motif of the lentivirus Vif protein.

1 . A method for inhibiting infectivity of a lentivirus, comprising contacting a cell which is producing the virus with an antiviral-effective amount of a membrane-permeable Zinc (Zn) chelator, wherein the antiviral-effective amount of the Zn chelator does not substantially inhibit proteins in the cell which contain Zn-binding motifs other than lentivirus Viral infectivity factor (Vif). 
     
     
         2 . The method of  claim 1 , wherein the lentivirus expresses Vif. 
     
     
         3 . The method of  claim 1 , wherein the membrane-permeable Zn chelator is N,N,N′,N′-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). 
     
     
         4 . The method of  claim 3 , wherein the antiviral-effective amount of TPEN is effectively between about 4 and about 12 μM. 
     
     
         5 . The method of  claim 3 , wherein the antiviral-effective amount of TPEN is effectively between about 4 and about 9 μM. 
     
     
         6 . The method of  claim 3 , wherein the antiviral-effective amount of TPEN is effectively about 7 μM. 
     
     
         7 . The method of  claim 2 , wherein the conditions are such that the membrane-permeable Zn chelator inhibits the interaction of the Vif protein with cellular Cullin 5-E3 ubiquitin ligase, thereby preventing the degradation of the viral inhibitor, Apobec3G, and thus allowing the Apobec3G to carry out its viral inhibiting function. 
     
     
         8 . The method of  claim 2 , wherein the membrane-permeable Zn chelator interacts with the Zn-stabilized domain/motif H— x2 —YF— x —CF- x4 -Φ- x2 -AΦ- x7-8 —C— x3-5 —H (SEQ ID NO:1) in the Vif protein. 
     
     
         9 . The method of  claim 1 , which is carried out in cell culture. 
     
     
         10 . The method of  claim 1 , which is carried out in a subject. 
     
     
         11 . The method  claim 1 , wherein the lentivirus is SIV, SHIV, or HIV. 
     
     
         12 . The method of  claim 1 , wherein the lentivirus which is inhibited is resistant to an inhibitor of reverse transcriptase, protease, integrase, entry of the virus into a cell, and/or assembly/maturation of the virus. 
     
     
         13 . A method for inhibiting Vif protein activity in a cell, comprising contacting the protein with an inhibitory-effective amount of a membrane-permeable Zn chelator, wherein the inhibitory-effective amount of the Zn chelator does not substantially inhibit proteins in the cell which contain Zn-binding motifs other than Vif. 
     
     
         14 . A method for treating lentiviral infection in a subject, comprising administering to the subject an antiviral-effective amount of a membrane-permeable Zinc (Zn) chelator, wherein the antiviral-effective amount of the Zn chelator does not substantially inhibit proteins in the cell which contain Zn-binding motifs other than Vif. 
     
     
         15 . The method of  claim 14 , wherein the membrane-permeable Zn chelator is TPEN, and antiviral-effective amount of the chelator is between about 0.5 mg/kg and about 30 mg/kg. 
     
     
         16 . The method of  claim 14 , wherein the membrane-permeable Zn chelator is TPEN, and antiviral-effective amount of the chelator is between about between about 0.5 mg/kg and about 10 mg/kg. 
     
     
         17 . A kit for treating a lentiviral infection, comprising a single dosage unit which comprises an antiviral-effective amount of a membrane-permeable Zn chelator, wherein the amount of the membrane-permeable Zn chelator is not sufficient to substantially inhibit proteins in the cell which contain Zn-binding motifs other than Vif, optionally packaged in a container. 
     
     
         18 . A method for identifying an agent that inhibits the infectivity of a lentivirus, comprising
 (a) contacting a putative inhibitory agent with a peptide comprising the Vif Zn-binding motif represented by SEQ ID NO:1 (Hx (2) YFxCFx (4) Φx (2) AΦx (7-8) Cx (3-5) H), in the presence of Cul5, under conditions that are effective for specific binding of the putative agent and the peptide; and   (b) detecting whether the agent disrupts the binding of the peptide to Cul5, wherein an agent that significantly disrupts the binding is expected to inhibit lentiviral infectivity.   
     
     
         19 . The method of  claim 18 , which is a high throughput method. 
     
     
         20 . A kit for identifying an agent that acts as an inhibitor of lentivirus infectivity, comprising
 a. a peptide comprising a Zn-binding motif as represented by SEQ ID NO:1;   b. Cul5; and c. one or more reagents for detecting whether the peptide binds to the Cul5,   wherein, optionally, a, b and/or c are packaged in a container.   
     
     
         21 . A complex comprising a membrane-permeable zinc chelator and a Vif protein. 
     
     
         22 . A method for using a membrane-permeable Zn chelator as a virucidal agent, comprising contacting the chelator with a sample containing, or suspected of containing, a lentivirus. 
     
     
         23 . The method of  claim 22 , which is a method for treating ex vivo a bodily fluid or tissue, comprising contacting the bodily fluid or tissue with a viral inhibitory effective amount of a membrane permeable Zn chelator. 
     
     
         24 . The method of  claim 22 , which is a method for inhibiting contamination of an inanimate object, such as surgical or medical tubing or laboratory supplies, by a lentivirus, comprising contacting the inanimate object with an antiviral effective amount of a membrane permeable Zn chelator. 
     
     
         25 . The method of  claim 22 , which is a method for inhibiting sexual transmission of infection by a lentivirus, comprising vaginal, rectal, oral, penile, or other topical, insertional or instillational treatment with an antiviral effective amount of a membrane-permeable Zn chelator of the invention. 
     
     
         26 . A pharmaceutical composition comprising an amount of a membrane-permeable Zn chelator TPEN that is effective to inhibit lentiviral infection, but that does not inhibit other Zn-finger containing proteins, and a pharmaceutically acceptable carrier. 
     
     
         27 . The pharmaceutical composition of  claim 26 , wherein the membrane-permeable Zn chelator is TPEN, and the anti lentiviral-effective amount of the TPEN is effectively between about 4 and about 12 μM.