Structure of the hepatitis C NS5A protein
The present invention provides a crystallized N-terminal domain of an NS5A protein of hepatitis C virus, methods of producing the same and methods of use thereof. The present invention also relates to structural elements of the N-terminal domain of hepatitis C virus NS5A protein, and methods of inhibiting hepatitis C virus infection, replication and/or pathogenesis, by interacting with the same.
1. A method of identifying a Hepatitis C virus (HCV) inhibitor in an inhibitor screening assay comprising:
(a) determining the three-dimensional structure of the N-terminal domain I-Δh of an NS5A protein crystal, wherein said NS5A N-terminal domain I-Δh protein comprises a protein with at least 90% identity to amino acids 36-199 of SEQ ID NO: 4 and wherein said crystal has space group P4 1 22 with unit cell dimensions of a=b=55.28Å, c=312.30 Å and α,β,γ=90°,
(b) selecting or designing a potential inhibitor by performing rational drug design with said three-dimensional structure determined in part (a) wherein said selecting or designing is performed in conjunction with computer modeling;
(c) contacting said potential inhibitor with an N-terminal domain of an NS5A protein of hepatitis C virus; and
(d) detecting an ability of said potential inhibitor for inhibiting infection or replication of a hepatitis C virus.
2. The method of claim 1 , wherein said inhibitor interferes with zinc binding to said N-terminal domain of an NS5A protein.
3. The method of claim 1 , wherein said inhibitor interferes with disulfide bridge formation between cysteine residues at positions 142 and 190 of said N-terminal domain of an NS5A protein.
4. The method of claim 1 , wherein said inhibitor interferes with dimerization of NS5A proteins of said virus.
5. The method of claim 1 , wherein said inhibitor interferes with the formation of a groove between two subdomain IB regions of N-terminal domains of dimerized NS5A.
6. The method of claim 1 , wherein said inhibitor is positioned within a groove formed between two subdomain IB regions of N-terminal domains of dimerized NS5A.
7. The method of claim 1 , wherein said inhibitor interacts with a binding site for zinc comprising a cysteine residue at positions 39, 57, 59, 80 or combinations thereof.
8. The method of claim 1 , wherein said ability for inhibiting infection or replication of a hepatitis C virus is assayed in vivo or in vitro.