Structural determinants of the anti-HIV activity of a CCR5 antagonist derived from Toxoplasma gondii.
The protozoan parasite Toxoplasma gondii possesses a protein, cyclophilin-18 (C-18), which binds to the chemokine receptor CCR5, induces interleukin-12 production from murine dendritic cells, and inhibits fusion and infectivity of human immunodeficiency virus 1 (HIV-1) R5 viruses by co-receptor antagonism. Site-directed mutagenesis was employed to identify the domains in C-18 responsible for its CCR5 binding and antiviral functions. To do so we focused on amino acid differences with Plasmodium falciparum cyclophilin, which, although 53% identical with C-18, has minimal binding activity for CCR5, and we generated 22 mutants with substitutions in the regions of non-homology located on the putative surface of the molecule. Two mutations situated on the face of C-18, predicted to be involved in its interaction with the ligand cyclosporin A, were shown to be critical for CCR5-binding and the inhibition of HIV-1 fusion and infectivity. In contrast, four mutations in C-18 specifically designed to abolish the peptidyl-prolyl cis-trans-isomerase activity of the protein failed to inactivate its CCR5 binding and HIV inhibitory activities. Interleukin-12 induction by C-18, on the other hand, was abrogated by mutations effecting either the CCR5 binding or enzymatic function of the molecule. These findings shed light on the structural basis of the molecular mimicry of the chemokine function by a pathogen-derived protein and provide a basis for further modification of C-18 into an antiviral agent.
Immunobiology Section, Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Felix
F
Yarovinsky
John F
JF
Andersen
Lisa R
LR
King
Patricia
P
Caspar
Julio
J
Aliberti
Hana
H
Golding
Alan
A
Sher
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001667:Binding, Competitive; D065100:CCR5 Receptor Antagonists; D021983:Cyclophilins / Q000737:chemistry / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D006678:HIV / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D010963:Plasmodium falciparum / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013154:Spleen / Q000378:metabolism; D014122:Toxoplasma / Q000378:metabolism*; D014616:Vaccinia virus / Q000378:metabolism
D000998:Antiviral Agents; D065100:CCR5 Receptor Antagonists; D008024:Ligands; D011994:Recombinant Proteins; C437224:cyclophilin hCyp-18; D018664:Interleukin-12; D016572:Cyclosporine; D021983:Cyclophilins