title: Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein.
abstract: BACKGROUND
  Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1.
  
  
  RESULTS
  A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank(GAG)1D4 (16.5 kDa) was isolated. Ank(GAG)1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K(d) ~ 1 μM, and the Ank(GAG)1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank(GAG)1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank(GAG)1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank(GAG)1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank(GAG)1D4-CA with the Gag assembly and budding pathway.
  
  
  CONCLUSIONS
  The resistance of Ank(GAG)1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank(GAG)1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules.
authors.affiliation: Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
authors.forename: Sawitree
authors.initials: S
authors.lastname: Nangola
authors.forename: Agathe
authors.initials: A
authors.lastname: Urvoas
authors.forename: Marie
authors.initials: M
authors.lastname: Valerio-Lepiniec
authors.forename: Wannisa
authors.initials: W
authors.lastname: Khamaikawin
authors.forename: Supachai
authors.initials: S
authors.lastname: Sakkhachornphop
authors.forename: Saw-See
authors.initials: SS
authors.lastname: Hong
authors.forename: Pierre
authors.initials: P
authors.lastname: Boulanger
authors.forename: Philippe
authors.initials: P
authors.lastname: Minard
authors.forename: Chatchai
authors.initials: C
authors.lastname: Tayapiwatana
mesh_terms: D000595:Amino Acid Sequence; D017487:Ankyrins / Q000494:pharmacology*; D019380:Anti-HIV Agents / Q000494:pharmacology*; D002460:Cell Line; D015497:HIV-1 / Q000187:drug effects* / Q000254:growth & development; D006801:Humans; D009052:Leukemia Virus, Murine / Q000187:drug effects / Q000254:growth & development; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000494:pharmacology; D019065:Virus Assembly / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors*
chemical_list: D017487:Ankyrins; D019380:Anti-HIV Agents; D011994:Recombinant Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus