Abstract
BackgroundAnkyrins 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.ResultsA 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 AnkGAG1D4 (16.5 kDa) was isolated. AnkGAG1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of Kd ~ 1 μM, and the AnkGAG1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing AnkGAG1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. AnkGAG1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The AnkGAG1D4-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 AnkGAG1D4-CA with the Gag assembly and budding pathway.ConclusionsThe resistance of AnkGAG1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin AnkGAG1D4 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.
Highlights
Ankyrins are cellular mediators of a number of essential protein-protein interactions
This study demonstrated the potential of ankyrin-repeat proteins as a novel class of intracellular antivirals and suggested that AnkGAG1D4 could serve as a protein platform for the design of efficient intracellular inhibitors of HIV-1 assembly
Design of artificial ankyrin-repeat motifs and construction of an ankyrin library The construction of an artificial ankyrin library implies the randomization of amino acid residues localized on the accessible surface of ankyrin which has a potential interaction with the desired target, while maintaining intact the conserved residues of the consensus repeat modules which form the rigid framework of ankyrin
Summary
Ankyrins are cellular mediators of a number of essential protein-protein interactions. Several strategies for anti-HIV gene therapy are currently under development, and certain ones have been tested in hematopoietic cells [6,7,8]. They can be classified into two major categories: (i) RNA-based agents including antisense, ribozymes, aptamers and RNA interference [9]; (ii) protein-based agents including dominant-negative mutant proteins, intrabodies, intrakines, fusion inhibitors and zinc-finger nucleases [10,11]. The complex structure of these molecules limits their antiviral function within cells, since their stability relies on disulfide bond(s) which rarely occur(s) in the reducing conditions of the intracellular milieu [12,13,14,15,16]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
