Enhancement of Ankyrin scaffold binding avidity against HIV-1 Capsid by molecular dimerization strategy

Abstract

Antiretroviral therapy (ART) regiment is the most common cure for human immunodeficiency virus type 1 (HIV-1). Although it suppresses the level of HIV-1 to undetectable viral load, ART is not effective eliminate HIV-1 viral reservoirs. Several studies have reported the possibility of side effects and potential drug resistance. Hence, alternative treatment has been vastly developed, especially gene therapy. Late stage of HIV-1 life cycle i.e.. assembly and budding relies on the polymerization of Gag protein at the inner leaf of plasma membrane. Earlier study demonstrated that CAp24-specific Ank1D4 reduced viral production. However, Ank1D4 cannot completely interfere assembly and budding processes. Eventually, binding activity of monomeric Ank1 D4 is improved by generating dimeric AnklD4 via a flexible (G4S)4 linker. Two orientations of dimers consist of C-terminus of the first monomer module linked with the N-terminus of second monomer (AnklD4NC-NC) or its inverted form (AnklD4NC-NC). Dimeric AnklD4NC-CN and AnklD4Nc-Nc have similar capsid binding characteristics to monomers proven by the competitive ELISA. The recognition of AnklD4NC-CN with CAp24 is significantly greater than that of Ank1D4NC-NC and the original AnklD4 and exhibit bifunctional property for capture and sandwich ELISA, respectively. Interestingly, the binding kinetic of Ank1D4NC-CN prominently enhances binding efficiency determined by BLI. The Ank1D4NC-CN recognized CAp24 with Kp of 3.5 nM, making it 36-fold more potent than parental Ank1D4 (Ko of 126.2 nM). Dual binding sites are investigated by molecular dynamics simulation. It illustrated that Ank1D4NC-CN is more favorable in capturing CAp24 as 1:2 interaction than that of Ank1D4Nc-Nc. This phenomenon is according to the longer distance of Ank1D4xc-cN interactive domains, which increases the possibility to interact with two CAp24 molecules at the same time. Circular dichroism was used to confirm that alpha-helical retains in dimer forms as comparable to parental Ank1D4. Furthermore, HEK293T cells stably expressing Myr (+) dimeric Ank1D4 mCherry showed antiviral effect to pseudotyped HIV-1. This study is the first report to experimentally demonstrate the possibility to generate active dimer by reversing peptide sequence of ankyrin scaffold. In addition, the significant enhancement of AnkID4NC-CN binding activity in avidity format will be an alternative scaffold for applying in stem cell- based therapy.