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Fulltext Broad-Spectrum Antiviral Activity of an Ankyrin Repeat Protein on Viral Assembly against Chimeric NL4-3 Viruses Carrying Gag/PR Derived from Circulating Strains among Northern Thai Patients

Sakkhachornphop S, Hadpech S, Wisitponchai T, Panto C, Kantamala D, Utaipat U, et al.

Viruses, 2018 11 13;10(11).
PMID: 30428529 DOI: 10.3390/v10110625

Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, AnkGAG1D4, showed a negative effect on the viral assembly of the HIV-1NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of AnkGAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-naïve HIV-1 infected individuals in northern Thailand during 2001⁻2012. SupT1, a stable T-cell line expressing AnkGAG1D4 and ankyrin non-binding control (AnkA32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/AnkGAG1D4 demonstrated significantly lower levels of p24CA than SupT1/AnkA32D3, which was found to correlate with the syncytia formation. This result suggests that AnkGAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.
Fulltext Deciphering critical amino acid residues to modify and enhance the binding affinity of ankyrin scaffold specific to capsid protein of human immunodeficiency virus type 1

Saoin S, Wisitponchai T, Intachai K, Chupradit K, Moonmuang S, Nangola S, et al.

Asian Pac J Allergy Immunol, 2018 06;36(2):126-135.
PMID: 28802032 DOI: 10.12932/AP-280217-0037

BACKGROUND: AnkGAG1D4 is an artificial ankyrin repeat protein which recognizes the capsid protein (CA) of the human immunodeficiency virus type 1 (HIV-1) and exhibits the intracellular antiviral activity on the viral assembly process. Improving the binding affinity of AnkGAG1D4 would potentially enhance the AnkGAG1D4-mediated antiviral activity.
OBJECTIVE: To augment the affinity of AnkGAG1D4 scaffold towards its CA target, through computational predictions and experimental designs.
METHOD: Three dimensional structure of the binary complex formed by AnkGAG1D4 docked to the CA was used as a model for van der Waals (vdW) binding energy calculation. The results generated a simple guideline to select the amino acids for modifications. Following the predictions, modified AnkGAG1D4 proteins were produced and further evaluated for their CA-binding activity, using ELISA-modified method and bio-layer interferometry (BLI).
RESULTS: Tyrosine at position 56 (Y56) in AnkGAG1D4 was experimentally identified as the most critical residue for CA binding. Rational substitutions of this residue diminished the binding affinity. However, vdW calculation preconized to substitute serine for tyrosine at position 45. Remarkably, the affinity for the viral CA was significantly enhanced in AnkGAG1D4-S45Y mutant, with no alteration of the target specificity.
CONCLUSIONS: The S-to-Y mutation at position 45, based on the prediction of interacting amino acids and on vdW binding energy calculation, resulted in a significant enhancement of the affinity of AnkGAG1D4 ankyrin for its CA target. AnkGAG1D4-S45Y mutant represented the starting point for further construction of variants with even higher affinity towards the viral CA, and higher therapeutic potential in the future.

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