Dimeric Ankyrin with Inverted Module Promotes Bifunctional Property in Capturing Capsid to Impede HIV-1 Replication.

Several anti-HIV scaffolds have been proposed as complementary treatments to highly active antiretroviral therapy. AnkGAG1D4, a designed ankyrin repeat protein, formerly demonstrated anti-HIV-1 replication by interfering with HIV-1 Gag polymerization. However, the improvement of the effectiveness was considered. Recently, the dimeric molecules of AnkGAG1D4 were accomplished in enhancing the binding activity against HIV-1 capsid (CAp24). In this study, the interaction of CAp24 against the dimer conformations was elucidated to elaborate the bifunctional property. The accessibility of the ankyrin binding domains was inspected by bio-layer interferometry. By inverting the second module of dimeric ankyrin (AnkGAG1D4NC-CN), the CAp24 interaction KD was significantly reduced. This reflects the capability of AnkGAG1D4NC-CN in simultaneously capturing CAp24. On the contrary, the binding activity of dimeric AnkGAG1D4NC-NC was indistinguishable from the monomeric AnkGAG1D4. The bifunctional property of AnkGAG1D4NC-CN was subsequently confirmed in the secondary reaction with additional p17p24. This data correlates with the MD simulation, which suggested the flexibility of the AnkGAG1D4NC-CN structure. The CAp24 capturing capacity was influenced by the distance of the AnkGAG1D4 binding domains to introduce the avidity mode of AnkGAG1D4NC-CN. Consequently, AnkGAG1D4NC-CN showed superior potency in interfering with HIV-1 NL4-3 WT and HIV-1 NL4-3 MIRCAI201V replication than AnkGAG1D4NC-NC and an affinity improved AnkGAG1D4-S45Y.

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.

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.

AnkPlex: algorithmic structure for refinement of near-native ankyrin-protein docking.

BACKGROUND: Computational analysis of protein-protein interaction provided the crucial information to increase the binding affinity without a change in basic conformation. Several docking programs were used to predict the near-native poses of the protein-protein complex in 10 top-rankings. The universal criteria for discriminating the near-native pose are not available since there are several classes of recognition protein. Currently, the explicit criteria for identifying the near-native pose of ankyrin-protein complexes (APKs) have not been reported yet. RESULTS: In this study, we established an ensemble computational model for discriminating the near-native docking pose of APKs named "AnkPlex". A dataset of APKs was generated from seven X-ray APKs, which consisted of 3 internal domains, using the reliable docking tool ZDOCK. The dataset was composed of 669 and 44,334 near-native and non-near-native poses, respectively, and it was used to generate eleven informative features. Subsequently, a re-scoring rank was generated by AnkPlex using a combination of a decision tree algorithm and logistic regression. AnkPlex achieved superior efficiency with ≥1 near-native complexes in the 10 top-rankings for nine X-ray complexes compared to ZDOCK, which only obtained six X-ray complexes. In addition, feature analysis demonstrated that the van der Waals feature was the dominant near-native pose out of the potential ankyrin-protein docking poses. CONCLUSION: The AnkPlex model achieved a success at predicting near-native docking poses and led to the discovery of informative characteristics that could further improve our understanding of the ankyrin-protein complex. Our computational study could be useful for predicting the near-native poses of binding proteins and desired targets, especially for ankyrin-protein complexes. The AnkPlex web server is freely accessible at http://ankplex.ams.cmu.ac.th .

Crystal structure of an antiviral ankyrin targeting the HIV-1 capsid and molecular modeling of the ankyrin-capsid complex.

Ankyrins are cellular repeat proteins, which can be genetically modified to randomize amino-acid residues located at defined positions in each repeat unit, and thus create a potential binding surface adaptable to macromolecular ligands. From a phage-display library of artificial ankyrins, we have isolated Ank(GAG)1D4, a trimodular ankyrin which binds to the HIV-1 capsid protein N-terminal domain (NTD(CA)) and has an antiviral effect at the late steps of the virus life cycle. In this study, the determinants of the Ank(GAG)1D4-NTD(CA) interaction were analyzed using peptide scanning in competition ELISA, capsid mutagenesis, ankyrin crystallography and molecular modeling. We determined the Ank(GAG)1D4 structure at 2.2 Å resolution, and used the crystal structure in molecular docking with a homology model of HIV-1 capsid. Our results indicated that NTD(CA) alpha-helices H1 and H7 could mediate the formation of the capsid-Ank(GAG)1D4 binary complex, but the interaction involving H7 was predicted to be more stable than with H1. Arginine-18 (R18) in H1, and R132 and R143 in H7 were found to be the key players of the Ank(GAG)1D4-NTD(CA) interaction. This was confirmed by R-to-A mutagenesis of NTD(CA), and by sequence analysis of trimodular ankyrins negative for capsid binding. In Ank(GAG)1D4, major interactors common to H1 and H7 were found to be S45, Y56, R89, K122 and K123. Collectively, our ankyrin-capsid binding analysis implied a significant degree of flexibility within the NTD(CA) domain of the HIV-1 capsid protein, and provided some clues for the design of new antivirals targeting the capsid protein and viral assembly.

Antiviral effect of melatonin on Japanese encephalitis virus infection involves inhibition of neuronal apoptosis and neuroinflammation in SH-SY5Y cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes high mortality rates in humans and it is the most clinically important and common cause of viral encephalitis in Asia. To date, there is no specific treatment for JEV infection. Melatonin, a neurotropic hormone, is reported to be effective in combating various bacterial and viral infections. However, the effects of melatonin on JEV infection have not yet been studied. The investigation tested the antiviral effects of melatonin against JEV infection and elucidated the possible molecular mechanisms of inhibition. Melatonin inhibited the viral production in JEV-infected SH-SY5Y cells in a time- and dose-dependent manner. Time-of-addition assays demonstrated a potent inhibitory effect of melatonin at the post-entry stage of viral replication. Molecular docking analysis revealed that melatonin negatively affected viral replication by interfering with physiological function and/or enzymatic activity of both JEV nonstructural 3 (NS3) and NS5 protein, suggesting a possible underlying mechanism of JEV replication inhibition. Moreover, treatment with melatonin reduced neuronal apoptosis and inhibited neuroinflammation induced by JEV infection. The present findings reveal a new property of melatonin as a potential molecule for the further development of anti-JEV agents and treatment of JEV infection.

Determination of a distinguished interferon gamma epitope recognized by monoclonal antibody relating to autoantibody associated immunodeficiency.

Anti-interferon gamma autoantibodies (anti-IFN-γ autoAbs) neutralize the IFN-γ-mediated functions, contributing to immunodeficiency. A particular autoAb in patient serum had been previously demonstrated to recognize the same determinant on IFN-γ as the neutralizing anti-IFN-γ monoclonal antibody clone B27 (B27 mAb). This study explored the epitope recognized by B27 mAb. The specific peptide sequence recognized by B27 mAb, TDFLRMMLQEER, was retrieved from a phage display random peptide library. Sequence alignment and homology modeling demonstrated that the queried phage peptide sequence and structure were similar to amino acids at position 27-40 (TLFLGILKNWKEES) of the human IFN-γ. This determinant resides in the contact surface of IFN-γ and interferon gamma receptor 1. To elucidate the crucial amino acids, mutations were introduced by substituting T27 and T27F29L30 with alanine or deleting the amino acid residues T27-L33. The binding of B27 mAb to IFN-γ T27A using western blotting was lesser than that to wild-type. The interaction with triple mutant and T27-L33 deletion mutant using western blotting and sandwich ELISA was abolished. The finding demonstrated that T27, F29, and L30 are critical residues in the B27 antigenic determinant. Identification of the functional domain of IFN-γ decrypted the relevance of neutralizing autoAb in adult-onset immunodeficiency.

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.

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.