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Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among Marburgvirus and Dianlovirus

Disease Intervention and Prevention, Texas Biomedical Research Institute, 8715 W. Military Dr., San Antonio, TX 78227-5302, USA
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Viruses 2019, 11(4), 364; https://doi.org/10.3390/v11040364
Received: 2 April 2019 / Revised: 17 April 2019 / Accepted: 18 April 2019 / Published: 20 April 2019
(This article belongs to the Collection Advances in Ebolavirus, Marburgvirus, and Cuevavirus Research)
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Abstract

We explore evolved soybean ascorbate peroxidase (APEX2) as a reporter when fused to the C-termini of llama nanobodies (single-domain antibodies, sdAb; variable domains of heavy chain-only antibodies, VHH) targeted to the E. coli periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous E. coli outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the Marburgvirus genus and cross-reactive within the Ebolavirus genus, we show that periplasmic sdAb–APEX2 fusion proteins are easily purified at multi-mg amounts. The fusions were used in Western blotting, ELISA, and microscopy to visualize NPs using colorimetric and fluorescent imaging. Dimeric sdAb–APEX2 fusions were superior at binding NPs from viruses that were evolutionarily distant to that originally used to select the sdAb. Partial conservation of the anti-Marburgvirus sdAb epitope enabled the recognition of a novel NP encoded by the recently discovered Mĕnglà virus genome. Antibody–antigen interactions were rationalized using monovalent nanoluciferase titrations and contact mapping analysis of existing crystal structures, while molecular modelling was used to reveal the potential landscape of the Mĕnglà NP C-terminal domain. The sdAb–APEX2 fusions also enabled live Marburgvirus and Ebolavirus detection 24 h post-infection of Vero E6 cells within a BSL-4 laboratory setting. The simple and inexpensive mining of large amounts of periplasmic sdAb–APEX2 fusion proteins should help advance studies of past, contemporary, and perhaps Filovirus species yet to be discovered. View Full-Text
Keywords: peroxidase; APEX2; VHH; sdAb; nanobody; Filovirus; Ebola; Marburg; nucleoprotein; nanoluciferase peroxidase; APEX2; VHH; sdAb; nanobody; Filovirus; Ebola; Marburg; nucleoprotein; nanoluciferase
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Sherwood, L.J.; Hayhurst, A. Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among Marburgvirus and Dianlovirus. Viruses 2019, 11, 364.

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