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

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 <i>E. coli</i> periplasm. Periplasmic expression preserves authenti...

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Main Authors: Laura J. Sherwood, Andrew Hayhurst
Format: Article
Language:English
Published: MDPI AG 2019-04-01
Series:Viruses
Subjects:
VHH
Online Access:https://www.mdpi.com/1999-4915/11/4/364
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spelling doaj-fb616e9dbcea46d9be7da6329e32fac82020-11-25T00:08:14ZengMDPI AGViruses1999-49152019-04-0111436410.3390/v11040364v11040364Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among <i>Marburgvirus</i> and <i>Dianlovirus</i>Laura J. Sherwood0Andrew Hayhurst1Disease Intervention and Prevention, Texas Biomedical Research Institute, 8715 W. Military Dr., San Antonio, TX 78227-5302, USADisease Intervention and Prevention, Texas Biomedical Research Institute, 8715 W. Military Dr., San Antonio, TX 78227-5302, USAWe 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 <i>E. coli</i> periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous <i>E. coli</i> outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the <i>Marburgvirus</i> genus and cross-reactive within the <i>Ebolavirus</i> genus, we show that periplasmic sdAb&#8211;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&#8211;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-<i>Marburgvirus</i> sdAb epitope enabled the recognition of a novel NP encoded by the recently discovered Mĕngl&#224; virus genome. Antibody&#8211;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 Mĕngl&#224; NP C-terminal domain. The sdAb&#8211;APEX2 fusions also enabled live <i>Marburgvirus</i> and <i>Ebolavirus</i> 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&#8211;APEX2 fusion proteins should help advance studies of past, contemporary, and perhaps Filovirus species yet to be discovered.https://www.mdpi.com/1999-4915/11/4/364peroxidaseAPEX2VHHsdAbnanobodyFilovirusEbolaMarburgnucleoproteinnanoluciferase
collection DOAJ
language English
format Article
sources DOAJ
author Laura J. Sherwood
Andrew Hayhurst
spellingShingle Laura J. Sherwood
Andrew Hayhurst
Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among <i>Marburgvirus</i> and <i>Dianlovirus</i>
Viruses
peroxidase
APEX2
VHH
sdAb
nanobody
Filovirus
Ebola
Marburg
nucleoprotein
nanoluciferase
author_facet Laura J. Sherwood
Andrew Hayhurst
author_sort Laura J. Sherwood
title Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among <i>Marburgvirus</i> and <i>Dianlovirus</i>
title_short Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among <i>Marburgvirus</i> and <i>Dianlovirus</i>
title_full Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among <i>Marburgvirus</i> and <i>Dianlovirus</i>
title_fullStr Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among <i>Marburgvirus</i> and <i>Dianlovirus</i>
title_full_unstemmed Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among <i>Marburgvirus</i> and <i>Dianlovirus</i>
title_sort periplasmic nanobody-apex2 fusions enable facile visualization of ebola, marburg, and mĕnglà virus nucleoproteins, alluding to similar antigenic landscapes among <i>marburgvirus</i> and <i>dianlovirus</i>
publisher MDPI AG
series Viruses
issn 1999-4915
publishDate 2019-04-01
description 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 <i>E. coli</i> periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous <i>E. coli</i> outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the <i>Marburgvirus</i> genus and cross-reactive within the <i>Ebolavirus</i> genus, we show that periplasmic sdAb&#8211;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&#8211;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-<i>Marburgvirus</i> sdAb epitope enabled the recognition of a novel NP encoded by the recently discovered Mĕngl&#224; virus genome. Antibody&#8211;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 Mĕngl&#224; NP C-terminal domain. The sdAb&#8211;APEX2 fusions also enabled live <i>Marburgvirus</i> and <i>Ebolavirus</i> 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&#8211;APEX2 fusion proteins should help advance studies of past, contemporary, and perhaps Filovirus species yet to be discovered.
topic peroxidase
APEX2
VHH
sdAb
nanobody
Filovirus
Ebola
Marburg
nucleoprotein
nanoluciferase
url https://www.mdpi.com/1999-4915/11/4/364
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