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Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection

DC-SIGN, a C-type lectin mainly expressed in dendritic cells (DCs), has been reported to mediate several viral infections. We previously reported that DC-SIGN mediated H5N1 influenza A virus (AIVs) infection, however, the important DC-SIGN interaction with N-glycosylation sites remain unknown. This...

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Autores principales: Yang, Zih-Syuan, Huang, Szu-Wei, Wang, Wen-Hung, Lin, Chih-Yen, Wang, Chu-Feng, Urbina, Aspiro Nayim, Thitithanyanont, Arunee, Tseng, Sung-Pin, Lu, Po-Liang, Chen, Yen-Hsu, Wang, Sheng-Fan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828482/
https://www.ncbi.nlm.nih.gov/pubmed/33451024
http://dx.doi.org/10.3390/ijms22020743
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author Yang, Zih-Syuan
Huang, Szu-Wei
Wang, Wen-Hung
Lin, Chih-Yen
Wang, Chu-Feng
Urbina, Aspiro Nayim
Thitithanyanont, Arunee
Tseng, Sung-Pin
Lu, Po-Liang
Chen, Yen-Hsu
Wang, Sheng-Fan
author_facet Yang, Zih-Syuan
Huang, Szu-Wei
Wang, Wen-Hung
Lin, Chih-Yen
Wang, Chu-Feng
Urbina, Aspiro Nayim
Thitithanyanont, Arunee
Tseng, Sung-Pin
Lu, Po-Liang
Chen, Yen-Hsu
Wang, Sheng-Fan
author_sort Yang, Zih-Syuan
collection PubMed
description DC-SIGN, a C-type lectin mainly expressed in dendritic cells (DCs), has been reported to mediate several viral infections. We previously reported that DC-SIGN mediated H5N1 influenza A virus (AIVs) infection, however, the important DC-SIGN interaction with N-glycosylation sites remain unknown. This study aims to identify the optimal DC-SIGN interacting N-glycosylation sites in HA proteins of H5N1-AIVs. Results from NetNGlyc program analyzed the H5 hemagglutinin sequences of isolates during 2004–2020, revealing that seven and two conserved N-glycosylation sites were detected in HA1 and HA2 domain, respectively. A lentivirus pseudotyped A/Vietnam/1203/04 H5N1 envelope (H5N1-PVs) was generated which displayed an abundance of HA5 proteins on the virions via immuno-electron microscope observation. Further, H5N1-PVs or reverse-genetics (H5N1-RG) strains carrying a serial N-glycosylated mutation was generated by site-directed mutagenesis assay. Human recombinant DC-SIGN (rDC-SIGN) coated ELISA showed that H5N1-PVs bound to DC-SIGN, however, mutation on the N27Q, N39Q, and N181Q significantly reduced this binding (p < 0.05). Infectivity and capture assay demonstrated that N27Q and N39Q mutations significantly ameliorated DC-SIGN mediated H5N1 infection. Furthermore, combined mutations (N27Q&N39Q) significantly waned the interaction on either H5N1-PVs or -RG infection in cis and in trans (p < 0.01). This study concludes that N27 and N39 are two essential N-glycosylation contributing to DC-SIGN mediating H5N1 infection.
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spelling pubmed-78284822021-01-25 Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection Yang, Zih-Syuan Huang, Szu-Wei Wang, Wen-Hung Lin, Chih-Yen Wang, Chu-Feng Urbina, Aspiro Nayim Thitithanyanont, Arunee Tseng, Sung-Pin Lu, Po-Liang Chen, Yen-Hsu Wang, Sheng-Fan Int J Mol Sci Article DC-SIGN, a C-type lectin mainly expressed in dendritic cells (DCs), has been reported to mediate several viral infections. We previously reported that DC-SIGN mediated H5N1 influenza A virus (AIVs) infection, however, the important DC-SIGN interaction with N-glycosylation sites remain unknown. This study aims to identify the optimal DC-SIGN interacting N-glycosylation sites in HA proteins of H5N1-AIVs. Results from NetNGlyc program analyzed the H5 hemagglutinin sequences of isolates during 2004–2020, revealing that seven and two conserved N-glycosylation sites were detected in HA1 and HA2 domain, respectively. A lentivirus pseudotyped A/Vietnam/1203/04 H5N1 envelope (H5N1-PVs) was generated which displayed an abundance of HA5 proteins on the virions via immuno-electron microscope observation. Further, H5N1-PVs or reverse-genetics (H5N1-RG) strains carrying a serial N-glycosylated mutation was generated by site-directed mutagenesis assay. Human recombinant DC-SIGN (rDC-SIGN) coated ELISA showed that H5N1-PVs bound to DC-SIGN, however, mutation on the N27Q, N39Q, and N181Q significantly reduced this binding (p < 0.05). Infectivity and capture assay demonstrated that N27Q and N39Q mutations significantly ameliorated DC-SIGN mediated H5N1 infection. Furthermore, combined mutations (N27Q&N39Q) significantly waned the interaction on either H5N1-PVs or -RG infection in cis and in trans (p < 0.01). This study concludes that N27 and N39 are two essential N-glycosylation contributing to DC-SIGN mediating H5N1 infection. MDPI 2021-01-13 /pmc/articles/PMC7828482/ /pubmed/33451024 http://dx.doi.org/10.3390/ijms22020743 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yang, Zih-Syuan
Huang, Szu-Wei
Wang, Wen-Hung
Lin, Chih-Yen
Wang, Chu-Feng
Urbina, Aspiro Nayim
Thitithanyanont, Arunee
Tseng, Sung-Pin
Lu, Po-Liang
Chen, Yen-Hsu
Wang, Sheng-Fan
Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection
title Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection
title_full Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection
title_fullStr Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection
title_full_unstemmed Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection
title_short Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection
title_sort identification of important n-linked glycosylation sites in the hemagglutinin protein and their functional impact on dc-sign mediated avian influenza h5n1 infection
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828482/
https://www.ncbi.nlm.nih.gov/pubmed/33451024
http://dx.doi.org/10.3390/ijms22020743
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