Molecular identification of dengue virus binding protein on filopodia of vero cells

Despite much efforts to control dengue virus, there is still neither scientifically proven effective drug nor an effective internationally licensed vaccine to treat or control dengue. Recent evidence has demonstrated that dengue virus requires an active filopodia formation for successful infection....

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Main Author:	Aliyu, Isah Abubakar
Format:	Thesis
Language:	English
Published:	2017
Subjects:	Dengue Virus Dengue Vaccines
Online Access:	http://psasir.upm.edu.my/id/eprint/85535/1/FPSK%28p%29%202019%2010%20UPM%20IR.pdf
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spelling	my-upm-ir.855352021-06-03T12:53:27Z Molecular identification of dengue virus binding protein on filopodia of vero cells 2017-05 Aliyu, Isah Abubakar Despite much efforts to control dengue virus, there is still neither scientifically proven effective drug nor an effective internationally licensed vaccine to treat or control dengue. Recent evidence has demonstrated that dengue virus requires an active filopodia formation for successful infection. However, the precise mechanism for dengue virus interaction with filopodia to enhance virus infection is not fully understood. Therefore, the present study was designed to identify the dengue virus 2 binding protein that could be responsible for enhanced virus infection upon filopodia formation. Filopodia formation in Vero cells was induced with bradykinin and a combination of virus overlay protein binding assay (VOPBA) and LC-MS/MS was employed to identify annexin II as a dengue virus 2 binding protein on filopodia-induced cells. Translocation of annexin II to the external leaflet of plasma membrane following filopodia formation was demonstrated by immunocytochemistry staining and western blotting analysis. Down-regulation of plasma membrane annexin II was observed in response to dengue virus 2 infection. Moreover, anti-annexin II antibody inhibited virus binding in an antibody-dependent binding inhibition assay and a dose-dependent reduction in both dengue virus infection level and virus production was demonstrated in an antibody-mediated infection inhibition assay. Similarly, siRNA-mediated knockdown of annexin II gene significantly reduced virus infection level as well as virus production. Confocal microscopic examination showed extracellular and intracellular colocalization between annexin II and dengue virus 2 E glycoprotein, and co-immunoprecipitation assay confirmed that annexin II interacted with dengue virus 2 E glycoprotein. Furthermore, the putative annexin II interaction site on dengue virus 2 E glycoprotein was predicted using molecular docking and the interaction was likely to occur at residues Ile-380 to Leu-389 of E glycoprotein, with Ser-22 to Asn-32, Leu-300 to Tyr-317 and Lys-328 to Asp-338 of annexin II. Data from the present study reported for the first time the expression of a dengue virus 2 binding protein upon filopodia formation, which might be involved in dengue virus 2 binding, internalization and intracellular trafficking. Thus, the results could explain the likely reason behind enhanced virus infection observed upon filopodia formation and could be a potential target for the development of a novel antiviral. Dengue Virus Dengue Vaccines 2017-05 Thesis http://psasir.upm.edu.my/id/eprint/85535/ http://psasir.upm.edu.my/id/eprint/85535/1/FPSK%28p%29%202019%2010%20UPM%20IR.pdf text en public doctoral Universiti Putra Malaysia Dengue Virus Dengue Vaccines Chee, Hui Yee
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
advisor	Chee, Hui Yee
topic	Dengue Virus Dengue Vaccines
spellingShingle	Dengue Virus Dengue Vaccines Aliyu, Isah Abubakar Molecular identification of dengue virus binding protein on filopodia of vero cells
description	Despite much efforts to control dengue virus, there is still neither scientifically proven effective drug nor an effective internationally licensed vaccine to treat or control dengue. Recent evidence has demonstrated that dengue virus requires an active filopodia formation for successful infection. However, the precise mechanism for dengue virus interaction with filopodia to enhance virus infection is not fully understood. Therefore, the present study was designed to identify the dengue virus 2 binding protein that could be responsible for enhanced virus infection upon filopodia formation. Filopodia formation in Vero cells was induced with bradykinin and a combination of virus overlay protein binding assay (VOPBA) and LC-MS/MS was employed to identify annexin II as a dengue virus 2 binding protein on filopodia-induced cells. Translocation of annexin II to the external leaflet of plasma membrane following filopodia formation was demonstrated by immunocytochemistry staining and western blotting analysis. Down-regulation of plasma membrane annexin II was observed in response to dengue virus 2 infection. Moreover, anti-annexin II antibody inhibited virus binding in an antibody-dependent binding inhibition assay and a dose-dependent reduction in both dengue virus infection level and virus production was demonstrated in an antibody-mediated infection inhibition assay. Similarly, siRNA-mediated knockdown of annexin II gene significantly reduced virus infection level as well as virus production. Confocal microscopic examination showed extracellular and intracellular colocalization between annexin II and dengue virus 2 E glycoprotein, and co-immunoprecipitation assay confirmed that annexin II interacted with dengue virus 2 E glycoprotein. Furthermore, the putative annexin II interaction site on dengue virus 2 E glycoprotein was predicted using molecular docking and the interaction was likely to occur at residues Ile-380 to Leu-389 of E glycoprotein, with Ser-22 to Asn-32, Leu-300 to Tyr-317 and Lys-328 to Asp-338 of annexin II. Data from the present study reported for the first time the expression of a dengue virus 2 binding protein upon filopodia formation, which might be involved in dengue virus 2 binding, internalization and intracellular trafficking. Thus, the results could explain the likely reason behind enhanced virus infection observed upon filopodia formation and could be a potential target for the development of a novel antiviral.
format	Thesis
qualification_level	Doctorate
author	Aliyu, Isah Abubakar
author_facet	Aliyu, Isah Abubakar
author_sort	Aliyu, Isah Abubakar
title	Molecular identification of dengue virus binding protein on filopodia of vero cells
title_short	Molecular identification of dengue virus binding protein on filopodia of vero cells
title_full	Molecular identification of dengue virus binding protein on filopodia of vero cells
title_fullStr	Molecular identification of dengue virus binding protein on filopodia of vero cells
title_full_unstemmed	Molecular identification of dengue virus binding protein on filopodia of vero cells
title_sort	molecular identification of dengue virus binding protein on filopodia of vero cells
granting_institution	Universiti Putra Malaysia
publishDate	2017
url	http://psasir.upm.edu.my/id/eprint/85535/1/FPSK%28p%29%202019%2010%20UPM%20IR.pdf
_version_	1747813554862424064

Molecular identification of dengue virus binding protein on filopodia of vero cells

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