Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers

ABSTRACT We recently described the architecture of the Epstein-Barr virus (EBV) fusion-triggering complex consisting of the EBV B cell receptor human leukocyte antigen (HLA) class II and the EBV-encoded proteins gp42 and gH/gL. The architecture of this structure positioned the main body of gp42, com...

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Autores principales:	Cynthia L. Rowe, Jia Chen, Theodore S. Jardetzky, Richard Longnecker
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/019c7f60d651462384bd50685775d6dd
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spelling	oai:doaj.org-article:019c7f60d651462384bd50685775d6dd2021-11-15T15:41:18ZMembrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers10.1128/mBio.02285-142150-7511https://doaj.org/article/019c7f60d651462384bd50685775d6dd2015-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02285-14https://doaj.org/toc/2150-7511ABSTRACT We recently described the architecture of the Epstein-Barr virus (EBV) fusion-triggering complex consisting of the EBV B cell receptor human leukocyte antigen (HLA) class II and the EBV-encoded proteins gp42 and gH/gL. The architecture of this structure positioned the main body of gp42, comprising the C-type lectin domain (CTLD), away from the membrane and distant from where the membrane-bound form of gp42 might be tethered. gp42 is a type II membrane glycoprotein, with functional gp42 formed by cleavage near the gp42 amino-terminal transmembrane domain. This cleavage results in an approximately 50-amino-acid unstructured region that is responsible for binding gH/gL with nanomolar affinity. Our previous studies had shown that membrane-bound gp42 is not functional in B cell fusion. To investigate whether we could restore gp42 function by extending it from the membrane, we introduced one, two, and four structured immunoglobulin-like domains from muscle protein titin into a membrane-bound form of gp42 and tested function in binding to gHgL and HLA class II and function in fusion. We hypothesized that cleavage of gp42 generates a soluble functional form that relieves steric hindrance imposed on gHgL by membrane-bound gp42. All of the linker mutants had a dominant-negative effect on gp42 function, indicating that gp42 fusion function could not be restored simply by the addition of one to four titin domains. IMPORTANCE Epstein-Barr virus (EBV) is associated with numerous diseases from benign mononucleosis to Burkitt’s and Hodgkin’s lymphoma, nasopharyngeal and gastric carcinoma, and lymphoproliferative disorders in patients with immune dysfunction resulting from immune suppression. Among the glycoproteins important for fusion, gp42, along with gH/gL, determines EBV tropism between epithelial and B cells. The function of gp42 is dependent on N-terminal cleavage, since membrane-bound gp42 cannot mediate fusion. We further investigated whether insertion of a linker into membrane-bound gp42 would relieve steric hindrance imposed on membrane-bound gp42 and restore fusion function. However, adding one, two, or four structured immunoglobulin-like domains to membrane gp42 did not restore fusion activity, indicating that the architecture and membrane orientation of the B cell fusion-triggering complex of EBV may be easily perturbed and that gp42 cleavage is essential for B cell fusion.Cynthia L. RoweJia ChenTheodore S. JardetzkyRichard LongneckerAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 1 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Cynthia L. Rowe Jia Chen Theodore S. Jardetzky Richard Longnecker Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers
description	ABSTRACT We recently described the architecture of the Epstein-Barr virus (EBV) fusion-triggering complex consisting of the EBV B cell receptor human leukocyte antigen (HLA) class II and the EBV-encoded proteins gp42 and gH/gL. The architecture of this structure positioned the main body of gp42, comprising the C-type lectin domain (CTLD), away from the membrane and distant from where the membrane-bound form of gp42 might be tethered. gp42 is a type II membrane glycoprotein, with functional gp42 formed by cleavage near the gp42 amino-terminal transmembrane domain. This cleavage results in an approximately 50-amino-acid unstructured region that is responsible for binding gH/gL with nanomolar affinity. Our previous studies had shown that membrane-bound gp42 is not functional in B cell fusion. To investigate whether we could restore gp42 function by extending it from the membrane, we introduced one, two, and four structured immunoglobulin-like domains from muscle protein titin into a membrane-bound form of gp42 and tested function in binding to gHgL and HLA class II and function in fusion. We hypothesized that cleavage of gp42 generates a soluble functional form that relieves steric hindrance imposed on gHgL by membrane-bound gp42. All of the linker mutants had a dominant-negative effect on gp42 function, indicating that gp42 fusion function could not be restored simply by the addition of one to four titin domains. IMPORTANCE Epstein-Barr virus (EBV) is associated with numerous diseases from benign mononucleosis to Burkitt’s and Hodgkin’s lymphoma, nasopharyngeal and gastric carcinoma, and lymphoproliferative disorders in patients with immune dysfunction resulting from immune suppression. Among the glycoproteins important for fusion, gp42, along with gH/gL, determines EBV tropism between epithelial and B cells. The function of gp42 is dependent on N-terminal cleavage, since membrane-bound gp42 cannot mediate fusion. We further investigated whether insertion of a linker into membrane-bound gp42 would relieve steric hindrance imposed on membrane-bound gp42 and restore fusion function. However, adding one, two, or four structured immunoglobulin-like domains to membrane gp42 did not restore fusion activity, indicating that the architecture and membrane orientation of the B cell fusion-triggering complex of EBV may be easily perturbed and that gp42 cleavage is essential for B cell fusion.
format	article
author	Cynthia L. Rowe Jia Chen Theodore S. Jardetzky Richard Longnecker
author_facet	Cynthia L. Rowe Jia Chen Theodore S. Jardetzky Richard Longnecker
author_sort	Cynthia L. Rowe
title	Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers
title_short	Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers
title_full	Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers
title_fullStr	Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers
title_full_unstemmed	Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers
title_sort	membrane anchoring of epstein-barr virus gp42 inhibits fusion with b cells even with increased flexibility allowed by engineered spacers
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/019c7f60d651462384bd50685775d6dd
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Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers

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