The Cytoplasmic Tail Domain of Epstein-Barr Virus gH Regulates Membrane Fusion Activity through Altering gH Binding to gp42 and Epithelial Cell Attachment.

Jia Chen,Theodore S Jardetzky,Richard Longnecker,Peter Palese

doi:10.1128/mbio.01871-16

Jia Chen, Theodore S Jardetzky + Show 2 more

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https://doi.org/10.1128/mbio.01871-16

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Journal: mBio	Publication Date: Nov 15, 2016
Citations: 14	License type: CC BY 4.0

Affiliation: Northwestern University, Stanford University

Abstract

ABSTRACTEpstein-Barr virus (EBV) is associated with infectious mononucleosis and a variety of cancers as well as lymphoproliferative disorders in immunocompromised patients. EBV mediates viral entry into epithelial and B cells using fusion machinery composed of four glycoproteins: gB, the gH/gL complex, and gp42. gB and gH/gL are required for both epithelial and B cell fusion. The specific role of gH/gL in fusion has been the most elusive among the required herpesvirus entry glycoproteins. Previous mutational studies have focused on the ectodomain of EBV gH and not on the gH cytoplasmic tail domain (CTD). In this study, we chose to examine the function of the gH CTD by making serial gH truncation mutants as well as amino acid substitution mutants to determine the importance of the gH CTD in epithelial and B cell fusion. Truncation of 8 amino acids (aa 698 to 706) of the gH CTD resulted in diminished fusion activity using a virus-free syncytium formation assay and fusion assay. The importance of the amino acid composition of the gH CTD was also investigated by amino acid substitutions that altered the hydrophobicity or hydrophilicity of the CTD. These mutations also resulted in diminished fusion activity. Interestingly, some of the gH CTD truncation mutants and hydrophilic tail substitution mutants lost the ability to bind to gp42 and epithelial cells. In summary, our studies indicate that the gH CTD is an important functional domain.

Highlights

Epstein-Barr virus (EBV) is associated with infectious mononucleosis and a variety of cancers as well as lymphoproliferative disorders in immunocompromised patients
We found that the deletion of the entire gH cytoplasmic tail domain (CTD) results in an approximately 50% reduction in cell surface expression (Fig. 1B) compared to wild-type (WT) gH/gL, indicating that the gH CTD is important for cell surface expression
Consistent with the results that soluble EBV gH cannot function in fusion and the observation that deletion, replacement, or insertion mutants inhibit the fusion activity of herpes simplex virus (HSV) gH [15, 16], we found that the EBV gH CTD is important for fusion activity (Fig. 1C and D)

Summary

Introduction

Epstein-Barr virus (EBV) is associated with infectious mononucleosis and a variety of cancers as well as lymphoproliferative disorders in immunocompromised patients. We chose to examine the function of the gH CTD by making serial gH truncation mutants as well as amino acid substitution mutants to determine the importance of the gH CTD in epithelial and B cell fusion. Entry into target cells is the first step for viral infection and is important for EBV to cause disease. Four viral-membrane-associated proteins have been determined as the minimal glycoproteins for B cell entry using virus-free cell-cell fusion. These are glycoprotein 42 (gp42), gH, gL, and gB. Plex comprised of gH/gL, gp, and HLA class II that is required for the infection of B cells by EBV [10] This structure provided a unique opportunity to further understand herpesvirus-induced membrane fusion. Our mutagenesis studies have shown that this groove participates in both B and epithelial cell fusion and indicate that flexibility between D-I and D-II in gH/gL is required for epithelial cell but not B cell fusion [12]

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