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Journal of Virology, January 2009, p. 734-747, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01817-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Analysis of Epstein-Barr Virus Glycoprotein B Functional Domains via Linker Insertion Mutagenesis{triangledown}

Jessica J. Reimer,1 Marija Backovic,2 Charuhas G. Deshpande,3 Theodore Jardetzky,4 and Richard Longnecker1*

Department of Microbiology and Immunology, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611,1 Departement de Virologie, Institut Pasteur, 75015 Paris, France,2 Department of Pathology, Northwestern Memorial Hospital, Northwestern University, Chicago, Illinois 60611,3 Department of Structural Biology, Stanford University School of Medicine, Stanford, California 943054

Received 29 August 2008/ Accepted 27 October 2008

Epstein-Barr Virus (EBV) glycoprotein B (gB) is essential for viral fusion events with epithelial and B cells. This glycoprotein has been studied extensively in other herpesvirus family members, but functional domains outside of the cytoplasmic tail have not been characterized in EBV gB. In this study, a total of 28 linker insertion mutations were generated throughout the length of gB. In general, the linker insertions did not disrupt intracellular expression and variably altered cell surface expression. Oligomerization was disrupted by insertions located between residues 561 and 620, indicating the location of a potential site of oligomer contacts between EBV gB monomers. In addition, a novel N-glycosylated form of wild-type gB was identified under nonreducing Western blot conditions that likely represents a mature form of the protein. Fusion activity was abolished in all but three variants containing mutations in the N-terminal region (gB30), within the ectodomain (gB421), and in the intracellular C-terminal domain (gB832) of the protein. Fusion activity with variants gB421 and gB832 was comparable to that of the wild type with epithelial and B cells, and only these two mutants, but not gB30, were able to complement gB-null virus and subsequently function in virus entry. The mutant gB30 exhibited a low level of fusion activity with B cells and was unable to complement gB-null virus. The mutations generated here indicate important structural domains, as well as regions important for function in fusion, within EBV gB.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Northwestern University, 303 E. Chicago Avenue, Chicago, IL 60611. Phone: (312) 503-0467. Fax: (312) 503-1339. E-mail: r-longnecker{at}northwestern.edu

{triangledown} Published ahead of print on 5 November 2008.

Journal of Virology, January 2009, p. 734-747, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01817-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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