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Journal of Virology, August 2009, p. 7495-7506, Vol. 83, No. 15
0022-538X/09/$08.00+0     doi:10.1128/JVI.00400-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mutagenesis of Varicella-Zoster Virus Glycoprotein B: Putative Fusion Loop Residues Are Essential for Viral Replication, and the Furin Cleavage Motif Contributes to Pathogenesis in Skin Tissue In Vivo{triangledown}

Stefan L. Oliver,1* Marvin Sommer,1 Leigh Zerboni,1 Jaya Rajamani,1 Charles Grose,2 and Ann M. Arvin1

Departments of Pediatrics and Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305,1 Departments of Microbiology and Pediatrics, University of Iowa Hospital, Iowa City, Iowa 522422

Received 23 February 2009/ Accepted 18 May 2009

Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-|-X, where | indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Substitutions in the primary loop, W180G and Y185G, plus the deletion mutation {Delta}491RSRR494 and point mutation 491GSGG494 in the furin recognition motif did not affect gB expression or cellular localization in transfected cells. Infectious VZV was recovered from parental Oka (pOka)-bacterial artificial chromosomes that had either the {Delta}491RSRR494 or 491GSGG494 mutation but not the point mutations W180G and Y185G, demonstrating that residues in the primary loop of gB were essential but gB cleavage was not required for VZV replication in vitro. Virion morphology, protein localization, plaque size, and replication were unaffected for the pOka-gB{Delta}491RSRR494 or pOka-gB491GSGG494 virus compared to pOka in vitro. However, deletion of the furin recognition motif caused attenuation of VZV replication in human skin xenografts in vivo. This is the first evidence that cleavage of a herpesvirus fusion protein contributes to viral pathogenesis in vivo, as seen for fusion proteins in other virus families.

* Corresponding author. Mailing address: S366, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305. Phone: (650) 725-6555. Fax: (650) 725-9828. E-mail: sloliver{at}stanford.edu

{triangledown} Published ahead of print on 27 May 2009.

Journal of Virology, August 2009, p. 7495-7506, Vol. 83, No. 15
0022-538X/09/$08.00+0     doi:10.1128/JVI.00400-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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