Research Article: Insertion of a ligand to HER2 in gB retargets HSV tropism and obviates the need for activation of the other entry glycoproteins

Date Published: April 19, 2017

Publisher: Public Library of Science

Author(s): Biljana Petrovic, Tatiana Gianni, Valentina Gatta, Gabriella Campadelli-Fiume, Peter Palese.

http://doi.org/10.1371/journal.ppat.1006352

Abstract

Herpes simplex virus (HSV) entry into the cells requires glycoproteins gD, gH/gL and gB, activated in a cascade fashion by conformational modifications induced by cognate receptors and intermolecular signaling. The receptors are nectin1 and HVEM (Herpes virus entry mediator) for gD, and αvβ6 or αvβ8 integrin for gH. In earlier work, insertion of a single chain antibody (scFv) to the cancer receptor HER2 (human epidermal growth factor receptor 2) in gD, or in gH, resulted in HSVs specifically retargeted to the HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Here, the scFv to HER2 was inserted in gB (gBHER2). The insertion re-targeted the virus tropism to the HER2-positive cancer cells. This was unexpected since gB is known to be a fusogenic glycoprotein, not a tropism determinant. The gB-retargeted recombinant offered the possibility to investigate how HER2 mediated entry. In contrast to wt-gB, the activation of the chimeric gBHER2 did not require the activation of the gD and of gH/gL by their respective receptors. Furthermore, a soluble form of HER2 could replace the membrane-bound HER2 in mediating virus entry, hinting that HER2 acted by inducing conformational changes to the chimeric gB. This study shows that (i) gB can be modified and become the major determinant of HSV tropism; (ii) the chimeric gBHER2 bypasses the requirement for receptor-mediated activation of other essential entry glycoproteins.

Partial Text

Herpes simplex virus encodes a multipartite entry apparatus made of four essential glycoproteins, named gD, the heterodimer gH/gL and gB, with distinct functions [1–4]. gD, whose structure includes an Ig-folded core with extensions, serves as a typical receptor-binding glycoprotein, and the major determinant of HSV tropism [5–7]. The heterodimer gH/gL is a multidomain protein, with no structural resemblance to any known protein [8–10]. gB is a trimer with structural features typical of viral fusion glycoproteins [11–13]. gH/gL and gB form the conserved fusion apparatus across the Herpesviridae family. The quartet assembles in complexes [14, 15, 16–18]. Contact regions among the glycoproteins were identified [10,17–20]. The system of receptors for the quartet of glycoproteins appears to be more and more complex, and affects the process of glycoprotein activation at virus entry. gD interacts with three alternative receptors, nectin1, HVEM, and modified heparan sulphate [21–24]. gH/gL interact with the αvβ subfamily of integrins [25,26]. αvβ6 and αvβ8 are required for entry, in that their depletion, or block with antibodies, results in block to virus infection [26]. Three co-receptors for gB were reported. They are PILRα (paired immunoglobulin-like type 2 receptor-alpha), myelin associated glycoprotein, and isoforms IIA and IIB of non-muscle myosin heavy chain [27–30]. Little is known about the role they play in HSV entry. In particular, there is no evidence that they contribute to define the host range of the virus. PILRα was reported to be expressed, and possibly to play a role in HSV infection of monocytes, a cell type not usually targeted by HSV [27]. The effect, if any, of depleting this receptor in epithelial cells, the targets of wt-HSV in vivo, was not investigated. The lack of contribution of gB receptors to overall viral tropism is highlighted by the finding that abrogation of the gD interaction with one of its receptors abrogates virus entry in virtually any cell.

In this study we addressed the question whether gB is a suitable glycoprotein for HSV retargeting. The aim was twofold, i.e., to better understand the biology of HSV entry through the lesson of retargeting studies, and to expand and improve the toolkit for the design of retargeted oncolytic HSVs. We report on three findings. First, the insertion of a scFv to HER2 in gB re-addresses HSV tropism to the HER2 receptor. In essence, when gB carries a ligand, the ligand receptor contributes to define the host range of the virus. This was an unexpected result since gB is responsible to execute the virion-cell fusion in HSV and across the Herpesviridae family. Second, we asked how does the novel HER2 receptor enable entry of the recombinant carrying the HER2-retargeted gB. We deleted a portion of gD, so that the virus lost the ability to use and be activated by the natural gD receptors; we further depleted cells of β6 and β8 integrins, which activate gH. We found that the entry of the gB-retargeted recombinant occurred in the absence of the cascade of gD and gH activation. Third, retargeting through the chimeric gBHER2 was intriguing since, the same scFv ligand to HER2 mediates entry when engineered in gD [45,46], gH [48], or gB (this work). This raised the question of how can the same receptor—HER2—enable virus entry by targeting alternatively gD, gH or gB, despite the fact that these glycoproteins carry out specific, non-interchangeable functions in wt-virus. We discuss these issues separately.

 

Source:

http://doi.org/10.1371/journal.ppat.1006352

 

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