The Murid Herpesvirus-4 gL Regulates an Entry- Associated Conformation Change in gH Laurent Gillet¤, Susanna Colaco, Philip G. Stevenson* Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom Abstract The glycoprotein H (gH)/gL heterodimer is crucial for herpesvirus membrane fusion. Yet how it functions is not well understood. The Murid Herpesvirus-4 gH, like that of other herpesviruses, adopts its normal virion conformation by associating with gL. However, gH switched back to a gL-independent conformation after virion endocytosis. This switch coincided with a conformation switch in gB and with capsid release. Virions lacking gL constitutively expressed the down- stream form of gH, prematurely switched gB to its down-stream form, and showed premature capsid release with poor infectivity. These data argue that gL plays a key role in regulating a gH and gB functional switch from cell binding to membrane fusion. Citation: Gillet L, Colaco S, Stevenson PG (2008) The Murid Herpesvirus-4 gL Regulates an Entry-Associated Conformation Change in gH. PLoS ONE 3(7): e2811. doi:10.1371/journal.pone.0002811 Editor: Douglas F. Nixon, University of California San Francisco, United States of America Received April 29, 2008; Accepted July 2, 2008; Published July 30, 2008 Copyright: 2008 Gillet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: LG is a Postdoctoral Researcher of the Fonds National Belge de la Recherche Scientifique. PGS is a Wellcome Trust Senior Clinical Fellow (GR076956MA). This work was also supported by Medical Research Council grants G0400427 and G9800903 and by CR-UK grant C19612/A6189. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pgs27@cam.ac.uk ¤ Current address: Immunology-Vaccinology, Faculty of Veterinary Medicine, University of Lie`ge, Lie`ge, Belgium Introduction Enveloped viruses deliver their capsids to the cytoplasm by fusing with either the plasma membrane or the limiting membranes of endocytic vesicles. Many viruses use a single glycoprotein for both binding and membrane fusion. Herpesvi- ruses, by contrast, use at least four [1,2]; their entry mechanisms are implicitly more complex. Proteins specific to individual herpesviruses can modulate fusion, for example the Herpes simplex virus gD brings together gB and gH/gL after ligand engagement to trigger fusion [3,4], and the Epstein-Barr virus gp42 binds gH/gL to promote B cell over epithelial cell fusion [5]. But the core fusion machinery of gB, gH and gL is conserved. Down-stream fusion events are therefore likely to be common to all herpesviruses. We have analyzed gB and gH/gL using the MHV-68 isolate of Murid Herpesvirus-4 (MuHV-4) [6–8]. This gamma-herpesvirus has subtle differences from the more extensively studied Herpes simplex virus, for example it lacks an obvious homolog of gD and fuses after endocytosis rather than at the plasma membrane [9]. Nevertheless, the conservation of gB and gH/gL implies that they operate similarly in these and other herpesviruses once the fusion mechanism is engaged. Incoming MuHV-4 virions first bind to cell surface heparan sulphate proteoglycans via gH/gL or gp70 [10,11]. Gp150 displacement then allows further, heparan sulphate- independent binding, probably via gB [10,12]. Virions are endocytosed and transported to LAMP-1+ late endosomes [13]; membrane fusion then releases virion capsids into the cytoplasm; and these move to the nuclear margin for genome delivery [13]. Both gB and gH have hydrophobic loops that could participate directly in fusion [14,15]. Structural analysis of the Herpes simplex virus gB has [14] shown how these might be made available. Comparison with the Vesicular stomatitis virus glycoprotein G [16] suggests that the crystallized form of gB, with its distinct crown formed mainly from C-terminal domains, is a down-stream form. Consistent with this, the Murid Herpesvirus-4 (MuHV-4) gB switches during entry from having readily accessible N-terminal epitopes and poorly accessible epitopes involving C-terminal domains to the converse [13]. The N-terminal domains are involved in cell binding [10]. Thus, gB on extracellular MuHV-4 virions adopts a conformation that allows cell-binding, then following endocytosis undergoes a pH-dependent conformation switch that leads to membrane fusion. Whether this conformation change itself drives membrane fusion, as for Vesicular stomatitis virus, or whether further conformation changes are required along the lines suggested by Heldwein et al. [14], remains unclear. While quite a lot is known about gB, much less is known about gH/gL. Understanding gH/gL therefore presents the major obstacle to understanding herpesvirus entry. Unlike gH, gL lacks obvious fusion loops or a trans-membrane domain. Its contribu- tion to membrane fusion is therefore likely to be indirect. So far, gL has been defined only as a chaperone for gH [9,17,18]. Some herpesviruses need gL to get gH into virions [17,18]; Pseudor- abiesvirus does not, but remains non-infectious without gL [19]; MuHV-4 lacking gL both incorporates gH into virions and remains infectious [20]. These differences may reflect how gB and gH associate. The Herpes simplex virus gB and gH come together only when gD is ligated [3,4], so their interaction is likely to involve extracellular domains; for gH, this would imply a requirement for gL. The MuHV-4 gB and gH associate constitutively and independently of gL, probably via an intra- membrane interaction [21]. Because MuHV-4 does not require gH/gL for viability, it provides a convenient tool with which to explore gL-dependent down-stream events in viral entry. PLoS ONE | www.plosone.org 1 July 2008 | Volume 3 | Issue 7 | e2811