gL2 MuHV-4 virions do show some attenuation relative to the wild-type. One deficit is reduced binding to BHK-21 fibroblasts [20]. This reflects that gH/gL binds to GAGs, whereas gH alone does not [11]. However, gL-deficient virions infect NMuMG epithelial poorly even though they bind to them quite well [20]. gL must therefore have another function that is important for NMuMG cell infection. Here we identify an entry-associated conformation change in gH that was consistent with gL dissociation in late endosomes, the site of MuHV-4 capsid release. When virions lacked gL, gH and gB both adopted their down- stream forms prematurely. This led to premature capsid release and poor infectivity. An important function of gL is therefore to regulate the gB and gH conformation changes that lead to viral membrane fusion. Results The MuHV-4 gH changes conformation after endocytosis MuHV-4 infects adherent cells via endocytosis. Capsid release is pH-dependent and occurs when virions reach the late endosomes/ lysosomes marked by lysosome-associated membrane protein-1 (LAMP-1) [13]. Capsid release is associated with a conformation change in gB. To test whether gH also changes, we examined its antigenicity before and after endocytosis (Fig. 1) using mAbs that MuHV-4 gH distinguish gL-dependent and gL-independent gH conformations [9,20]. The gH on wild-type virions is mostly bound to gL [20]. MAbs requiring both gH and gL for recognition, such as 7E5 and T2C12, accordingly stained incoming virions strongly at the cell surface (Fig. 1A). However, this staining was lost after endocytosis. In contrast, the gH-only-specific mAb MG-9B10 [20] gave weak staining at the cell surface and much stronger staining after endocytosis. Other gH-only-specific mAbs such as MG-1A2 and MG-2E6 [20] gave similar results to MG-9B10. The correspond- ing gH and gH/gL epitopes are all conformational, as the mAbs do not recognize denatured virions, and can all be expressed in the absence of other virion proteins on transfected cells. Therefore gH did not appear to be denatured or disguised, but rather switched from a gL-dependent to a gL-independent conformation. The MuHV-4 gH changes conformation in LAMP-1+ endosomes The disappearance of gH/gL epitopes coincided with virions reaching LAMP-1+ late endosomes; any residual gH/gL-specific staining after incubation at 37uC was confined to LAMP-12 compartments (Fig. 1B, arrow). In contrast, gH-only staining co- localized with LAMP-1. Most virion gH therefore switched from gH/gL on extracellular virions to gH-only in late endosomes. Figure 1. Glycoprotein H changes its antigenicity after endocytosis. A. NMuMG cells were exposed to MuHV-4 virions (3 p.f.u./cell, 2 h, 4uC), washed 63 with PBS and either fixed immediately (4uC) or after a further 2 h incubation at 37uC (37uC). The cells were then stained for gH (MG-9B10), gH/gL (T2C12, 7E5) or gN (3F7) as an invariant control. Glycoprotein staining is green; nuclei are counterstained with DAPI (blue). The data are representative of 5 experiments, and similar results were obtained with 3 other gH-specific/gH/gL-specific mAb pairs. Single cells are shown for optimal resolution. In this as in subsequent figures, each individual cell is fully representative of at least 75% of the total examined (n.100). B. Cells were infected as in A, then stained for LAMP-1 (red) plus either gH (mAb MG-9B10) or gH/gL (mAb 7E5) (green). Co-localization is yellow. The arrow indicates residual gH/gL staining that does not colocalize with LAMP-1. doi:10.1371/journal.pone.0002811.g001 PLoS ONE | www.plosone.org 2 July 2008 | Volume 3 | Issue 7 | e2811