Figure 2. The gH/gL conformation change is pH-dependent. A. MuHV-4 virions were bound to NMuMG cells (2 h, 4uC) with or without bafilomycin (500 nM) or concanamycin A (1 mM). The cells were then washed in PBS to remove unbound virions, further incubated with or without drugs (2 h, 37uC), fixed, permeabilized and stained for gH or gH/gL plus LAMP-1. Nuclei were counterstained with DAPI. B. In a similar experiment, MuHV-4 virions were bound to NMuMG cells (2 h, MuHV-4 gH 4uC) with or without NH4Cl (100 mM) or chlorpromazine (3 mg/ml). Unbound virus was removed by PBS wash and the cells incubated further (37uC, 2 h), again with or without drug treatment, before fixing, permeabilizing and staining for gH or gH/gL. Although NH4Cl is generally considered to block endosomal acidification, here it also seemed to block virion endocytosis. doi:10.1371/journal.pone.0002811.g002 Thus, the gB conformation switch, the gH conformation switch and capsid release all coincided in time and place. The gH/gL conformation changes are pH-dependent The localization of the gH conformation switch to LAMP-1+ late endosomes suggested that it required low pH. This was confirmed with inhibitors of lysosomal acidification (Fig. 2A). After incubating virions with cells at 37uC without drugs, gH-only staining predominated over gH/gL. But when the cells were treated with bafilomycin or Concanamycin A, although virions were still endocytosed and transported to LAMP-1+ endosomes, gH/gL staining was preserved and gH-only staining correspond- ingly reduced. In particular, gH/gL staining now co-localized with LAMP-1, something never observed without drug treatment. Blocking endocytosis with chlorpromazine also blocked the gH conformation change, as did ammonium chloride treatment (Fig. 2B). Although the latter classically blocks endosomal acidification, it also blocked MuHV-4 endocytosis. Such an effect has been observed before [22]. Thus, in contrast to bafilomycin and Concanamycin A, the effect of NH4Cl could not be attributed to a rise in endosomal pH. However, it seemed clear from the bafilomycin and Concanamycin A treatments that the MuHV-4 gH conformation switch required virion delivery to low pH endosomes rather than just endocytosis. A lack of gL causes a post-binding entry deficit The apparent dissociation of gL from gH during MuHV-4 entry suggested that the gH-only conformation is functionally important, and therefore that gL has a more complicated role than merely folding gH for cell binding. One prediction of this would be that removing gL has consequences for post-binding entry events. We looked for evidence of a gL-dependent post-binding infection deficit by binding gL+ and gL2 virions to NMuMG cells for 3 h, washing off any unbound virions, and then comparing the initial virion uptake with subsequent viral eGFP expression (Fig. 3). We compared first a gL2 mutant with the wild-type, and then an independent gL2 mutant with its revertant. Cell binding was similar between the gL2 and gL+ viruses, but virus-driven eGFP expression was markedly different. After 6 h, eGFP expression by the gL2 viruses was virtually undetectable: for an equivalent level of binding, the infectivity of gL+ MuHV-4 for NMuMG cells at 6 h was at least 100 times greater than that of gL2 mutants. Even after 18 h, gL+ viruses showed at least 10-fold more eGFP expression for an equivalent level of binding. gL2 virions therefore showed a marked post-binding infection deficit in NMuMG cells. gL2 virions show premature capsid release We tracked the entry of gL2 virions into NMuMG cells by immunofluorescence (Fig. 4A). MAb MG-12B8 recognizes an ORF65 capsid epitope that is inaccessible on virions until they have uncoated [23]. There was accordingly no MG-12B8 staining of either wild-type or gL2 virions after binding to NMuMG cells at 4uC (Fig. 4A). After incubation at 37uC, the capsids of wild-type virions had become visible and reached the nuclear margin. In contrast, gL2 virion capsids, although accessible, remained scattered throughout the cytoplasm. The same abnormal capsid PLoS ONE | www.plosone.org 3 July 2008 | Volume 3 | Issue 7 | e2811