MuHV-4 gH Figure 3. gL2 virions show a post-binding deficit in NMuMG cell infection. NMuMG cells were exposed to gL+ or gL2 MuHV-4 virions (3 h, 37uC), then washed 63 in PBS. Equivalent samples were then either fixed, permeabilized and stained for gN with mAb 3F7 to determine virion uptake by flow cytometry; or incubated for a further 3 h or 15 h, after which viral eGFP expression was assayed by flow cytometry of intact cells. Each point shows the result for 10,000 cells. Equivalent results - the infectivity of gL2 viruses was ,1% that of gL+ viruses for an equivalent level of cell binding - were obtained in a repeat experiment. doi:10.1371/journal.pone.0002811.g003 distribution after incubation at 37uC was observed for a range of different gL mutants, but not for their revertants or the wild-type (Fig. 4B). Since capsids are normally released only from perinuclear late endosomes, the gL2 virions must have released their capsids prematurely in the entry pathway. The glycoproteins of gL2 virions were still internalized rather than incorporated into the plasma membrane. Surprisingly, infection by gL2 virions also remained at least as sensitive to inhibitors of endocytosis and lysosomal acidification as that of the wild-type or gL+ revertants (Fig. 5). This may reflect that low pH has a wider importance for MuHV-4 entry than switching the conformation of virion glycoproteins. For example, it could be required to reveal a key cellular ligand. Simply exposing cell- bound virions to low pH triggered the gH conformation change at best poorly (data not shown). We have observed this also for gB [13]. Therefore the MuHV-4 gH and gB conformation switches were not simply pH-dependent as for Vesicular stomatitis virus glycoprotein G [16]: other features of the endosome environment were required. Nevertheless, low pH was essential for efficient gH and gB switching and for capsid release. It is important to note that most gL2 virions were non-infectious (Fig. 3). Therefore most of the prematurely capsids of gL2 virions were destined not to reach nuclear pores. What gL2 infection there was may have depended on a few of these capsids being transported, or on some virions still managing to follow the normal pathway to late endosomes without premature membrane fusion. gL2 virions show gB conformational instability Since MuHV-4 membrane fusion, as defined by capsid release, is associated with a conformation change in gB [13], we further analyzed incoming gL2 virions for changes in gB antigenicity. The gB of wild-type virions changed from BN-1A7+MG-1A122 (pre- fusion) at the cell surface to BN-1A72MG-1A12+ (post-fusion) in late endosomes (Fig. 4A). gL2 virions attached to the plasma membrane were also BN-1A7+MG-1A122. However, unlike the wild-type, some acquired the MG-1A12 epitope before reaching LAMP-1+ endosomes. A premature gB switch to MG-1A12+ was also observed for gL2 virions in NIH-3T3 cells (Fig. 6). The gL- dependent conformational instability of gB was not marked, as considerable BN-1A7 staining remained outside LAMP-1+ endosomes. Nevertheless, as judged by capsid release this degree of instability was functionally important. Flow cytometry of infected BHK-21 cells provided further evidence of gB conformational instability in the absence of gL PLoS ONE | www.plosone.org 4 July 2008 | Volume 3 | Issue 7 | e2811