Figure 5. gL-deficient MuHV-4 remains dependent on endocy- tosis and lysosomal acidification. BHK-21 cells were treated as indicated for 2 h, then infected with gL- (gL2DEL, gL 2 DEL-STOP) or gL+ (wild-type, revertant) MuHV-4 (1 p.f.u./cell). Drug treatment was maintained throughout the course of infection. Infection rates were determined by flow cytometric assay of viral eGFP expression after 18 h. Each point shows the result for 10,000 cells. Equivalent results were obtained in a repeat experiment. doi:10.1371/journal.pone.0002811.g005 (Fig. 7). For an equivalent level of gN expression, BN-1A7 gB staining (pre-fusion) was weaker on cells infected with gL knockout viruses than with wild-type or revertant viruses, and MG-1A12 staining (post-fusion) was stronger. Thus, the premature capsid release of gL2 virions reflected gH being in its down-stream gH- only form from the start, and gB engaging prematurely in membrane fusion after endocytosis. gL deficiency compromises host entry A key point with MuHV-4 is that in vitro molecular deficits can be linked to in vivo host colonization phenotypes. We have shown before that MuHV-4 gL knockouts colonize mice remarkably normally after intranasal inoculation of 104 plaque-forming units [20]. However, the in vitro entry deficit of gL2 virions (Fig. 3) suggested that initial host entry might be impaired. Since host colonization by MuHV-4 is relatively dose-independent [24], such a deficit might not be apparent except at very low inoculation doses. We therefore compared gL2 (2 independent mutants) and gL+ (revertant and wild-type) viruses for their capacity to establish MuHV-4 gH infection after intranasal inoculation of 1–100 p.f.u. (Table 1). gL disruption reduced significantly the rate of infection after low dose inoculation. Thus, while gL deficiency has little impact on MuHV- 4 intra-host spread, it compromised severely the efficiency with which cell-free virions first entered naive hosts. Discussion Sheer complexity has made herpesvirus membrane fusion hard to unravel, for example Herpes simplex virus can undergo endosomal or plasma membrane fusion in different cell types [25]. However, the underlying molecular events are likely to be similar. Endocytic entry is easier to dissect because binding and fusion remain anatomically distinct. MuHV-4 capsid release after endocytic entry was associated with conformation switches in both gB and gH. Low pH was a key trigger of these switches and gL was a key negative regulator. Thus, by associating with newly synthesized gH in the endoplasmic reticulum gL sets virions for cell binding, and by dissociating again in late endosomes of the next cell it triggers membrane fusion. Analogy with the Vesicular stomatitis virus glycoprotein G [15,16] would suggest that the conformation changes in the MuHV-4 gB are intrinsic to its fusion mechanism, with the stable BN-1A7+ or MG-1A12+ forms representing the respective pre- and post-fusion states. This general model does not apply so easily to gH (gH/gL being pre-fusion and gH-only post-fusion), as gL2 virions fail to express gH/gL yet fuse more readily than the wild- type rather than less. It is possible that gL2 virions express another, as yet unidentified, pre-fusion form of gH. However, we have found no antigenic evidence of other forms (data not shown). Our working hypothesis is therefore that gH/gL and gH-only are both pre-fusion - gH/gL being up-stream of gH-only - and that further gH conformation changes accompany fusion itself. In support of this, gH-only epitopes tended to disappear after a 4 h 37uC incubation, whereas MG-1A12-type gB epitopes were maintained (data not shown). Thus, gL dissociation from gH appears to prime gH for membrane fusion rather than being a part of the fusion reaction per se. A pre-fusion conformation change cannot be ruled out for gB either. Heldwein et al. [15] proposed that their gB conformation - which is probably a down-stream form [26] - undergoes further changes to reveal fusion loops. The neutralization epitopes mapped onto this structure [15] would also be difficult to explain if it were simply a post-fusion form. Thus, although the default explanation has to be that gB functions like the Vesicular stomatitis virus glycoprotein G because of their structural homology, it is also possible that the herpesvirus membrane fusion mechanism is different. gB and gH conformation changes must drive fusion, but this does not necessarily mean that every conformation change is part of the fusion mechanism. It is possible that the conformation changes described for Vesicular stomatits virus have for herpesviruses acquired a different function. But whatever the precise fusion mechanism of herpesviruses, a central role for the MuHV-4 gL in stabilizing gH and gB against engaging in fusion seemed clear. The influence of gL on the conformation of gB was presumably indirect. A loss of gL would change gH/gL to gH-only and would therefore disrupt the interaction [27] between gH/gL and the gB N-terminus (gB-NT). This would not separate gH from gB, since their association is independent of both gL [21] and gB-NT [27], but could promote a conformation change in the gB extracellular domain. Consistent with such a scheme, MuHV-4 lacking gB-NT shows a gB conformational instability much like that of gL2 mutants [27]. The gB-NT-gH/gL interaction does not appear to PLoS ONE | www.plosone.org 6 July 2008 | Volume 3 | Issue 7 | e2811