MuHV-4 gH Figure 6. gL-deficient virions show abnormal entry in NIH-3T3 fibroblasts. gL+ and gL2 virions were bound to adherent NIH-3T3 cells (2 h, 4uC). Unbound virions were then removed by PBS wash. The cells were then either fixed (4uC) or first incubated (2 h, 37uC) to allow virion endocytosis and then fixed (37uC). All cells were then permeabilized and stained for MuHV-4 virion components (green) and LAMP-1 (red) as shown. Nuclei were counter-stained with DAPI (blue). In the absence of gL, both capsids and glycoproteins remained peripheral. gB conformation changes were also affected. Notably, MG-1A12+ gB appeared in peripheral, LAMP-12 endosomes. Representative cells are shown. doi:10.1371/journal.pone.0002811.g006 Figure 7. gL-dependent gB conformational instability in MuHV- 4-infected BHK-21 cells. BHK-21 cells were left uninfected (UI) or infected with gL2 (gL2STOP, gL2DEL) or gL+ (wild-type, gL2DEL revertant) viruses (2 p.f.u./cell, 18 h). The cells were then trypsinized and stained with secondary antibody only (nil, solid lines) or for gN (mAb 3F7, dotted lines), gH-only (mAb MG-9B10, solid lines), gH/gL (mAb T2C12, dotted lines), the mAb BN-1A7+ virion gB conformation (gB vir, dotted lines) or the mAb MG-1A12+ pro-fusion gB conformation (gB fus, solid lines). The data are from 1 of 3 equivalent experiments. doi:10.1371/journal.pone.0002811.g007 play a significant role in stabilizing gH/gL, since gB-NT-deficient mutants maintain gH/gL expression and infect normally [27]. Therefore the gB conformation change alone is not sufficient for fusion: gL rather than gB-NT is the key fusion regulator. We envisage that the late endosomal milieu disrupts the gH/gL interaction, with low pH playing an important permissive role; gH then changes its conformation to allow hemi-fusion [28] and Table 1. Low dose in vivo infection by gL+ and gL2 MuHV-4. virus wild-type gL2DEL gL2STOP revertant 1 p.f.u. 6/121 1/15 2/15 4/12 infecting dose 10 p.f.u. 100 p.f.u. 12/12 7/12 7/12 12/12 12/12 10/12 10/12 12/12 1infected mice/total exposed. Mice were infected significantly better by wild-type MuHV-4 than by either the gL2DEL or gL2STOP mutants at 10 p.f.u./mouse (p,0.02 by Fisher’s exact test) and 1 p.f.u./mouse (p,0.05). The revertant was not significantly different to wild-type. Comparison between 1 p.f.u. and 10 p.f.u. suggested that the in vivo infectivity of gL knockout virions is about 1/10 that of the wild-type. doi:10.1371/journal.pone.0002811.t001 displace gB-NT; this then allows gB to switch to its pro-fusion form and complete the fusion reaction. In this way, the whole of membrane fusion can be regulated by gL. The neatness of this mechanism for regulating virion entry suggests that it will prove common to many herpesviruses. Materials and Methods Cells, viruses, mice BHK-21 fibroblasts, NIH-3T3 fibroblasts, NMuMG epithelial cells, and MCCD epithelial cells were propagated as described [27]. Wild-type and gL2 viruses were derived from a cloned MuHV-4 BAC [29] and grown in BHK-21 cells [29]. Female BALB/c mice (Harlan, Bicester, U.K.), were infected intranasally with MuHV-4 under general anaesthesia when 6–8 weeks old, in accordance with the animal care guidelines of Home Office Project Licence 80/1992 [30]. For virus titrations, lungs were removed post-mortem, freeze-thawed, and homogenized in 1 ml PBS. Serial dilutions were then plated onto BHK-21 cell monolayers. These were fixed with 4% formaldehyde after 4 days and stained with 0.1% toluidine blue for plaque counting [30]. Immunofluorescence Cells on glass coverslips [31] were exposed to MuHV-4 virions (3 p.f.u./cell) to allow binding, in most experiments at 4uC. The cells were then washed 63 in PBS to remove unbound virions and shifted to 37uC to allow endocytosis. They were fixed in 4% paraformaldehyde (30 min) either before or after endocytosis, then permeabilized with 0.1% Triton X-100. Where indicated, bafilomycin, concanamycin A, NH4Cl or chlorpromazine was added from 2 h pre-infection to the time of fixation. Viral glycoproteins were detected with murine mAbs (Table 2) at 1– 10 mg/ml plus Alexa488- or Alexa568-labeled anti-mouse IgG (Invitrogen, Paisley, U.K.) or Alexa488- or Alexa633- labeled anti- mouse IgG1 plus Alexa568- labeled anti-mouse IgG2a at 1 mg/ml. LAMP-1 was detected with mAb 104B (BD-Pharmingen, Oxford, U.K.) plus Alexa488- or Alexa568- labeled anti-rat IgG (Invitro- gen). Nuclei were counterstained with DAPI. Fluorescence was visualized on a Leica SP2 confocal microscope. Table 2. MAbs used in this study. MAb BN-1A7 MG-1A12 LS-B11 7E5 T2C12 MG-9B10 3F7 MG-12B8 Target gB gB gp150 gH/gL gH/gL gH gN ORF65 (capsid) Isotype Epitope1 IgG2a IgG2a IgG1 IgG2a IgG2a IgG2b IgG2a IgG2a conformational conformational linear conformational conformational conformational linear linear 1As defined by recognition or not of denatured protein on immunoblots. doi:10.1371/journal.pone.0002811.t002 PLoS ONE | www.plosone.org 8 July 2008 | Volume 3 | Issue 7 | e2811