The gH/gL conformation change is pH-dependent.

<p>A. MuHV-4 virions were bound to NMuMG cells (2 h, 4°C) with or without bafilomycin (500 nM) or concanamycin A (1 µM). The cells were then washed in PBS to remove unbound virions, further incubated with or without drugs (2 h, 37°C), 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, 4°C) with or without NH₄Cl (100 mM) or chlorpromazine (3 µg/ml). Unbound virus was removed by PBS wash and the cells incubated further (37°C, 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.</p

gL-deficient virions show abnormal entry in NIH-3T3 fibroblasts.

<p>gL⁺ and gL⁻ virions were bound to adherent NIH-3T3 cells (2 h, 4°C). Unbound virions were then removed by PBS wash. The cells were then either fixed (4°C) or first incubated (2 h, 37°C) to allow virion endocytosis and then fixed (37°C). 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-1⁻ endosomes. Representative cells are shown.</p

Infectivity of gp150 and gp70 MHV-68 knockout viruses.

<p>A. Wild-type and ORF4-deficient (ORF4⁻) viruses were tested for growth in BHK-21 and NMuMG cells after low multiplicity infection (0.01 PFU/cell). The data are from 1 of 2 equivalent experiments. B. BHK-21 or NMuMG cells were exposed to wild-type, ORF4-deficient (ORF4⁻) or gp150-deficient (gp150⁻) virions for different times before washing with PBS. 18 h later viral eGFP expression was assayed by flow cytometry. Each value is expressed as a percentage of the eGFP expression of unwashed cells. The data are from 1 of 2 equivalent experiments. C. Wild-type, ORF4-deficient (ORF4⁻) and gp150-deficient (gp150⁻) virions were preincubated with heparin then added to BHK-21 or NMuMG cells. 18 h later, viral eGFP expression was assayed by flow cytometry. Each value is expressed as a percentage of the eGFP expression with untreated virus. The data are from 1 of 2 equivalent experiments. D. BHK-21, CHO GAG⁺ or CHO GAG⁻ cells were infected overnight with wild-type (WT), gp150-deficient (gp150⁻) or ORF4-deficient viruses (ORF4⁻) in the presence of 10 µg/ml phosphonoacetic acid to inhibit any viral spread. Each virus expressed eGFP from an HCMV IE1 promoter in the BAC cassette at the left end of the genome. The number of infected cells was assessed by flow cytometric counting of eGFP⁺ cells. The data are from 1 of 2 equivalent experiments. E. CHO GAG⁺ or CHO GAG⁻ cells were exposed to gp150⁺ (WT) or gp150⁻ virions that were fluorescent by virtue of an eGFP tag on glycoprotein M. At the times shown, the cells were washed with PBS to remove unbound virions. Virion binding was then quantitated by flow cytometry. The data are from 1 of 2 equivalent experiments.</p

MAbs used in this study.

1<p>As defined by recognition or not of denatured protein on immunoblots.</p

GAG binding by gp150 truncations fused to GST.

<p>A. BHK-21 or L929 cells were incubated with GST alone or GST fused to gp150 ( = M7) amino acid residues 21-151, 41-151, 81-151 or 108-151. Binding was detected with a biotinylated anti-GST pAb plus phycoerythrin-conjugated streptavidin and analyzed by flow cytometry. 1 of 5 equivalent experiments is shown. B. The GST-M7:41-151 fusion was preincubated with soluble heparin as shown before staining BHK-21 cells. These cells were pre-digested or not with heparitinase III. The effect of heparitinase III treatment on cell surface GAGs is shown in the inset. Protein binding was analyzed by flow cytometry as in A. 1 of 3 equivalent experiments is shown. C. Binding of the different GST-M7 fusions to GAG⁺ and GAG⁻ CHO cells was quantitated by flow cytometry. 1 of 3 equivalent experiments is shown.</p

Inhibition of MuHV-4 infection by gp70-Fc and gHL-Fc blocking mAbs.

<p>A. Wild-type virions (200 p.f.u.) were incubated with mAbs as shown (2h, 37°C) then plaque-assayed on BHK-21 cells. We compared pairs of treatment arms by scoring (+ or -) for each antibody dilution whether the titer of a given treatment arm was more or less than the mean of both. We then performed an exact Chi-squared test on the comparisons. By this measure, mAb 230-4A2 but not mAb LT-6E8 gave significant neutralization, and 230-4A2 plus LT-6E8 was significantly better than 230-4A2 alone (p<0.0001). Equivalent data were obtained in 2 further experiments. B. Wild-type virions were incubated with mAbs (2 h, 37°C, 1 µg/1000 p.f.u.) then bound to either BHK-21 or NMuMG cells (2 h, 37°C, 3 p.f.u./cell). Bound virions were detected by washing, fixing and permeabilizing the cells, then staining them for gN with mAb 3F7. Secondary detection was with Alexa488-conjugated goat anti-mouse IgG pAb. This would have detected mAbs LT-6E8 and 230-4A2 even if the gN epitope were masked. The cells were scored simply as positive or negative on staining. Each bar shows the % positive of 10,000 cells, based on a gate where unexposed cells were 0% positive. The differences in binding between no mAb block/single mAb block, and between single mAb block/dual mAb block were highly significant (p<0.0001 by Student's t test). Equivalent data were obtained in 1 further experiment. C. Wild-type, gp70-deficient or gL-deficient virions (200 p.f.u.) were each incubated with mAbs (2h, 37°C, 1 µg/1000 p.f.u.), then with BHK-21 cells (2h, 37°C, 3 p.f.u./cell) as in B. The cells were then washed x3 in PBS, fixed and permeabilized, stained for gN with mAb 3F7 (green), counterstained with DAPI (blue) and examined microscopically for virion uptake. As in B, the anti-mouse secondary antibody would also have detected bound 230-4A2 or LT-6E8, so we could be sure no virions were missed because mAb binding had masked gN. D. Wild-type, gp70-deficient or gL-deficient virions (200 p.f.u.) were each incubated with mAbs as shown (2h, 37°C) then plaque assayed on BHK-21 cells. 230-4A2 and 230-5B2 both block gHL-Fc binding; LT-6E8 blocks gp70-Fc binding. Titers are plotted as % of p.f.u. without mAb for each virus. We compared treatment arms by an exact Chi-squared test of the pairwise comparisons at each antibody dilution, as in A. By this measure, mAb LT-6E8 neutralized the gL knockout but not the wild-type or gp70 knockout, and mAbs 230-4A2 and 230-5B2 neutralized the wild-type and gp70 knockout-the gp70 knockout significantly better than the wild-type-but not the gL knockout (p<0.004). The data are from 1 of 5 equivalent experiments.</p

Expression of gH/gL as a single Fc fusion protein.

<p>A. 293T cells were transfected with different expression plasmids. 48h later they were trypsinized and analyzed by flow cytometry for expression of gH with mAb 8C1 (short dashes) or gH/gL with mAb 7E5 (long dashes). Control = secondary antibody only (solid lines). “gH+gL ORF” used the full-length genomic ORF47; “gH+gL” used the RACE-mapped gL, which starts at the 5th in-frame ORF47 AUG codon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001669#pone.0001669-ShannonLowe1" target="_blank">[9]</a>; “gH+gL-GPI” used a GPI-linked form of the RACE-mapped gL. In each case, gH was expressed from the full-length genomic ORF22. nil = untransfected. B. 293T cells were transfected with diferent gH expression plasmids or left untransfected (nil), and 48h later analyzed for gH expression with mAb 8C1 (short dashes, white arrow) and for gH/gL expresion with mAb 7E5 (long dashes, grey arrow), as in A. Solid lines/black arrow = secondary antibody only. gHL-GPI and gHL-Fc are the same fusion protein with either a C-terminal GPI anchor or human IgG₁-Fc. Each histogram shows 10,000 cells. Both gH-specific and gH/gL-specific staining was significantly increased after gHL-Fc transfection compared with controls (p<0.00001 by Student's t test). Equivalent results were obtained in a repeat experiment.</p

Identification of ORF4 gene products.

<p>A. NMuMG epithelial cells or BHK-21 fibroblasts were infected with wild-type (WT) or ORF4-deficient (ORF4⁻) MHV-68 (2 PFU/cell). 48 h later, cells were removed by low speed centrifugation (400×g, 10 min) and virions then removed by high speed centrifugation (20,000×g, 3 h). Supernatants were immunoblotted with mAbs 16D2 or 9C7. B. The wild-type virions from A were similarly analyzed by SDS-PAGE and immunoblotting. Neat and 1/3 diluted lysates are shown. C. Virus from BHK-21 (B) or NMuMG cells (N) was denatured and then left undigested (nil) or treated with PNGase F (N-gase) or sialidase plus O-glycanase (O-gase). All samples were resolved by SDS-PAGE and immunoblotted with mAbs 9C7 or 16D2.</p

Heparin binding by ORF4 gene products.

<p>A. Gp70 truncations were fused to C-terminal human IgG₁ Fc and expressed by transient transfection of 293T cells. An equivalent fusion of gp150 amino acids 1-151 was used as a control. 293T cell supernatants were immunoblotted for fusion protein content with a human IgG Fc-specific pAb. B. The same supernatants were used to stain unfixed BHK-21 cells or NMuMG cells. Protein binding was assayed by flow cytometry. nil = supernatant from untransfected cells. 1 of 3 equivalent experiments is shown. C. The binding of each fusion protein to L929 cells was tested with or without soluble heparin. The fusion proteins were pre-incubated with heparin (1 h, 4°C) and then added to the cells. The graph summarizes data similar to those shown in B, from 1 of 3 equivalent experiments, each acquiring 30,000 events per sample. D. The binding of each Fc fusion protein to GAG⁺ or GAG⁻ CHO cells was assayed by flow cytometry. 1 of 2 equivalent experiments is shown. E. Each ORF4-specific mAb was tested for its capacity to inhibit BHK-21 cell binding by each Fc fusion protein. The mAbs and Fc fusion proteins were incubated together (1 h, 4°C) then added to cells. The cells stained with a human IgG-Fc-specific pAb. 1 of 2 equivalent experiments is shown, again acquiring 30,000 events per sample.</p

gL⁻ virions show a post-binding deficit in NMuMG cell infection.

<p>NMuMG cells were exposed to gL⁺ or gL⁻ MuHV-4 virions (3 h, 37°C), then washed ×3 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 gL⁻ viruses was <1% that of gL⁺ viruses for an equivalent level of cell binding - were obtained in a repeat experiment.</p