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Virion endocytosis is a major target for murid herpesvirus-4 neutralization.

Glauser DL, Gillet L, Stevenson PG - J. Gen. Virol. (2012)

Bottom Line: The MuHV-4 gH-gL binds to heparan sulfate.However, most gH-gL-specific neutralizing antibodies did not block this interaction; neither did they act directly on fusion.The poor endocytosis of gH-gL-neutralized virions was recapitulated precisely by virions genetically lacking gL.

View Article: PubMed Central - PubMed

Affiliation: Division of Virology, Department of Pathology, University of Cambridge, UK.

ABSTRACT
Herpesviruses consistently transmit from immunocompetent carriers, implying that their neutralization is hard to achieve. Murid herpesvirus-4 (MuHV-4) exploits host IgG Fc receptors to bypass blocks to cell binding, and pH-dependent protein conformation changes to unveil its fusion machinery only after endocytosis. Nevertheless, neutralization remains possible by targeting the virion glycoprotein H (gH)-gL heterodimer, and the neutralizing antibody responses of MuHV-4 carriers are improved by boosting with recombinant gH-gL. We analysed here how gH-gL-directed neutralization works. The MuHV-4 gH-gL binds to heparan sulfate. However, most gH-gL-specific neutralizing antibodies did not block this interaction; neither did they act directly on fusion. Instead, they blocked virion endocytosis and transport to the late endosomes, where membrane fusion normally occurs. The poor endocytosis of gH-gL-neutralized virions was recapitulated precisely by virions genetically lacking gL. Therefore, driving virion uptake appears to be an important function of gH-gL that provides a major target for antibody-mediated neutralization.

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Related in: MedlinePlus

gH–gL-neutralized virions show defective endocytosis. (a) WT MuHV-4 virions (3 p.f.u. per cell) were left untreated or incubated (2 h, 37 °C) with mAb T2C12 (anti-gH–gL, IgG2a, 400 µg ml−1). These and gL− virions (50 p.f.u. per cell to give equivalent binding) were then bound to NMuMG cells (2 h, 4 °C). The cells were washed with PBS and incubated (2 h, 37 °C) to allow virion endocytosis, washed, fixed, and stained for the ORF75c virion tegument protein with mAb BN-8C3 (IgG1, green), for the early endosomal marker EEA-1 (red) and with DAPI (blue). Significant co-localization (yellow) was not observed. The zoomed images show virion and endosome distributions in more detail. (b) eGFP-expressing MuHV-4 virions (0.2 p.f.u. per cell) were left untreated or incubated (2 h, 37 °C) with mAb SE-9E8 (anti-gB, blocks fusion), mAb T2C12 (anti-gH–gL), mAb 7D6 (anti-gH–gL) or MuHV-4 immune serum (blocks binding). The virus was then added to BHK-21 cells and incubated (2 h, 37 °C) to allow virus endocytosis. The cells were then washed with PBS to remove unbound virions or with pH 3 citrate/phosphate buffer to inactivate non-endocytosed virions, then incubated (16 h, 37 °C) in complete medium with 100 µg phosphonoacetic acid ml−1 and analysed for viral eGFP expression by flow cytometry. Each point shows the mean±sem of two experiments. Comparison across each dilution by Fisher’s exact test established that acid washing reduced infection significantly for mAbs T2C12 and 7D6 (P<0.02), but not for mAb SC-9E8 or for immune serum (P>0.5). (c) A schematic diagram shows where neutralization seems to act during MuHV-4 entry. Immune sera block virion binding to the plasma membrane; mAb T2C12 blocks the endocytosis of bound virions, and so presumably prevents the delivery of an endocytic signal by its target, gH–gL; gB-directed neutralization blocks membrane fusion and so strands virions in late endosomes.
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f5: gH–gL-neutralized virions show defective endocytosis. (a) WT MuHV-4 virions (3 p.f.u. per cell) were left untreated or incubated (2 h, 37 °C) with mAb T2C12 (anti-gH–gL, IgG2a, 400 µg ml−1). These and gL− virions (50 p.f.u. per cell to give equivalent binding) were then bound to NMuMG cells (2 h, 4 °C). The cells were washed with PBS and incubated (2 h, 37 °C) to allow virion endocytosis, washed, fixed, and stained for the ORF75c virion tegument protein with mAb BN-8C3 (IgG1, green), for the early endosomal marker EEA-1 (red) and with DAPI (blue). Significant co-localization (yellow) was not observed. The zoomed images show virion and endosome distributions in more detail. (b) eGFP-expressing MuHV-4 virions (0.2 p.f.u. per cell) were left untreated or incubated (2 h, 37 °C) with mAb SE-9E8 (anti-gB, blocks fusion), mAb T2C12 (anti-gH–gL), mAb 7D6 (anti-gH–gL) or MuHV-4 immune serum (blocks binding). The virus was then added to BHK-21 cells and incubated (2 h, 37 °C) to allow virus endocytosis. The cells were then washed with PBS to remove unbound virions or with pH 3 citrate/phosphate buffer to inactivate non-endocytosed virions, then incubated (16 h, 37 °C) in complete medium with 100 µg phosphonoacetic acid ml−1 and analysed for viral eGFP expression by flow cytometry. Each point shows the mean±sem of two experiments. Comparison across each dilution by Fisher’s exact test established that acid washing reduced infection significantly for mAbs T2C12 and 7D6 (P<0.02), but not for mAb SC-9E8 or for immune serum (P>0.5). (c) A schematic diagram shows where neutralization seems to act during MuHV-4 entry. Immune sera block virion binding to the plasma membrane; mAb T2C12 blocks the endocytosis of bound virions, and so presumably prevents the delivery of an endocytic signal by its target, gH–gL; gB-directed neutralization blocks membrane fusion and so strands virions in late endosomes.

Mentions: T2C12-neutralized and gL− virions not only failed to reach late endosomes, but also failed to reach the early endosomes marked by staining for early endosome antigen 1 (EEA-1) (Fig. 5a). Thus, they appeared not to be endocytosed. This was confirmed by analysing the susceptibility to PBS and acid washes of virions exposed to sub-neutralizing antibody concentrations (Fig. 5b). PBS wash removes unbound virions; acid wash also inactivates virions bound to the cell surface. SC-9E8-exposed virions incubated with cells for 2 h at 37 °C showed similar sensitivity to PBS and acid washes, consistent with a post-endocytic infection block. In contrast, virions exposed to T2C12 or to 7D6, another gH–gL-specific neutralizing mAb that does not block HS binding (Gillet et al., 2008a), showed significantly greater sensitivity to acid wash. Thus, they were bound but not endocytosed. Immune sera mainly block cell binding (Gill et al., 2006) and gave equal sensitivity to either wash. Immune sera have low gH–gL-specific antibody titres (Gillet et al., 2007b) so, if cell binding is achieved, little further block to infection remains. Fig. 5(c) shows a schematic diagram of where we envisage that the different blocks occur.


Virion endocytosis is a major target for murid herpesvirus-4 neutralization.

Glauser DL, Gillet L, Stevenson PG - J. Gen. Virol. (2012)

gH–gL-neutralized virions show defective endocytosis. (a) WT MuHV-4 virions (3 p.f.u. per cell) were left untreated or incubated (2 h, 37 °C) with mAb T2C12 (anti-gH–gL, IgG2a, 400 µg ml−1). These and gL− virions (50 p.f.u. per cell to give equivalent binding) were then bound to NMuMG cells (2 h, 4 °C). The cells were washed with PBS and incubated (2 h, 37 °C) to allow virion endocytosis, washed, fixed, and stained for the ORF75c virion tegument protein with mAb BN-8C3 (IgG1, green), for the early endosomal marker EEA-1 (red) and with DAPI (blue). Significant co-localization (yellow) was not observed. The zoomed images show virion and endosome distributions in more detail. (b) eGFP-expressing MuHV-4 virions (0.2 p.f.u. per cell) were left untreated or incubated (2 h, 37 °C) with mAb SE-9E8 (anti-gB, blocks fusion), mAb T2C12 (anti-gH–gL), mAb 7D6 (anti-gH–gL) or MuHV-4 immune serum (blocks binding). The virus was then added to BHK-21 cells and incubated (2 h, 37 °C) to allow virus endocytosis. The cells were then washed with PBS to remove unbound virions or with pH 3 citrate/phosphate buffer to inactivate non-endocytosed virions, then incubated (16 h, 37 °C) in complete medium with 100 µg phosphonoacetic acid ml−1 and analysed for viral eGFP expression by flow cytometry. Each point shows the mean±sem of two experiments. Comparison across each dilution by Fisher’s exact test established that acid washing reduced infection significantly for mAbs T2C12 and 7D6 (P<0.02), but not for mAb SC-9E8 or for immune serum (P>0.5). (c) A schematic diagram shows where neutralization seems to act during MuHV-4 entry. Immune sera block virion binding to the plasma membrane; mAb T2C12 blocks the endocytosis of bound virions, and so presumably prevents the delivery of an endocytic signal by its target, gH–gL; gB-directed neutralization blocks membrane fusion and so strands virions in late endosomes.
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Related In: Results  -  Collection

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Show All Figures
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f5: gH–gL-neutralized virions show defective endocytosis. (a) WT MuHV-4 virions (3 p.f.u. per cell) were left untreated or incubated (2 h, 37 °C) with mAb T2C12 (anti-gH–gL, IgG2a, 400 µg ml−1). These and gL− virions (50 p.f.u. per cell to give equivalent binding) were then bound to NMuMG cells (2 h, 4 °C). The cells were washed with PBS and incubated (2 h, 37 °C) to allow virion endocytosis, washed, fixed, and stained for the ORF75c virion tegument protein with mAb BN-8C3 (IgG1, green), for the early endosomal marker EEA-1 (red) and with DAPI (blue). Significant co-localization (yellow) was not observed. The zoomed images show virion and endosome distributions in more detail. (b) eGFP-expressing MuHV-4 virions (0.2 p.f.u. per cell) were left untreated or incubated (2 h, 37 °C) with mAb SE-9E8 (anti-gB, blocks fusion), mAb T2C12 (anti-gH–gL), mAb 7D6 (anti-gH–gL) or MuHV-4 immune serum (blocks binding). The virus was then added to BHK-21 cells and incubated (2 h, 37 °C) to allow virus endocytosis. The cells were then washed with PBS to remove unbound virions or with pH 3 citrate/phosphate buffer to inactivate non-endocytosed virions, then incubated (16 h, 37 °C) in complete medium with 100 µg phosphonoacetic acid ml−1 and analysed for viral eGFP expression by flow cytometry. Each point shows the mean±sem of two experiments. Comparison across each dilution by Fisher’s exact test established that acid washing reduced infection significantly for mAbs T2C12 and 7D6 (P<0.02), but not for mAb SC-9E8 or for immune serum (P>0.5). (c) A schematic diagram shows where neutralization seems to act during MuHV-4 entry. Immune sera block virion binding to the plasma membrane; mAb T2C12 blocks the endocytosis of bound virions, and so presumably prevents the delivery of an endocytic signal by its target, gH–gL; gB-directed neutralization blocks membrane fusion and so strands virions in late endosomes.
Mentions: T2C12-neutralized and gL− virions not only failed to reach late endosomes, but also failed to reach the early endosomes marked by staining for early endosome antigen 1 (EEA-1) (Fig. 5a). Thus, they appeared not to be endocytosed. This was confirmed by analysing the susceptibility to PBS and acid washes of virions exposed to sub-neutralizing antibody concentrations (Fig. 5b). PBS wash removes unbound virions; acid wash also inactivates virions bound to the cell surface. SC-9E8-exposed virions incubated with cells for 2 h at 37 °C showed similar sensitivity to PBS and acid washes, consistent with a post-endocytic infection block. In contrast, virions exposed to T2C12 or to 7D6, another gH–gL-specific neutralizing mAb that does not block HS binding (Gillet et al., 2008a), showed significantly greater sensitivity to acid wash. Thus, they were bound but not endocytosed. Immune sera mainly block cell binding (Gill et al., 2006) and gave equal sensitivity to either wash. Immune sera have low gH–gL-specific antibody titres (Gillet et al., 2007b) so, if cell binding is achieved, little further block to infection remains. Fig. 5(c) shows a schematic diagram of where we envisage that the different blocks occur.

Bottom Line: The MuHV-4 gH-gL binds to heparan sulfate.However, most gH-gL-specific neutralizing antibodies did not block this interaction; neither did they act directly on fusion.The poor endocytosis of gH-gL-neutralized virions was recapitulated precisely by virions genetically lacking gL.

View Article: PubMed Central - PubMed

Affiliation: Division of Virology, Department of Pathology, University of Cambridge, UK.

ABSTRACT
Herpesviruses consistently transmit from immunocompetent carriers, implying that their neutralization is hard to achieve. Murid herpesvirus-4 (MuHV-4) exploits host IgG Fc receptors to bypass blocks to cell binding, and pH-dependent protein conformation changes to unveil its fusion machinery only after endocytosis. Nevertheless, neutralization remains possible by targeting the virion glycoprotein H (gH)-gL heterodimer, and the neutralizing antibody responses of MuHV-4 carriers are improved by boosting with recombinant gH-gL. We analysed here how gH-gL-directed neutralization works. The MuHV-4 gH-gL binds to heparan sulfate. However, most gH-gL-specific neutralizing antibodies did not block this interaction; neither did they act directly on fusion. Instead, they blocked virion endocytosis and transport to the late endosomes, where membrane fusion normally occurs. The poor endocytosis of gH-gL-neutralized virions was recapitulated precisely by virions genetically lacking gL. Therefore, driving virion uptake appears to be an important function of gH-gL that provides a major target for antibody-mediated neutralization.

Show MeSH
Related in: MedlinePlus