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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

gL− virions also show little conformation change in gB. (a) NMuMG cells were incubated (2 h, 4 °C) with WT (3 p.f.u. per cell) or gL− (50 p.f.u. per cell for equivalent binding) MuHV-4, then washed with PBS and either fixed immediately or first further incubated (2 h, 37 °C) to allow virion endocytosis. The cells were stained for pre-fusion gB with mAb BN-1A7 (IgG2a) or for post-fusion gB with mAb MG-1A12 (IgG2a) (both green), for LAMP-1 (red) and with DAPI (blue). Co-localization appears as yellow. Equivalent data were obtained in three further experiments. (b) Cells and viruses were incubated as in (a), then antibody binding was detected with an alkaline phosphatase-conjugated IgG2a-specific secondary antibody, p-nitrophenylphosphate substrate and A405. For each condition, the A405 was normalized to the value obtained at 4 °C. The bars show mean±sem values from six wells. After incubation at 37 °C, the WT BN-1A7 signal was reduced significantly relative to gL− (P<10−9 by Student’s t-test) and the WT MG-1A12 signal was increased significantly (P<10−5). Equivalent data were obtained in a repeat experiment.
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f7: gL− virions also show little conformation change in gB. (a) NMuMG cells were incubated (2 h, 4 °C) with WT (3 p.f.u. per cell) or gL− (50 p.f.u. per cell for equivalent binding) MuHV-4, then washed with PBS and either fixed immediately or first further incubated (2 h, 37 °C) to allow virion endocytosis. The cells were stained for pre-fusion gB with mAb BN-1A7 (IgG2a) or for post-fusion gB with mAb MG-1A12 (IgG2a) (both green), for LAMP-1 (red) and with DAPI (blue). Co-localization appears as yellow. Equivalent data were obtained in three further experiments. (b) Cells and viruses were incubated as in (a), then antibody binding was detected with an alkaline phosphatase-conjugated IgG2a-specific secondary antibody, p-nitrophenylphosphate substrate and A405. For each condition, the A405 was normalized to the value obtained at 4 °C. The bars show mean±sem values from six wells. After incubation at 37 °C, the WT BN-1A7 signal was reduced significantly relative to gL− (P<10−9 by Student’s t-test) and the WT MG-1A12 signal was increased significantly (P<10−5). Equivalent data were obtained in a repeat experiment.

Mentions: gL− virions (Fig. 7) similarly maintained gB in its ‘extracellular virion’ form (BN-1A7+MG-1A12−). Here, we used BN-1A7 (IgG2a) rather than SC-9A5 (IgG3) to detect pre-fusion gB because there was no need to allow for bound T2C12 (IgG2a); both epitopes are present on extracellular virions and lost after endocytosis (Glauser et al., 2011). Immunofluorescence (Fig. 7a) showed that the BN-1A7 signal was maintained much better with gL− than with wild-type virions. ELISA of gL− virions (Fig. 7b) showed some loss of BN-1A7 detection at 37 °C, presumably because these virions bind less well, but the loss was substantially greater for wild-type virions. Both immunofluorescence (Fig. 7a) and ELISA (Fig. 7b) showed gL− virions remaining largely MG-1A12−, while wild-type virions became strongly MG-1A12+. Tracking gB antigenic changes therefore supported the idea that having less functional gH–gL, through either mAb binding or gL disruption, impaired virion endocytosis.


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

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

gL− virions also show little conformation change in gB. (a) NMuMG cells were incubated (2 h, 4 °C) with WT (3 p.f.u. per cell) or gL− (50 p.f.u. per cell for equivalent binding) MuHV-4, then washed with PBS and either fixed immediately or first further incubated (2 h, 37 °C) to allow virion endocytosis. The cells were stained for pre-fusion gB with mAb BN-1A7 (IgG2a) or for post-fusion gB with mAb MG-1A12 (IgG2a) (both green), for LAMP-1 (red) and with DAPI (blue). Co-localization appears as yellow. Equivalent data were obtained in three further experiments. (b) Cells and viruses were incubated as in (a), then antibody binding was detected with an alkaline phosphatase-conjugated IgG2a-specific secondary antibody, p-nitrophenylphosphate substrate and A405. For each condition, the A405 was normalized to the value obtained at 4 °C. The bars show mean±sem values from six wells. After incubation at 37 °C, the WT BN-1A7 signal was reduced significantly relative to gL− (P<10−9 by Student’s t-test) and the WT MG-1A12 signal was increased significantly (P<10−5). Equivalent data were obtained in a repeat experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3755512&req=5

f7: gL− virions also show little conformation change in gB. (a) NMuMG cells were incubated (2 h, 4 °C) with WT (3 p.f.u. per cell) or gL− (50 p.f.u. per cell for equivalent binding) MuHV-4, then washed with PBS and either fixed immediately or first further incubated (2 h, 37 °C) to allow virion endocytosis. The cells were stained for pre-fusion gB with mAb BN-1A7 (IgG2a) or for post-fusion gB with mAb MG-1A12 (IgG2a) (both green), for LAMP-1 (red) and with DAPI (blue). Co-localization appears as yellow. Equivalent data were obtained in three further experiments. (b) Cells and viruses were incubated as in (a), then antibody binding was detected with an alkaline phosphatase-conjugated IgG2a-specific secondary antibody, p-nitrophenylphosphate substrate and A405. For each condition, the A405 was normalized to the value obtained at 4 °C. The bars show mean±sem values from six wells. After incubation at 37 °C, the WT BN-1A7 signal was reduced significantly relative to gL− (P<10−9 by Student’s t-test) and the WT MG-1A12 signal was increased significantly (P<10−5). Equivalent data were obtained in a repeat experiment.
Mentions: gL− virions (Fig. 7) similarly maintained gB in its ‘extracellular virion’ form (BN-1A7+MG-1A12−). Here, we used BN-1A7 (IgG2a) rather than SC-9A5 (IgG3) to detect pre-fusion gB because there was no need to allow for bound T2C12 (IgG2a); both epitopes are present on extracellular virions and lost after endocytosis (Glauser et al., 2011). Immunofluorescence (Fig. 7a) showed that the BN-1A7 signal was maintained much better with gL− than with wild-type virions. ELISA of gL− virions (Fig. 7b) showed some loss of BN-1A7 detection at 37 °C, presumably because these virions bind less well, but the loss was substantially greater for wild-type virions. Both immunofluorescence (Fig. 7a) and ELISA (Fig. 7b) showed gL− virions remaining largely MG-1A12−, while wild-type virions became strongly MG-1A12+. Tracking gB antigenic changes therefore supported the idea that having less functional gH–gL, through either mAb binding or gL disruption, impaired virion endocytosis.

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