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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

Monitoring virion entry by envelope/tegument co-localization. WT MuHV-4 virions (3 p.f.u. per cell) were left untreated or pre-incubated (2 h, 37 °C) with mAb T2C12 (anti-gH–gL, IgG2a, 400 µg ml−1). WT (3 p.f.u. per cell) and gL− (50 p.f.u. per cell to give equivalent binding) virions were then bound to NMuMG cells for 2 h at 4 °C. The cells were washed with PBS and either fixed immediately or first incubated (2 h, 37 °C) to allow virion endocytosis. They were then stained for the ORF75c tegument component with mAb BN-8C3 (IgG1, green), for the gp150 envelope protein with mAb BH-6H2 (IgG2b, red) and with DAPI (blue). The numbers give the fraction of green signal co-localizing with red signal. The zoomed images show this relationship in more detail.
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f4: Monitoring virion entry by envelope/tegument co-localization. WT MuHV-4 virions (3 p.f.u. per cell) were left untreated or pre-incubated (2 h, 37 °C) with mAb T2C12 (anti-gH–gL, IgG2a, 400 µg ml−1). WT (3 p.f.u. per cell) and gL− (50 p.f.u. per cell to give equivalent binding) virions were then bound to NMuMG cells for 2 h at 4 °C. The cells were washed with PBS and either fixed immediately or first incubated (2 h, 37 °C) to allow virion endocytosis. They were then stained for the ORF75c tegument component with mAb BN-8C3 (IgG1, green), for the gp150 envelope protein with mAb BH-6H2 (IgG2b, red) and with DAPI (blue). The numbers give the fraction of green signal co-localizing with red signal. The zoomed images show this relationship in more detail.

Mentions: We also used gp150/ORF75c co-localization to track virion entry (Fig. 4). After binding at 4 °C, wild-type virions showed extensive gp150/ORF75c co-localization; after incubation at 37 °C, little co-localization remained. Thus most, if not all, wild-type virions underwent membrane fusion. In contrast, T2C12-neutralized and gL− virions maintained extensive gp150/ORF75c co-localization, even after incubation at 37 °C.


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

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

Monitoring virion entry by envelope/tegument co-localization. WT MuHV-4 virions (3 p.f.u. per cell) were left untreated or pre-incubated (2 h, 37 °C) with mAb T2C12 (anti-gH–gL, IgG2a, 400 µg ml−1). WT (3 p.f.u. per cell) and gL− (50 p.f.u. per cell to give equivalent binding) virions were then bound to NMuMG cells for 2 h at 4 °C. The cells were washed with PBS and either fixed immediately or first incubated (2 h, 37 °C) to allow virion endocytosis. They were then stained for the ORF75c tegument component with mAb BN-8C3 (IgG1, green), for the gp150 envelope protein with mAb BH-6H2 (IgG2b, red) and with DAPI (blue). The numbers give the fraction of green signal co-localizing with red signal. The zoomed images show this relationship in more detail.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Monitoring virion entry by envelope/tegument co-localization. WT MuHV-4 virions (3 p.f.u. per cell) were left untreated or pre-incubated (2 h, 37 °C) with mAb T2C12 (anti-gH–gL, IgG2a, 400 µg ml−1). WT (3 p.f.u. per cell) and gL− (50 p.f.u. per cell to give equivalent binding) virions were then bound to NMuMG cells for 2 h at 4 °C. The cells were washed with PBS and either fixed immediately or first incubated (2 h, 37 °C) to allow virion endocytosis. They were then stained for the ORF75c tegument component with mAb BN-8C3 (IgG1, green), for the gp150 envelope protein with mAb BH-6H2 (IgG2b, red) and with DAPI (blue). The numbers give the fraction of green signal co-localizing with red signal. The zoomed images show this relationship in more detail.
Mentions: We also used gp150/ORF75c co-localization to track virion entry (Fig. 4). After binding at 4 °C, wild-type virions showed extensive gp150/ORF75c co-localization; after incubation at 37 °C, little co-localization remained. Thus most, if not all, wild-type virions underwent membrane fusion. In contrast, T2C12-neutralized and gL− virions maintained extensive gp150/ORF75c co-localization, even after incubation at 37 °C.

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