Limits...
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.

Show MeSH

Related in: MedlinePlus

Capsid migration of T2C12-neutralized and gL− virions. (a) 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. All cells were then stained for the ORF25 virion capsid component with mAb BH-6D3 (IgG1, green), for the late endosomal marker LAMP-1 (red), and with DAPI (blue). Equivalent data were obtained in a repeat experiment. (b) Cells were exposed to WT virions with or without T2C12 neutralization or to gL− virions as in (a), but antibody binding was detected with an alkaline phosphatase-conjugated IgG1-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 non-neutralized WT signal was significantly higher than that of gL− or T2C12-treated WT virions (P<0.0005 by Student’s t-test). Equivalent data were obtained in two further experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3755512&req=5

f3: Capsid migration of T2C12-neutralized and gL− virions. (a) 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. All cells were then stained for the ORF25 virion capsid component with mAb BH-6D3 (IgG1, green), for the late endosomal marker LAMP-1 (red), and with DAPI (blue). Equivalent data were obtained in a repeat experiment. (b) Cells were exposed to WT virions with or without T2C12 neutralization or to gL− virions as in (a), but antibody binding was detected with an alkaline phosphatase-conjugated IgG1-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 non-neutralized WT signal was significantly higher than that of gL− or T2C12-treated WT virions (P<0.0005 by Student’s t-test). Equivalent data were obtained in two further experiments.

Mentions: Peripheral virion arrest by mAb T2C12 was confirmed by staining for the ORF25 capsid component with mAb BH-6D3 (Fig. 3a). After incubation at 37 °C, the capsids of wild-type virions migrated to the nuclear margin, consistent with their release by membrane fusion. In contrast, the capsids of T2C12-neutralized and gL− virions remained outside late endosomes. Non-neutralized wild-type virions also showed an increase in ORF25 signal after incubation at 37 °C, consistent with released capsid making ORF25 epitopes more accessible (Fig. 3b), whereas T2C12-neutralized and gL− virions showed little such increase.


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

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

Capsid migration of T2C12-neutralized and gL− virions. (a) 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. All cells were then stained for the ORF25 virion capsid component with mAb BH-6D3 (IgG1, green), for the late endosomal marker LAMP-1 (red), and with DAPI (blue). Equivalent data were obtained in a repeat experiment. (b) Cells were exposed to WT virions with or without T2C12 neutralization or to gL− virions as in (a), but antibody binding was detected with an alkaline phosphatase-conjugated IgG1-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 non-neutralized WT signal was significantly higher than that of gL− or T2C12-treated WT virions (P<0.0005 by Student’s t-test). Equivalent data were obtained in two further experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Capsid migration of T2C12-neutralized and gL− virions. (a) 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. All cells were then stained for the ORF25 virion capsid component with mAb BH-6D3 (IgG1, green), for the late endosomal marker LAMP-1 (red), and with DAPI (blue). Equivalent data were obtained in a repeat experiment. (b) Cells were exposed to WT virions with or without T2C12 neutralization or to gL− virions as in (a), but antibody binding was detected with an alkaline phosphatase-conjugated IgG1-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 non-neutralized WT signal was significantly higher than that of gL− or T2C12-treated WT virions (P<0.0005 by Student’s t-test). Equivalent data were obtained in two further experiments.
Mentions: Peripheral virion arrest by mAb T2C12 was confirmed by staining for the ORF25 capsid component with mAb BH-6D3 (Fig. 3a). After incubation at 37 °C, the capsids of wild-type virions migrated to the nuclear margin, consistent with their release by membrane fusion. In contrast, the capsids of T2C12-neutralized and gL− virions remained outside late endosomes. Non-neutralized wild-type virions also showed an increase in ORF25 signal after incubation at 37 °C, consistent with released capsid making ORF25 epitopes more accessible (Fig. 3b), whereas T2C12-neutralized and gL− virions showed little such increase.

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