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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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Transport of neutralized virions. (a) Wild-type (WT) MuHV-4 virions (3 p.f.u. per cell) were left untreated or pre-incubated (2 h, 37 °C) with mAbs 8F10 (anti-gH–gL, IgG2a), T2C12 (anti-gH–gL, IgG2a) or SC-9E8 (anti-gB, IgG2a) (400 µg ml−1) before binding to NMuMG cells (2 h, 4 °C). For comparison, other NMuMG cells were similarly exposed to gL− virions (50 p.f.u. per cell, so as to get equivalent binding). The cells were then washed with PBS and either fixed immediately or first further incubated (2 h, 37 °C) to allow virion endocytosis. The cells were then stained for the ORF75c virion tegument protein with mAb BN-8C3 (IgG1, green) and for the late endosomal marker LAMP-1 (red), and with DAPI (blue). Red/green co-localization appears as yellow. Equivalent data were obtained in a repeat experiment. In this and all subsequent figures, the data shown are fully representative of at least 100 cells examined. The confocal settings were the same for the corresponding images at 4 °C and after 2 h at 37 °C. The numbers give the fraction of green signal co-localizing with red signal. The zoomed images show in more detail the relationship between virions (green) and endosomes (red). (b) As in (a), cells were exposed to virions for 2 h at 4 °C, washed in PBS, then either analysed immediately or first incubated for 1 or 2 h at 37 °C, but antibody binding was detected with an IgG1-specific alkaline phosphatase-conjugated secondary antibody and incubation with p-nitrophenylphosphate substrate, and quantified by measuring A405. For each condition, the A405 was normalized to the value obtained at 4 °C. The bars show mean±sem values from six wells. The ORF75c signal after incubation at 37 °C was significantly higher for non-neutralized WT virions than for gL− or neutralized WT virions (P<0.008 by Student’s t-test). The images show the distribution of ORF75c after virion binding at 4 °C, and after incubation at 37 °C for 1 or 2 h. (c) In a similar experiment to (b), virions were bound to cells for 2 h at 4 °C and detected with the gp150-specific IgG2b mAb BH-6H2 plus an alkaline phosphatase-conjugated IgG2b-specific secondary antibody. The bars show mean±semA405 values from six wells. The signal with 8F10-neutralized virions was reduced significantly relative to other treatments (P<10−5). Equivalent data were obtained in a repeat experiment.
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f2: Transport of neutralized virions. (a) Wild-type (WT) MuHV-4 virions (3 p.f.u. per cell) were left untreated or pre-incubated (2 h, 37 °C) with mAbs 8F10 (anti-gH–gL, IgG2a), T2C12 (anti-gH–gL, IgG2a) or SC-9E8 (anti-gB, IgG2a) (400 µg ml−1) before binding to NMuMG cells (2 h, 4 °C). For comparison, other NMuMG cells were similarly exposed to gL− virions (50 p.f.u. per cell, so as to get equivalent binding). The cells were then washed with PBS and either fixed immediately or first further incubated (2 h, 37 °C) to allow virion endocytosis. The cells were then stained for the ORF75c virion tegument protein with mAb BN-8C3 (IgG1, green) and for the late endosomal marker LAMP-1 (red), and with DAPI (blue). Red/green co-localization appears as yellow. Equivalent data were obtained in a repeat experiment. In this and all subsequent figures, the data shown are fully representative of at least 100 cells examined. The confocal settings were the same for the corresponding images at 4 °C and after 2 h at 37 °C. The numbers give the fraction of green signal co-localizing with red signal. The zoomed images show in more detail the relationship between virions (green) and endosomes (red). (b) As in (a), cells were exposed to virions for 2 h at 4 °C, washed in PBS, then either analysed immediately or first incubated for 1 or 2 h at 37 °C, but antibody binding was detected with an IgG1-specific alkaline phosphatase-conjugated secondary antibody and incubation with p-nitrophenylphosphate substrate, and quantified by measuring A405. For each condition, the A405 was normalized to the value obtained at 4 °C. The bars show mean±sem values from six wells. The ORF75c signal after incubation at 37 °C was significantly higher for non-neutralized WT virions than for gL− or neutralized WT virions (P<0.008 by Student’s t-test). The images show the distribution of ORF75c after virion binding at 4 °C, and after incubation at 37 °C for 1 or 2 h. (c) In a similar experiment to (b), virions were bound to cells for 2 h at 4 °C and detected with the gp150-specific IgG2b mAb BH-6H2 plus an alkaline phosphatase-conjugated IgG2b-specific secondary antibody. The bars show mean±semA405 values from six wells. The signal with 8F10-neutralized virions was reduced significantly relative to other treatments (P<10−5). Equivalent data were obtained in a repeat experiment.

Mentions: To investigate how gH–gL-directed neutralization inhibited MuHV-4 entry into NMuMG epithelial cells, we visualized virions by isotype-specific immunofluorescence for the ORF75c tegument protein (Fig. 2a). This allowed us to track both cell binding and membrane fusion, as ORF75c relocates rapidly to the cell nucleus when released by fusion (Gaspar et al., 2008). After incubation at 4 °C, ORF75c remained in virions bound to the plasma membrane; after 1 h at 37 °C, it was both in the cytoplasm – either in endocytosed virions or just released from them – and in the nucleus (Fig. 2b); after 2 h at 37 °C it was almost entirely in the nucleus. ORF75c staining increased during its relocation, presumably because release from the virion tegument made it more accessible to antibody. This increase was evident both by immunofluorescence of individual cells (Fig. 2a) and by ELISA of virus-exposed cell populations (Fig. 2b). The ELISA signal was consistently stronger after 1 h at 37 °C than after 2 h (Fig. 2b), suggesting that ORF75c is either masked or degraded with time.


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

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

Transport of neutralized virions. (a) Wild-type (WT) MuHV-4 virions (3 p.f.u. per cell) were left untreated or pre-incubated (2 h, 37 °C) with mAbs 8F10 (anti-gH–gL, IgG2a), T2C12 (anti-gH–gL, IgG2a) or SC-9E8 (anti-gB, IgG2a) (400 µg ml−1) before binding to NMuMG cells (2 h, 4 °C). For comparison, other NMuMG cells were similarly exposed to gL− virions (50 p.f.u. per cell, so as to get equivalent binding). The cells were then washed with PBS and either fixed immediately or first further incubated (2 h, 37 °C) to allow virion endocytosis. The cells were then stained for the ORF75c virion tegument protein with mAb BN-8C3 (IgG1, green) and for the late endosomal marker LAMP-1 (red), and with DAPI (blue). Red/green co-localization appears as yellow. Equivalent data were obtained in a repeat experiment. In this and all subsequent figures, the data shown are fully representative of at least 100 cells examined. The confocal settings were the same for the corresponding images at 4 °C and after 2 h at 37 °C. The numbers give the fraction of green signal co-localizing with red signal. The zoomed images show in more detail the relationship between virions (green) and endosomes (red). (b) As in (a), cells were exposed to virions for 2 h at 4 °C, washed in PBS, then either analysed immediately or first incubated for 1 or 2 h at 37 °C, but antibody binding was detected with an IgG1-specific alkaline phosphatase-conjugated secondary antibody and incubation with p-nitrophenylphosphate substrate, and quantified by measuring A405. For each condition, the A405 was normalized to the value obtained at 4 °C. The bars show mean±sem values from six wells. The ORF75c signal after incubation at 37 °C was significantly higher for non-neutralized WT virions than for gL− or neutralized WT virions (P<0.008 by Student’s t-test). The images show the distribution of ORF75c after virion binding at 4 °C, and after incubation at 37 °C for 1 or 2 h. (c) In a similar experiment to (b), virions were bound to cells for 2 h at 4 °C and detected with the gp150-specific IgG2b mAb BH-6H2 plus an alkaline phosphatase-conjugated IgG2b-specific secondary antibody. The bars show mean±semA405 values from six wells. The signal with 8F10-neutralized virions was reduced significantly relative to other treatments (P<10−5). Equivalent data were obtained in a repeat experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Transport of neutralized virions. (a) Wild-type (WT) MuHV-4 virions (3 p.f.u. per cell) were left untreated or pre-incubated (2 h, 37 °C) with mAbs 8F10 (anti-gH–gL, IgG2a), T2C12 (anti-gH–gL, IgG2a) or SC-9E8 (anti-gB, IgG2a) (400 µg ml−1) before binding to NMuMG cells (2 h, 4 °C). For comparison, other NMuMG cells were similarly exposed to gL− virions (50 p.f.u. per cell, so as to get equivalent binding). The cells were then washed with PBS and either fixed immediately or first further incubated (2 h, 37 °C) to allow virion endocytosis. The cells were then stained for the ORF75c virion tegument protein with mAb BN-8C3 (IgG1, green) and for the late endosomal marker LAMP-1 (red), and with DAPI (blue). Red/green co-localization appears as yellow. Equivalent data were obtained in a repeat experiment. In this and all subsequent figures, the data shown are fully representative of at least 100 cells examined. The confocal settings were the same for the corresponding images at 4 °C and after 2 h at 37 °C. The numbers give the fraction of green signal co-localizing with red signal. The zoomed images show in more detail the relationship between virions (green) and endosomes (red). (b) As in (a), cells were exposed to virions for 2 h at 4 °C, washed in PBS, then either analysed immediately or first incubated for 1 or 2 h at 37 °C, but antibody binding was detected with an IgG1-specific alkaline phosphatase-conjugated secondary antibody and incubation with p-nitrophenylphosphate substrate, and quantified by measuring A405. For each condition, the A405 was normalized to the value obtained at 4 °C. The bars show mean±sem values from six wells. The ORF75c signal after incubation at 37 °C was significantly higher for non-neutralized WT virions than for gL− or neutralized WT virions (P<0.008 by Student’s t-test). The images show the distribution of ORF75c after virion binding at 4 °C, and after incubation at 37 °C for 1 or 2 h. (c) In a similar experiment to (b), virions were bound to cells for 2 h at 4 °C and detected with the gp150-specific IgG2b mAb BH-6H2 plus an alkaline phosphatase-conjugated IgG2b-specific secondary antibody. The bars show mean±semA405 values from six wells. The signal with 8F10-neutralized virions was reduced significantly relative to other treatments (P<10−5). Equivalent data were obtained in a repeat experiment.
Mentions: To investigate how gH–gL-directed neutralization inhibited MuHV-4 entry into NMuMG epithelial cells, we visualized virions by isotype-specific immunofluorescence for the ORF75c tegument protein (Fig. 2a). This allowed us to track both cell binding and membrane fusion, as ORF75c relocates rapidly to the cell nucleus when released by fusion (Gaspar et al., 2008). After incubation at 4 °C, ORF75c remained in virions bound to the plasma membrane; after 1 h at 37 °C, it was both in the cytoplasm – either in endocytosed virions or just released from them – and in the nucleus (Fig. 2b); after 2 h at 37 °C it was almost entirely in the nucleus. ORF75c staining increased during its relocation, presumably because release from the virion tegument made it more accessible to antibody. This increase was evident both by immunofluorescence of individual cells (Fig. 2a) and by ELISA of virus-exposed cell populations (Fig. 2b). The ELISA signal was consistently stronger after 1 h at 37 °C than after 2 h (Fig. 2b), suggesting that ORF75c is either masked or degraded with time.

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