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The Murid Herpesvirus-4 gL regulates an entry-associated conformation change in gH.

Gillet L, Colaco S, Stevenson PG - PLoS ONE (2008)

Bottom Line: However, gH switched back to a gL-independent conformation after virion endocytosis.This switch coincided with a conformation switch in gB and with capsid release.Virions lacking gL constitutively expressed the down-stream form of gH, prematurely switched gB to its down-stream form, and showed premature capsid release with poor infectivity.

View Article: PubMed Central - PubMed

Affiliation: Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
The glycoprotein H (gH)/gL heterodimer is crucial for herpesvirus membrane fusion. Yet how it functions is not well understood. The Murid Herpesvirus-4 gH, like that of other herpesviruses, adopts its normal virion conformation by associating with gL. However, gH switched back to a gL-independent conformation after virion endocytosis. This switch coincided with a conformation switch in gB and with capsid release. Virions lacking gL constitutively expressed the down-stream form of gH, prematurely switched gB to its down-stream form, and showed premature capsid release with poor infectivity. These data argue that gL plays a key role in regulating a gH and gB functional switch from cell binding to membrane fusion.

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Glycoprotein H changes its antigenicity after endocytosis.A. NMuMG cells were exposed to MuHV-4 virions (3 p.f.u./cell, 2 h, 4°C), washed ×3 with PBS and either fixed immediately (4°C) or after a further 2 h incubation at 37°C (37°C). The cells were then stained for gH (MG-9B10), gH/gL (T2C12, 7E5) or gN (3F7) as an invariant control. Glycoprotein staining is green; nuclei are counterstained with DAPI (blue). The data are representative of 5 experiments, and similar results were obtained with 3 other gH-specific/gH/gL-specific mAb pairs. Single cells are shown for optimal resolution. In this as in subsequent figures, each individual cell is fully representative of at least 75% of the total examined (n>100). B. Cells were infected as in A, then stained for LAMP-1 (red) plus either gH (mAb MG-9B10) or gH/gL (mAb 7E5) (green). Co-localization is yellow. The arrow indicates residual gH/gL staining that does not colocalize with LAMP-1.
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pone-0002811-g001: Glycoprotein H changes its antigenicity after endocytosis.A. NMuMG cells were exposed to MuHV-4 virions (3 p.f.u./cell, 2 h, 4°C), washed ×3 with PBS and either fixed immediately (4°C) or after a further 2 h incubation at 37°C (37°C). The cells were then stained for gH (MG-9B10), gH/gL (T2C12, 7E5) or gN (3F7) as an invariant control. Glycoprotein staining is green; nuclei are counterstained with DAPI (blue). The data are representative of 5 experiments, and similar results were obtained with 3 other gH-specific/gH/gL-specific mAb pairs. Single cells are shown for optimal resolution. In this as in subsequent figures, each individual cell is fully representative of at least 75% of the total examined (n>100). B. Cells were infected as in A, then stained for LAMP-1 (red) plus either gH (mAb MG-9B10) or gH/gL (mAb 7E5) (green). Co-localization is yellow. The arrow indicates residual gH/gL staining that does not colocalize with LAMP-1.

Mentions: MuHV-4 infects adherent cells via endocytosis. Capsid release is pH-dependent and occurs when virions reach the late endosomes/lysosomes marked by lysosome-associated membrane protein-1 (LAMP-1) [13]. Capsid release is associated with a conformation change in gB. To test whether gH also changes, we examined its antigenicity before and after endocytosis (Fig. 1) using mAbs that distinguish gL-dependent and gL-independent gH conformations [9], [20]. The gH on wild-type virions is mostly bound to gL [20]. MAbs requiring both gH and gL for recognition, such as 7E5 and T2C12, accordingly stained incoming virions strongly at the cell surface (Fig. 1A). However, this staining was lost after endocytosis. In contrast, the gH-only-specific mAb MG-9B10 [20] gave weak staining at the cell surface and much stronger staining after endocytosis. Other gH-only-specific mAbs such as MG-1A2 and MG-2E6 [20] gave similar results to MG-9B10. The corresponding gH and gH/gL epitopes are all conformational, as the mAbs do not recognize denatured virions, and can all be expressed in the absence of other virion proteins on transfected cells. Therefore gH did not appear to be denatured or disguised, but rather switched from a gL-dependent to a gL-independent conformation.


The Murid Herpesvirus-4 gL regulates an entry-associated conformation change in gH.

Gillet L, Colaco S, Stevenson PG - PLoS ONE (2008)

Glycoprotein H changes its antigenicity after endocytosis.A. NMuMG cells were exposed to MuHV-4 virions (3 p.f.u./cell, 2 h, 4°C), washed ×3 with PBS and either fixed immediately (4°C) or after a further 2 h incubation at 37°C (37°C). The cells were then stained for gH (MG-9B10), gH/gL (T2C12, 7E5) or gN (3F7) as an invariant control. Glycoprotein staining is green; nuclei are counterstained with DAPI (blue). The data are representative of 5 experiments, and similar results were obtained with 3 other gH-specific/gH/gL-specific mAb pairs. Single cells are shown for optimal resolution. In this as in subsequent figures, each individual cell is fully representative of at least 75% of the total examined (n>100). B. Cells were infected as in A, then stained for LAMP-1 (red) plus either gH (mAb MG-9B10) or gH/gL (mAb 7E5) (green). Co-localization is yellow. The arrow indicates residual gH/gL staining that does not colocalize with LAMP-1.
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Related In: Results  -  Collection

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pone-0002811-g001: Glycoprotein H changes its antigenicity after endocytosis.A. NMuMG cells were exposed to MuHV-4 virions (3 p.f.u./cell, 2 h, 4°C), washed ×3 with PBS and either fixed immediately (4°C) or after a further 2 h incubation at 37°C (37°C). The cells were then stained for gH (MG-9B10), gH/gL (T2C12, 7E5) or gN (3F7) as an invariant control. Glycoprotein staining is green; nuclei are counterstained with DAPI (blue). The data are representative of 5 experiments, and similar results were obtained with 3 other gH-specific/gH/gL-specific mAb pairs. Single cells are shown for optimal resolution. In this as in subsequent figures, each individual cell is fully representative of at least 75% of the total examined (n>100). B. Cells were infected as in A, then stained for LAMP-1 (red) plus either gH (mAb MG-9B10) or gH/gL (mAb 7E5) (green). Co-localization is yellow. The arrow indicates residual gH/gL staining that does not colocalize with LAMP-1.
Mentions: MuHV-4 infects adherent cells via endocytosis. Capsid release is pH-dependent and occurs when virions reach the late endosomes/lysosomes marked by lysosome-associated membrane protein-1 (LAMP-1) [13]. Capsid release is associated with a conformation change in gB. To test whether gH also changes, we examined its antigenicity before and after endocytosis (Fig. 1) using mAbs that distinguish gL-dependent and gL-independent gH conformations [9], [20]. The gH on wild-type virions is mostly bound to gL [20]. MAbs requiring both gH and gL for recognition, such as 7E5 and T2C12, accordingly stained incoming virions strongly at the cell surface (Fig. 1A). However, this staining was lost after endocytosis. In contrast, the gH-only-specific mAb MG-9B10 [20] gave weak staining at the cell surface and much stronger staining after endocytosis. Other gH-only-specific mAbs such as MG-1A2 and MG-2E6 [20] gave similar results to MG-9B10. The corresponding gH and gH/gL epitopes are all conformational, as the mAbs do not recognize denatured virions, and can all be expressed in the absence of other virion proteins on transfected cells. Therefore gH did not appear to be denatured or disguised, but rather switched from a gL-dependent to a gL-independent conformation.

Bottom Line: However, gH switched back to a gL-independent conformation after virion endocytosis.This switch coincided with a conformation switch in gB and with capsid release.Virions lacking gL constitutively expressed the down-stream form of gH, prematurely switched gB to its down-stream form, and showed premature capsid release with poor infectivity.

View Article: PubMed Central - PubMed

Affiliation: Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
The glycoprotein H (gH)/gL heterodimer is crucial for herpesvirus membrane fusion. Yet how it functions is not well understood. The Murid Herpesvirus-4 gH, like that of other herpesviruses, adopts its normal virion conformation by associating with gL. However, gH switched back to a gL-independent conformation after virion endocytosis. This switch coincided with a conformation switch in gB and with capsid release. Virions lacking gL constitutively expressed the down-stream form of gH, prematurely switched gB to its down-stream form, and showed premature capsid release with poor infectivity. These data argue that gL plays a key role in regulating a gH and gB functional switch from cell binding to membrane fusion.

Show MeSH
Related in: MedlinePlus