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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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gL-deficient virions show abnormal entry in NIH-3T3 fibroblasts.gL+ and gL− virions were bound to adherent NIH-3T3 cells (2 h, 4°C). Unbound virions were then removed by PBS wash. The cells were then either fixed (4°C) or first incubated (2 h, 37°C) to allow virion endocytosis and then fixed (37°C). All cells were then permeabilized and stained for MuHV-4 virion components (green) and LAMP-1 (red) as shown. Nuclei were counter-stained with DAPI (blue). In the absence of gL, both capsids and glycoproteins remained peripheral. gB conformation changes were also affected. Notably, MG-1A12+ gB appeared in peripheral, LAMP-1− endosomes. Representative cells are shown.
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pone-0002811-g006: gL-deficient virions show abnormal entry in NIH-3T3 fibroblasts.gL+ and gL− virions were bound to adherent NIH-3T3 cells (2 h, 4°C). Unbound virions were then removed by PBS wash. The cells were then either fixed (4°C) or first incubated (2 h, 37°C) to allow virion endocytosis and then fixed (37°C). All cells were then permeabilized and stained for MuHV-4 virion components (green) and LAMP-1 (red) as shown. Nuclei were counter-stained with DAPI (blue). In the absence of gL, both capsids and glycoproteins remained peripheral. gB conformation changes were also affected. Notably, MG-1A12+ gB appeared in peripheral, LAMP-1− endosomes. Representative cells are shown.

Mentions: Since MuHV-4 membrane fusion, as defined by capsid release, is associated with a conformation change in gB [13], we further analyzed incoming gL− virions for changes in gB antigenicity. The gB of wild-type virions changed from BN-1A7+MG-1A12− (pre-fusion) at the cell surface to BN-1A7−MG-1A12+ (post-fusion) in late endosomes (Fig. 4A). gL− virions attached to the plasma membrane were also BN-1A7+MG-1A12−. However, unlike the wild-type, some acquired the MG-1A12 epitope before reaching LAMP-1+ endosomes. A premature gB switch to MG-1A12+ was also observed for gL− virions in NIH-3T3 cells (Fig. 6). The gL-dependent conformational instability of gB was not marked, as considerable BN-1A7 staining remained outside LAMP-1+ endosomes. Nevertheless, as judged by capsid release this degree of instability was functionally important.


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

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

gL-deficient virions show abnormal entry in NIH-3T3 fibroblasts.gL+ and gL− virions were bound to adherent NIH-3T3 cells (2 h, 4°C). Unbound virions were then removed by PBS wash. The cells were then either fixed (4°C) or first incubated (2 h, 37°C) to allow virion endocytosis and then fixed (37°C). All cells were then permeabilized and stained for MuHV-4 virion components (green) and LAMP-1 (red) as shown. Nuclei were counter-stained with DAPI (blue). In the absence of gL, both capsids and glycoproteins remained peripheral. gB conformation changes were also affected. Notably, MG-1A12+ gB appeared in peripheral, LAMP-1− endosomes. Representative cells are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481400&req=5

pone-0002811-g006: gL-deficient virions show abnormal entry in NIH-3T3 fibroblasts.gL+ and gL− virions were bound to adherent NIH-3T3 cells (2 h, 4°C). Unbound virions were then removed by PBS wash. The cells were then either fixed (4°C) or first incubated (2 h, 37°C) to allow virion endocytosis and then fixed (37°C). All cells were then permeabilized and stained for MuHV-4 virion components (green) and LAMP-1 (red) as shown. Nuclei were counter-stained with DAPI (blue). In the absence of gL, both capsids and glycoproteins remained peripheral. gB conformation changes were also affected. Notably, MG-1A12+ gB appeared in peripheral, LAMP-1− endosomes. Representative cells are shown.
Mentions: Since MuHV-4 membrane fusion, as defined by capsid release, is associated with a conformation change in gB [13], we further analyzed incoming gL− virions for changes in gB antigenicity. The gB of wild-type virions changed from BN-1A7+MG-1A12− (pre-fusion) at the cell surface to BN-1A7−MG-1A12+ (post-fusion) in late endosomes (Fig. 4A). gL− virions attached to the plasma membrane were also BN-1A7+MG-1A12−. However, unlike the wild-type, some acquired the MG-1A12 epitope before reaching LAMP-1+ endosomes. A premature gB switch to MG-1A12+ was also observed for gL− virions in NIH-3T3 cells (Fig. 6). The gL-dependent conformational instability of gB was not marked, as considerable BN-1A7 staining remained outside LAMP-1+ endosomes. Nevertheless, as judged by capsid release this degree of instability was functionally important.

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