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Regulation of herpes simplex virus gB-induced cell-cell fusion by mutant forms of gH/gL in the absence of gD and cellular receptors.

Atanasiu D, Cairns TM, Whitbeck JC, Saw WT, Rao S, Eisenberg RJ, Cohen GH - MBio (2013)

Bottom Line: Unexplainably, monoclonal antibodies (MAbs) with virus-neutralizing activity map to these residues.The absence of any of these proteins abolishes the entry process.Our study supports the concept that gB is the HSV fusogen and its activity is regulated by gH/gL.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

ABSTRACT

Unlabelled: Herpesvirus entry requires the viral glycoprotein triad of gB and gH/gL to carry out fusion between the virion envelope and a cellular membrane in order to release the nucleocapsid into the target cell. Herpes simplex virus (HSV) also requires glycoprotein gD to initiate the fusion cascade by binding a cell receptor such as nectin 1 or herpesvirus entry mediator (HVEM). While the structure of gB is that of a class III fusion protein, gH/gL has no features that resemble other viral fusion proteins. Instead, it is suggested that gH/gL acts as a regulator of gB. The crystal structure of HSV-2 gH/gL was obtained with a functional protein that had a deletion of 28 residues at the gH N terminus (gHΔ48/gL). Unexplainably, monoclonal antibodies (MAbs) with virus-neutralizing activity map to these residues. To reconcile these two disparate observations, we studied the ability of gHΔ48/gL to regulate fusion. Here, we show that the protein induces low (constitutive) levels of fusion by gB in the absence of gD and/or receptor. However, when gD and receptor are present, this mutant functions as well as does wild-type (wt) gH/gL for fusion. We propose that gHΔ48/gL has an intermediate structure on the pathway leading to full regulatory activation. We suggest that a key step in the pathway of fusion is the conversion of gH/gL to an activated state by receptor-bound gD; this activated gH/gL resembles gHΔ48/gL.

Importance: Herpes simplex viruses (HSVs) cause many human diseases, from mild cold sores to lethal neonatal herpes. As an enveloped virus, HSV must fuse its membrane with a host membrane in order for replication to take place. The virus uses four glycoproteins for this process, gD, gB, and gH/gL, and either of two cell receptors, herpesvirus entry mediator (HVEM) and nectin 1. Although the virus can enter the cell by direct fusion at the plasma membrane or via endocytosis, the same four glycoproteins are involved. The absence of any of these proteins abolishes the entry process. Here, we show that a mutant form of gH/gL, gHΔ48/gL, can induce fusion of gB-expressing cells in the absence of gD and a gD receptor. Our study supports the concept that gB is the HSV fusogen and its activity is regulated by gH/gL.

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Blocking of cell-cell fusion with gH1 and gL2 monoclonal antibodies. (A) Blocking of fusion triggered by gD306 in C10 cells transfected with gB and hybrid gH1Δ48/gL2. Both type 1 (52S and LP11) and type 2 (CHL18 and CHL34) gH/gL MAbs were used. (B) Effect of MAbs on the constitutive function of gH1Δ48/gL2. Fusion levels were expressed as percentages of the no-antibody sample.
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fig5: Blocking of cell-cell fusion with gH1 and gL2 monoclonal antibodies. (A) Blocking of fusion triggered by gD306 in C10 cells transfected with gB and hybrid gH1Δ48/gL2. Both type 1 (52S and LP11) and type 2 (CHL18 and CHL34) gH/gL MAbs were used. (B) Effect of MAbs on the constitutive function of gH1Δ48/gL2. Fusion levels were expressed as percentages of the no-antibody sample.

Mentions: Earlier, we postulated that there are two functional “faces” on gH/gL, one that interacts with gD and a second that interacts with gB. This argument was based on the ability of two gH/gL virus-neutralizing MAbs, 52S and LP11, to block cell fusion (15). The 52S epitope, with monoclonal antibody-resistant (MAR) residue 536 (26, 27), was proposed to be on the gD face, and the MAb blocks a step in entry initiated by the gD-gH/gL interaction. In contrast, LP11 (MAR residues 86, 168, and 329) (Fig. 1) was proposed to block the gH/gL interaction with gB (gB face) (15). If our arguments are correct, we postulate that gHΔ48/gL can act directly on the gB face, and as such, only the gB face is important. Here, we asked on which side of gHΔ48/gL these MAbs act. However, both LP11 and 52S are type 1 specific, while our anti-gL MAbs CΔ48L3, CHL18, and CHL26 are type 2 specific. We overcame this problem by using a hybrid molecule which contains a type 1 version of gHΔ48 and a type 2 version of gL. Our data show that the deletions produced in type 1 gH/gL (gH1Δ48/gL2) also function in cell-cell fusion and virus entry (23). We have used hybrid molecules in a fusion assay, where wt type 2 glycoproteins were replaced with the equivalent type 1 proteins, with no loss in activity (23, 28). Indeed, we found by immunofluorescence that the resulting hybrid, gH1Δ48/gL2, interacts with both the type 1-neutralizing MAbs 52S and LP11. LP11, 52S, CHL18, and CHL34 epitopes were present on the hybrid molecule (data not shown). When C10 cells were transfected with plasmids for gB along with gH1Δ48 and gL2, addition of gD306t triggered fusion. Moreover, both MAb 52S and MAb LP11 blocked this activity (Fig. 5A), showing that these two type 1-specific epitopes are presented the same way in the type 1/type 2 hybrid as in their respective type 1 counterpart. Importantly, the type-2-specific CHL18 MAb also blocked fusion when this hybrid form was used. Interestingly, CHL34 blocked fusion of cells transfected with gH2Δ48 and gL2 (Fig. 4C) but had no effect on fusion of cells bearing the hybrid gH1Δ48/gL2 (Fig. 5A). Thus, we conclude that this epitope was altered in the hybrid.


Regulation of herpes simplex virus gB-induced cell-cell fusion by mutant forms of gH/gL in the absence of gD and cellular receptors.

Atanasiu D, Cairns TM, Whitbeck JC, Saw WT, Rao S, Eisenberg RJ, Cohen GH - MBio (2013)

Blocking of cell-cell fusion with gH1 and gL2 monoclonal antibodies. (A) Blocking of fusion triggered by gD306 in C10 cells transfected with gB and hybrid gH1Δ48/gL2. Both type 1 (52S and LP11) and type 2 (CHL18 and CHL34) gH/gL MAbs were used. (B) Effect of MAbs on the constitutive function of gH1Δ48/gL2. Fusion levels were expressed as percentages of the no-antibody sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585445&req=5

fig5: Blocking of cell-cell fusion with gH1 and gL2 monoclonal antibodies. (A) Blocking of fusion triggered by gD306 in C10 cells transfected with gB and hybrid gH1Δ48/gL2. Both type 1 (52S and LP11) and type 2 (CHL18 and CHL34) gH/gL MAbs were used. (B) Effect of MAbs on the constitutive function of gH1Δ48/gL2. Fusion levels were expressed as percentages of the no-antibody sample.
Mentions: Earlier, we postulated that there are two functional “faces” on gH/gL, one that interacts with gD and a second that interacts with gB. This argument was based on the ability of two gH/gL virus-neutralizing MAbs, 52S and LP11, to block cell fusion (15). The 52S epitope, with monoclonal antibody-resistant (MAR) residue 536 (26, 27), was proposed to be on the gD face, and the MAb blocks a step in entry initiated by the gD-gH/gL interaction. In contrast, LP11 (MAR residues 86, 168, and 329) (Fig. 1) was proposed to block the gH/gL interaction with gB (gB face) (15). If our arguments are correct, we postulate that gHΔ48/gL can act directly on the gB face, and as such, only the gB face is important. Here, we asked on which side of gHΔ48/gL these MAbs act. However, both LP11 and 52S are type 1 specific, while our anti-gL MAbs CΔ48L3, CHL18, and CHL26 are type 2 specific. We overcame this problem by using a hybrid molecule which contains a type 1 version of gHΔ48 and a type 2 version of gL. Our data show that the deletions produced in type 1 gH/gL (gH1Δ48/gL2) also function in cell-cell fusion and virus entry (23). We have used hybrid molecules in a fusion assay, where wt type 2 glycoproteins were replaced with the equivalent type 1 proteins, with no loss in activity (23, 28). Indeed, we found by immunofluorescence that the resulting hybrid, gH1Δ48/gL2, interacts with both the type 1-neutralizing MAbs 52S and LP11. LP11, 52S, CHL18, and CHL34 epitopes were present on the hybrid molecule (data not shown). When C10 cells were transfected with plasmids for gB along with gH1Δ48 and gL2, addition of gD306t triggered fusion. Moreover, both MAb 52S and MAb LP11 blocked this activity (Fig. 5A), showing that these two type 1-specific epitopes are presented the same way in the type 1/type 2 hybrid as in their respective type 1 counterpart. Importantly, the type-2-specific CHL18 MAb also blocked fusion when this hybrid form was used. Interestingly, CHL34 blocked fusion of cells transfected with gH2Δ48 and gL2 (Fig. 4C) but had no effect on fusion of cells bearing the hybrid gH1Δ48/gL2 (Fig. 5A). Thus, we conclude that this epitope was altered in the hybrid.

Bottom Line: Unexplainably, monoclonal antibodies (MAbs) with virus-neutralizing activity map to these residues.The absence of any of these proteins abolishes the entry process.Our study supports the concept that gB is the HSV fusogen and its activity is regulated by gH/gL.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

ABSTRACT

Unlabelled: Herpesvirus entry requires the viral glycoprotein triad of gB and gH/gL to carry out fusion between the virion envelope and a cellular membrane in order to release the nucleocapsid into the target cell. Herpes simplex virus (HSV) also requires glycoprotein gD to initiate the fusion cascade by binding a cell receptor such as nectin 1 or herpesvirus entry mediator (HVEM). While the structure of gB is that of a class III fusion protein, gH/gL has no features that resemble other viral fusion proteins. Instead, it is suggested that gH/gL acts as a regulator of gB. The crystal structure of HSV-2 gH/gL was obtained with a functional protein that had a deletion of 28 residues at the gH N terminus (gHΔ48/gL). Unexplainably, monoclonal antibodies (MAbs) with virus-neutralizing activity map to these residues. To reconcile these two disparate observations, we studied the ability of gHΔ48/gL to regulate fusion. Here, we show that the protein induces low (constitutive) levels of fusion by gB in the absence of gD and/or receptor. However, when gD and receptor are present, this mutant functions as well as does wild-type (wt) gH/gL for fusion. We propose that gHΔ48/gL has an intermediate structure on the pathway leading to full regulatory activation. We suggest that a key step in the pathway of fusion is the conversion of gH/gL to an activated state by receptor-bound gD; this activated gH/gL resembles gHΔ48/gL.

Importance: Herpes simplex viruses (HSVs) cause many human diseases, from mild cold sores to lethal neonatal herpes. As an enveloped virus, HSV must fuse its membrane with a host membrane in order for replication to take place. The virus uses four glycoproteins for this process, gD, gB, and gH/gL, and either of two cell receptors, herpesvirus entry mediator (HVEM) and nectin 1. Although the virus can enter the cell by direct fusion at the plasma membrane or via endocytosis, the same four glycoproteins are involved. The absence of any of these proteins abolishes the entry process. Here, we show that a mutant form of gH/gL, gHΔ48/gL, can induce fusion of gB-expressing cells in the absence of gD and a gD receptor. Our study supports the concept that gB is the HSV fusogen and its activity is regulated by gH/gL.

Show MeSH
Related in: MedlinePlus