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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 gH2 and gL2 monoclonal antibodies. (A to C) C10 cells were transfected with gB and gH2/gL2 (A), gH2Δ29L2 (B), or gH2Δ48/gL2 (C). Cells were incubated with 100 µg/ml of the indicated MAb for 2 h before fusion was triggered with gD2306. Fusion levels were determined based on the number and size of syncytia identified by immunofluorescence. (D) Effect of MAbs on the constitutive function of gH2Δ48/gL2 (in the absence of gD). Fusion levels were expressed as percentages of the no-antibody sample.
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fig4: Blocking of cell-cell fusion with gH2 and gL2 monoclonal antibodies. (A to C) C10 cells were transfected with gB and gH2/gL2 (A), gH2Δ29L2 (B), or gH2Δ48/gL2 (C). Cells were incubated with 100 µg/ml of the indicated MAb for 2 h before fusion was triggered with gD2306. Fusion levels were determined based on the number and size of syncytia identified by immunofluorescence. (D) Effect of MAbs on the constitutive function of gH2Δ48/gL2 (in the absence of gD). Fusion levels were expressed as percentages of the no-antibody sample.

Mentions: B78H1-C10 cells expressing gB and wild-type gH2/gL2, gH2Δ29/gL2, or gH2Δ48/gL2 were incubated with the indicated antibody for 2 h before fusion was triggered with gD306t. As expected, the gH MAb CHL25 failed to block cell-cell fusion and MAb CHL2 blocked fusion, regardless of the form of gH/gL present in the transfected cells (Fig. 4A to C). Critically, as previously shown (24), none of the gL MAbs had any effect on fusion of cells transfected with wt gH2/gL2 (Fig. 4A). In contrast, three gL MAbs, CHL18, CHL34, and CΔ48L3, each of which recognizes a different stretch of amino acids of gL (Table 1), blocked fusion of cells transfected with either gH2Δ29/gL2 (Fig. 4B) or gH2Δ48/gL2 (Fig. 4C). Thus, the properties of these MAbs fit with our rationale that deletion of portions of the gH N terminus affects the C terminus of gL so that the MAbs now block. Interestingly, anti-gL MAb CHL26 had an intermediate effect, possibly because its epitope is partially uncovered in the two truncated forms (Fig. 4B and C). We propose that region 146 to 203 of gL is hidden in wt gH/gL and exposed when gH residues 19 to 47 are absent (Fig. 4B and C). We conclude that once these sites of gL (in the context of wt gH/gL) are bound by antibodies, triggering of downstream events is blocked at that step.


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 gH2 and gL2 monoclonal antibodies. (A to C) C10 cells were transfected with gB and gH2/gL2 (A), gH2Δ29L2 (B), or gH2Δ48/gL2 (C). Cells were incubated with 100 µg/ml of the indicated MAb for 2 h before fusion was triggered with gD2306. Fusion levels were determined based on the number and size of syncytia identified by immunofluorescence. (D) Effect of MAbs on the constitutive function of gH2Δ48/gL2 (in the absence of gD). 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

fig4: Blocking of cell-cell fusion with gH2 and gL2 monoclonal antibodies. (A to C) C10 cells were transfected with gB and gH2/gL2 (A), gH2Δ29L2 (B), or gH2Δ48/gL2 (C). Cells were incubated with 100 µg/ml of the indicated MAb for 2 h before fusion was triggered with gD2306. Fusion levels were determined based on the number and size of syncytia identified by immunofluorescence. (D) Effect of MAbs on the constitutive function of gH2Δ48/gL2 (in the absence of gD). Fusion levels were expressed as percentages of the no-antibody sample.
Mentions: B78H1-C10 cells expressing gB and wild-type gH2/gL2, gH2Δ29/gL2, or gH2Δ48/gL2 were incubated with the indicated antibody for 2 h before fusion was triggered with gD306t. As expected, the gH MAb CHL25 failed to block cell-cell fusion and MAb CHL2 blocked fusion, regardless of the form of gH/gL present in the transfected cells (Fig. 4A to C). Critically, as previously shown (24), none of the gL MAbs had any effect on fusion of cells transfected with wt gH2/gL2 (Fig. 4A). In contrast, three gL MAbs, CHL18, CHL34, and CΔ48L3, each of which recognizes a different stretch of amino acids of gL (Table 1), blocked fusion of cells transfected with either gH2Δ29/gL2 (Fig. 4B) or gH2Δ48/gL2 (Fig. 4C). Thus, the properties of these MAbs fit with our rationale that deletion of portions of the gH N terminus affects the C terminus of gL so that the MAbs now block. Interestingly, anti-gL MAb CHL26 had an intermediate effect, possibly because its epitope is partially uncovered in the two truncated forms (Fig. 4B and C). We propose that region 146 to 203 of gL is hidden in wt gH/gL and exposed when gH residues 19 to 47 are absent (Fig. 4B and C). We conclude that once these sites of gL (in the context of wt gH/gL) are bound by antibodies, triggering of downstream events is blocked at that step.

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