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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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Epitope mapping of gL monoclonal antibodies. (A) Denaturing Western blotting. C10 cells were transfected with control pCAGS plasmid (untfd) or plasmids expressing wt gH2 and either wt gL2 or gL2 deletion mutants gL208 and gL194. Cell lysates were examined for their reactivity with the indicated MAbs. Numbers at left are molecular masses in kilodaltons. (B) Peptide mapping. Each peptide was added to a well of a 96-well streptavidin-coated plate. The wells were blocked and probed with CΔ48L3 MAb. Bound IgG was visualized with goat anti-mouse horseradish peroxidase.
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fig3: Epitope mapping of gL monoclonal antibodies. (A) Denaturing Western blotting. C10 cells were transfected with control pCAGS plasmid (untfd) or plasmids expressing wt gH2 and either wt gL2 or gL2 deletion mutants gL208 and gL194. Cell lysates were examined for their reactivity with the indicated MAbs. Numbers at left are molecular masses in kilodaltons. (B) Peptide mapping. Each peptide was added to a well of a 96-well streptavidin-coated plate. The wells were blocked and probed with CΔ48L3 MAb. Bound IgG was visualized with goat anti-mouse horseradish peroxidase.

Mentions: IgGs were purified from the murine ascitic fluid and tested for gH/gL reactivity. Of these, only the CΔ48L3 MAb bound specifically to gL2 as detected by electrophoresis under denaturing conditions (Fig. 3A). By Western blotting, the other two MAbs either were completely nonreactive (CΔ48L1) or recognized a similar pattern with the untransfected samples (CΔ48L2) and were therefore excluded from future experiments. To fine map this MAb, we made two additional C-terminal gL truncations: gL208t (deleting residues 209 to 224) and gL194t (deleting residues 194 to 224) (Fig. 1B). We also included previously studied gL MAbs with linear epitopes within the C terminus (Fig. 1B) (24). These included MAbs CHL34, CHL18, and CHL26. In particular, CHL34 was previously shown to bind two disparate stretches of gL sequence (residues 146 to 165 and 205 to 219) (24). Here, we found that CΔ48L3 and CHL34 MAbs bound both C-terminal gL truncations, indicating that their epitopes lie upstream of gL amino acid 194. We thus narrowed the CHL34 epitope to gL residues 146 to 165. MAb CHL26 bound gL208t but not the short protein gL194t, indicating that its epitope lies between amino acids 195 and 208. Finally, MAb CHL18, which had been characterized as binding gL peptide 182 to 219, was unable to bind either gL C-terminal truncation. Taken together with previously published peptide binding data (24), this approach localized the CHL18 epitope to gL residues 209 to 219 (Fig. 1A and Table 1).


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)

Epitope mapping of gL monoclonal antibodies. (A) Denaturing Western blotting. C10 cells were transfected with control pCAGS plasmid (untfd) or plasmids expressing wt gH2 and either wt gL2 or gL2 deletion mutants gL208 and gL194. Cell lysates were examined for their reactivity with the indicated MAbs. Numbers at left are molecular masses in kilodaltons. (B) Peptide mapping. Each peptide was added to a well of a 96-well streptavidin-coated plate. The wells were blocked and probed with CΔ48L3 MAb. Bound IgG was visualized with goat anti-mouse horseradish peroxidase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Epitope mapping of gL monoclonal antibodies. (A) Denaturing Western blotting. C10 cells were transfected with control pCAGS plasmid (untfd) or plasmids expressing wt gH2 and either wt gL2 or gL2 deletion mutants gL208 and gL194. Cell lysates were examined for their reactivity with the indicated MAbs. Numbers at left are molecular masses in kilodaltons. (B) Peptide mapping. Each peptide was added to a well of a 96-well streptavidin-coated plate. The wells were blocked and probed with CΔ48L3 MAb. Bound IgG was visualized with goat anti-mouse horseradish peroxidase.
Mentions: IgGs were purified from the murine ascitic fluid and tested for gH/gL reactivity. Of these, only the CΔ48L3 MAb bound specifically to gL2 as detected by electrophoresis under denaturing conditions (Fig. 3A). By Western blotting, the other two MAbs either were completely nonreactive (CΔ48L1) or recognized a similar pattern with the untransfected samples (CΔ48L2) and were therefore excluded from future experiments. To fine map this MAb, we made two additional C-terminal gL truncations: gL208t (deleting residues 209 to 224) and gL194t (deleting residues 194 to 224) (Fig. 1B). We also included previously studied gL MAbs with linear epitopes within the C terminus (Fig. 1B) (24). These included MAbs CHL34, CHL18, and CHL26. In particular, CHL34 was previously shown to bind two disparate stretches of gL sequence (residues 146 to 165 and 205 to 219) (24). Here, we found that CΔ48L3 and CHL34 MAbs bound both C-terminal gL truncations, indicating that their epitopes lie upstream of gL amino acid 194. We thus narrowed the CHL34 epitope to gL residues 146 to 165. MAb CHL26 bound gL208t but not the short protein gL194t, indicating that its epitope lies between amino acids 195 and 208. Finally, MAb CHL18, which had been characterized as binding gL peptide 182 to 219, was unable to bind either gL C-terminal truncation. Taken together with previously published peptide binding data (24), this approach localized the CHL18 epitope to gL residues 209 to 219 (Fig. 1A and Table 1).

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