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Structure of a core fragment of glycoprotein H from pseudorabies virus in complex with antibody.

ABSTRACT: Compared with many well-studied enveloped viruses, herpesviruses use a more sophisticated molecular machinery to induce fusion of viral and cellular membranes during cell invasion. This essential function is carried out by glycoprotein B (gB), a class III viral fusion protein, together with the heterodimer of glycoproteins H and L (gH/gL). In pseudorabies virus (PrV), a porcine herpesvirus, it was shown that gH/gL can be substituted by a chimeric fusion protein gDgH, containing the receptor binding domain (RBD) of glycoprotein D fused to a truncated version of gH lacking its N-terminal domain. We report here the 2.1-Å resolution structure of the core fragment of gH present in this chimera, bound to the Fab fragment of a PrV gH-specific monoclonal antibody. The structure strongly complements the information derived from the recently reported structure of gH/gL from herpes simplex virus type 2 (HSV-2). Together with the structure of Epstein-Barr virus (EBV) gH/gL reported in parallel, it provides insight into potentially functional conserved structural features. One feature is the presence of a syntaxin motif, and the other is an extended "flap" masking a conserved hydrophobic patch in the C-terminal domain, which is closest to the viral membrane. The negative electrostatic surface potential of this domain suggests repulsive interactions with the lipid heads. The structure indicates the possible unmasking of an extended hydrophobic patch by movement of the flap during a receptor-triggered conformational change of gH, exposing a hydrophobic surface to interact with the viral membrane during the fusion process.

SUBMITTER: Backovic M

PROVIDER: S-EPMC3012528 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Structure of a core fragment of glycoprotein H from pseudorabies virus in complex with antibody.

Backovic Marija M DuBois Rebecca M RM Cockburn Joseph J JJ Sharff Andrew J AJ Vaney Marie-Christine MC Granzow Harald H Klupp Barbara G BG Bricogne Gerard G Mettenleiter Thomas C TC Rey Felix A FA

Proceedings of the National Academy of Sciences of the United States of America 20101213 52

Compared with many well-studied enveloped viruses, herpesviruses use a more sophisticated molecular machinery to induce fusion of viral and cellular membranes during cell invasion. This essential function is carried out by glycoprotein B (gB), a class III viral fusion protein, together with the heterodimer of glycoproteins H and L (gH/gL). In pseudorabies virus (PrV), a porcine herpesvirus, it was shown that gH/gL can be substituted by a chimeric fusion protein gDgH, containing the receptor bind ...[more]

PMID: 21149698

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Project description:Conserved across the family Herpesviridae, glycoprotein B (gB) is responsible for driving fusion of the viral envelope with the host cell membrane for entry upon receptor binding and activation by the viral gH/gL complex. Although crystal structures of the gB ectodomains of several herpesviruses have been reported, the membrane fusion mechanism has remained elusive. Here, we report the X-ray structure of the pseudorabies virus (PrV) gB ectodomain, revealing a typical class III postfusion trimer that binds membranes via its fusion loops (FLs) in a cholesterol-dependent manner. Mutagenesis of FL residues allowed us to dissect those interacting with distinct subregions of the lipid bilayer and their roles in membrane interactions. We tested 15 gB variants for the ability to bind to liposomes and further investigated a subset of them in functional assays. We found that PrV gB FL residues Trp187, Tyr192, Phe275, and Tyr276, which were essential for liposome binding and for fusion in cellular and viral contexts, form a continuous hydrophobic patch at the gB trimer surface. Together with results reported for other alphaherpesvirus gBs, our data suggest a model in which Phe275 from the tip of FL2 protrudes deeper into the hydrocarbon core of the lipid bilayer, while the side chains of Trp187, Tyr192, and Tyr276 form a rim that inserts into the more superficial interfacial region of the membrane to catalyze the fusion process. Comparative analysis with gBs from beta- and gamma-herpesviruses suggests that this membrane interaction model is valid for gBs from all herpesviruses.IMPORTANCE Herpesviruses are common human and animal pathogens that infect cells by entering via fusion of viral and cellular membranes. Central to the membrane fusion event is glycoprotein B (gB), which is the most conserved envelope protein across the herpesvirus family. Like other viral fusion proteins, gB anchors itself in the target membrane via two polypeptide segments called fusion loops (FLs). The molecular details of how gB FLs insert into the lipid bilayer have not been described. Here, we provide structural and functional data regarding key FL residues of gB from pseudorabies virus, a porcine herpesvirus of veterinary concern, which allows us to propose, for the first time, a molecular model to understand how the initial interactions by gBs from all herpesviruses with target membranes are established.

Project description:Enveloped viruses utilize membrane fusion for entry into, and release from, host cells. For entry, members of the Herpesviridae require at least three envelope glycoproteins: the homotrimeric gB and a heterodimer of gH and gL. The crystal structures of three gH homologues, including pseudorabies virus (PrV) gH, revealed four conserved domains. Domain II contains a planar ?-sheet ("fence") and a syntaxin-like bundle of three ?-helices (SLB), similar to those found in eukaryotic fusion proteins, potentially executing an important role in gH function. To test this hypothesis, we introduced targeted mutations into the PrV gH gene, which either disrupt the helices of the SLB by introduction of proline residues or covalently join them by artificial intramolecular disulfide bonds between themselves, to the adjacent fence region, or to domain III. Disruption of either of the three ?-helices of the SLB (A250P, V275P, V298P) severely affected gH function in in vitro fusion assays and replication of corresponding PrV mutants. Considerable defects in fusion activity of gH, as well as in penetration kinetics and cell-to-cell spread of PrV mutants, were also observed after disulfide linkage of two ?-helices within the SLB (A284C-S291C) or between SLB and domain III (H251C-L432C), as well as by insertions of additional cysteine pairs linking fence, SLB, and domain III. In vitro fusion activity of mutated gH could be partly restored by reduction of the artificial disulfide bonds. Our results indicate that the structure and flexibility of the SLB are relevant for the function of PrV gH in membrane fusion.Mutational analysis based on crystal structures of proteins is a powerful tool to understand protein function. Here, we continued our study of pseudorabies virus gH, a part of the core fusion machinery of herpesviruses. We previously showed that the "flap" region in domain IV of PrV gH is important for its function. We now demonstrate that mutations within domain II that interfere with integrity or flexibility of a syntaxin-like three-helix bundle also significantly impair gH function during fusion. These studies provide important insights into the structural requirements of gH for function in fusion.

Dataset Information

Structure of a core fragment of glycoprotein H from pseudorabies virus in complex with antibody.

Publications

Structure of a core fragment of glycoprotein H from pseudorabies virus in complex with antibody.

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