Structure-Function Dissection of Pseudorabies Virus Glycoprotein B Fusion Loops

Abstract

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.

Crystal structure of Mokola virus glycoprotein in its post-fusion conformation

“…This low pH-induced local reorganization of the glycoprotein network at the surface of the virion, outside the contact zone with the target membrane, might drive pore enlargement as suggested for VSV G [28]. The formation of such networks of spikes in their post-fusion conformation is very general among class III fusion glycoproteins as it has been also observed with pseudorabies virus fusion glycoprotein gB [41]. It is also reminiscent of the behavior of class II fusion glycoproteins, which, in their post-fusion conformation, form more or less regular networks that are different from their initial icosahedral organization [42][43][44].…”

Gammaherpesvirus entry and fusion: A tale how two human pathogenic viruses enter their host cells

“…Crystal structure studies of gB across Herpesviridae revealed that the herpesvirus postfusion form exists as spike-like homotrimer formed by five individual domains that adopt an elongated conformation. The EBV gB structure is very similar to the postfusion structures of HSV-1, PrV, and HCMV gB but the domain orientations vary suggesting species-specific functional adaptations (Backovic et al, 2009, Burke and Heldwein, 2015, Chandramouli et al, 2015, Heldwein, 2016, Vallbracht et al, 2017.…”

“…Based on previous studies, a molecular model of herpesvirus gB postulates that the tip of the second fusion loop can further penetrate into the hydrophobic core of the cellular lipid bilayer enabling the side chains forming a rim structure. Thereby, the fusion loops form a stable interaction with the host cell membrane ensuring that the two membranes will fuse (Vallbracht et al, 2017). The collapse and refolding of this extended pre-hairpin intermediate to a trimer of hairpins catalyzes enough free energy to overcome the high kinetic barrier for merging membranes (Connolly et al, 2011, Harrison, 2015, Sathiyamoorthy et al, 2017.…”

“…In contrast, a larger perturbation of this region next to the DII/III interface that binds to gp42 disturbs the formation of the B cell entry-triggering complex (Möhl et al, 2014. Despite the striking structural similarity of the postfusion forms of gB conserved across Herpesviridae, the differences in domain orientations indicate virusspecific functional adaptations regarding its receptor-binding partners (Backovic et al, 2009, Burke and Heldwein, 2015, Chandramouli et al, 2015, Connolly et al, 2011, Heldwein et al, 2006, Stampfer and Heldwein, 2012, Vallbracht et al, 2017.…”

Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules

“…Glycoprotein B is a critical factor in herpesvirus infections. Since numerous reports have described gB as the virus entry mediator for example, [52][53][54][55][56], the main goal of our study was to provide new data on the roles of alphaherpesvirus gB expressed in the cells, which should correspond to the situation in of the post-entry phases of infection, i.e., when it appears as a product of an early-late gene [57][58][59]. We could address its localization in the endosomal and lysosomal compartments, including CD63-and MHC II-positive structures, its targeting to EVs and its immunomodulatory effect on the cellular MHC II.…”