Cell entry of enveloped viruses requires specialized viral proteins that mediate fusion with the host membrane by substantial structural rearrangements from a metastable pre- to a stable postfusion conformation. This metastability renders the herpes simplex virus 1 (HSV-1) fusion glycoprotein B (gB) highly unstable such that it readily converts into the postfusion form, thereby precluding structural elucidation of the pharmacologically relevant prefusion conformation. By identification of conserved sequence signatures and molecular dynamics simulations, we devised a mutation that stabilized this form. Functionally locking gB allowed the structural determination of its membrane-embedded prefusion conformation at sub-nanometer resolution and enabled the unambiguous fit of all ectodomains. The resulting pseudo-atomic model reveals a notable conservation of conformational domain rearrangements during fusion between HSV-1 gB and the vesicular stomatitis virus glycoprotein G, despite their very distant phylogeny. In combination with our comparative sequence-structure analysis, these findings suggest common fusogenic domain rearrangements in all class III viral fusion proteins.

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.

Entry of herpesviruses depends on the combined action of viral glycoprotein B (gB) and the heterodimeric gH/gL complex, which are activated by binding of the virion to specific cellular receptors. While gB carries signatures of a bona fide fusion protein, efficient membrane fusion requires gH/gL. However, although gB and gH/gL are essential for entry, the alphaherpesvirus pseudorabies virus (PrV) is capable of limited cell-to-cell spread in the absence of gL. To understand gH/gL function in more detail, the limited spread of PrV-⌬gL was used for reversion analyses by serial cell culture passages. In a first experiment, an infectious gL-negative mutant in which gL function was replaced by generation of a gD-gH hybrid protein was isolated (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014 -3022, 1999). In a second, independent experiment PrV-⌬gLPassB4.1, which also replicated productively without gL, was isolated. Sequence analysis revealed mutations in gH but also in gB and gD. In a transfection-based fusion assay, two amino acid substitutions in the N-terminal part of gH B4.1 (L 70 P and W 103 R) were found to be sufficient to compensate for lack of gL, while mutations present in gB B4.1 enhanced fusogenicity. Coexpression of gB B4.1 with the homologous gH B4.1 resulted in strongly increased syncytium formation, which was further augmented by truncation of the gB B4.1 C-terminal 29 amino acids. Nevertheless, gH was still required for membrane fusion. Surprisingly, coexpression of gD B4.1 blocked syncytium formation in the fusion assays, which could be attributed to a V 106 A substitution within the ectodomain of gD B4.1 . IMPORTANCEIn contrast to many other enveloped viruses, herpesviruses rely on the concerted action of four viral glycoproteins for membrane fusion during infectious entry. Although the highly conserved gB shows signatures of a fusion protein, for fusion induction it requires the gH/gL complex, whose role is still elusive. Here we demonstrated fusion activation by gH in the absence of gL after reversion analysis of gL-deleted pseudorabies virus. This gL-independent fusion activity depended on single amino acid exchanges affecting the gL-binding domain in gH, increasing fusogenicity in gB and allowing negative fusion regulation by gD. Thus, our results provide novel information on the interplay in the fusion machinery of herpesviruses.

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Several envelope glycoproteins are involved in herpesvirus entry into cells, direct cell-to-cell spread, and induction of cell fusion. The membrane fusion protein glycoprotein B (gB) and the presumably gB-activating heterodimer gH/gL are essential for these processes and conserved throughout the Herpesviridae. However, after extended cell culture passage of gL-negative mutants of the alphaherpesvirus pseudorabies virus (PrV), phenotypic revertants could be isolated which had acquired spontaneous mutations affecting the gL-interacting N-terminal part of the gH ectodomain (gDH and gH 2264 -2272, 2016). To investigate the functional relevance of this part of gH in more detail, we introduced an in-frame deletion of 66 codons at the 5= end of the plasmid-cloned gH gene (gH 32/98 ). The N-terminal signal peptide was retained, and the deletion did not affect expression or processing of gH but abrogated its function in in vitro fusion assays. Insertion of the engineered gH gene into the PrV genome resulted in a defective mutant (pPrV-gH 32/98 K), which was incapable of entry and spread. Interestingly, in vitro activity of mutated gH 32/98 was restored when it was coexpressed with hyperfusogenic gB B4.1 , obtained from a passaged gL deletion mutant of PrV. Moreover, the entry and spread defects of pPrV-gH 32/98 K were compensated by the mutations in gB B4.1 in cis, as well as in trans, independent of gL. Thus, PrV gL and the gL-interacting domain of gH are not strictly required for function. IMPORTANCE Membrane fusion is crucial for infectious entry and spread of enveloped viruses. While many enveloped viruses require only one or two proteins for receptor binding and membrane fusion, herpesvirus infection depends on several envelope glycoproteins. Besides subfamily-specific receptor binding proteins, the core fusion machinery consists of the conserved fusion protein gB and the gH/gL complex. The role of the latter is unclear, but it is hypothesized to interact with gB for fusion activation. Using isogenic virus recombinants, we demonstrate here that gL and the gL-binding domain of PrV gH are not strictly required for membrane fusion during virus entry and spread when concomitantly mutations in gB are present which increase its fusogenicity. Thus, our results strongly support the notion of a functional gB-gH interaction during the fusion process. Editor Richard M. Longnecker, Northwestern University

Herpesvirus entry and spread requires fusion of viral and host cell membranes, which is mediated by the conserved surface glycoprotein B (gB). Upon activation, gB undergoes a major conformational change and transits from a metastable prefusion to a stable postfusion conformation. Although gB is a structural homolog of low-pH-triggered class III fusogens, its fusion activity depends strictly on the presence of the conserved regulatory gH/gL complex and nonconserved receptor binding proteins, which ensure that fusion occurs at the right time and space. How gB maintains its prefusion conformation and how gB fusogenicity is controlled remain poorly understood. Here, we report the isolation and characterization of a naturally selected pseudorabies virus (PrV) gB able to mediate efficient gH/gL-independent virus-cell and cell-cell fusion. We found that the control exerted on gB by the accompanying viral proteins is mediated via its cytosolic domain (CTD). Whereas gB variants lacking the CTD are inactive, a single mutation of a conserved asparagine residue in an alpha-helical motif of the ectodomain recently shown to be at the core of the gB prefusion trimer compensated for CTD absence and uncoupled gB from regulatory viral proteins, resulting in a hyperfusion phenotype. This phenotype was transferred to gB homologs from different alphaherpesvirus genera. Overall, our data propose a model in which the central helix acts as a molecular switch for the gB pre-to-postfusion transition by conveying the structural status of the endo- to the ectodomain, thereby governing their cross talk for fusion activation, providing a new paradigm for herpesvirus fusion regulation. IMPORTANCE The class III fusion protein glycoprotein B (gB) drives membrane fusion during entry and spread of herpesviruses. To mediate fusion, gB requires activation by the conserved gH/gL complex by a poorly defined mechanism. A detailed molecular-level understanding of herpesvirus membrane fusion is of fundamental virological interest and has considerable potential for the development of new therapeutics blocking herpesvirus cell invasion and spread. Using in vitro evolution and targeted mutagenesis of three different animal alphaherpesviruses, we identified a single conserved amino acid in a regulatory helix in the center of the gB ectodomain that enables efficient gH/gL-independent entry and plays a crucial role in the pre-to-postfusion transition of gB. Our results propose that the central helix is a key regulatory element involved in the intrastructural signal transduction between the endo- and ectodomain for fusion activation. This study expands our understanding of herpesvirus membrane fusion and uncovers potential targets for therapeutic interventions.

Herpesvirus membrane fusion depends on the core fusion machinery, comprised of glycoproteins B (gB) and gH/gL. Although gB structurally resembles autonomous class III fusion proteins, it strictly depends on gH/gL to drive membrane fusion. Whether the gH/gL complex needs to be membrane anchored to fulfill its function and which role the gH cytoplasmic (CD) and transmembrane domains (TMD) play in fusion is unclear. While the gH CD and TMD play an important role during infection, soluble gH/gL of herpes simplex virus 1 (HSV-1) seems to be sufficient to mediate cell-cell fusion in transient assays, arguing against an essential contribution of the CD and TMD. To shed more light on this apparent discrepancy, we investigated the role of the CD and TMD of the related alphaherpesvirus pseudorabies virus (PrV) gH. For this purpose, we expressed C-terminally truncated and soluble gH and replaced the TMD with a glycosylphosphatidylinositol (gpi) anchor. We also generated chimeras containing the TMD and/or CD of PrV gD or HSV-1 gH. Proteins were characterized in cell-based fusion assays and during virus infection. Although truncation of the CD resulted in decreased membrane fusion activity, the mutant proteins still supported replication of gH-negative PrV, indicating that the PrV gH CD is dispensable for viral replication. In contrast, PrV gH lacking the TMD, membrane-anchored via a lipid linker, or comprising the PrV gD TMD were nonfunctional, highlighting the essential role of the gH TMD for function. Interestingly, despite low sequence identity, the HSV-1 gH TMD could substitute for the PrV gH TMD, pointing to functional conservation. Enveloped viruses depend on membrane fusion for virus entry. While this process can be mediated by only one or two proteins, herpesviruses depend on the concerted action of at least three different glycoproteins. Although gB has features of bona fide fusion proteins, it depends on gH and its complex partner, gL, for fusion. Whether gH/gL prevents premature fusion or actively triggers gB-mediated fusion is unclear, and there are contradictory results on whether gH/gL function requires stable membrane anchorage or whether the ectodomains alone are sufficient. Our results show that in pseudorabies virus gH, the transmembrane anchor plays an essential role for gB-mediated fusion while the cytoplasmic tail is not strictly required.