Entry of all herpesviruses requires fusion of the viral envelope with a host-cell membrane, executed by glycoprotein B (gB), a class III fusogen (one of the three known structural classes of viral membrane-fusion proteins) conserved across the family. Unlike other class III fusogens, gB refolding is strictly controlled by the regulatory gH/gL complex, by receptor-binding proteins such as gD, and by gB's own cytoplasmic domain. In vitro evolution of a pseudorabies virus (PrV) mutant lacking the gB cytoplasmic domain produced a single ectodomain substitution, N735S, in a conserved regulatory helix of gB domain V that confers gH/gL-independent ('autonomous') fusion; the phenotype transfers to bovine herpesvirus 1 (BoHV-1) and infectious laryngotracheitis virus (ILTV) gB, but not to HSV-1. Vallbracht et al. (2021) proposed the domain V (DV) helix as an allosteric switch coupling the cytoplasmic clamp to the ectodomain, with refolding triggered by as-yet-unidentified cellular factor(s). The open problem is to identify the cellular trigger(s) that activate gB refolding — the bovine-cell-specific trigger for autonomous BoHV-1 gB, the signals used by PrV and ILTV autonomous variants, and whether gB directly engages host receptors during normal gH/gL-dependent entry — and to determine the structural pathway by which trigger engagement propagates through the DV helix, membrane-proximal region, transmembrane segment, and cytoplasmic domain, jointly explaining why the conserved DV asparagine is not functionally interchangeable across alphaherpesvirus genera. Prefusion gB structures and prefusion-specific antibodies have been determined, but no cellular trigger has been identified and the activating transition has not been observed.

Background

Herpesviruses are large enveloped DNA viruses that enter host cells by fusing the viral envelope with a cellular membrane. Entry depends on a conserved core fusion machine: glycoprotein B (gB), a trimeric class III viral fusion protein (a structural class it shares with, for example, vesicular stomatitis virus glycoprotein G), together with the heterodimeric regulatory complex gH/gL. In the alphaherpesvirus subfamily — which includes herpes simplex viruses 1 and 2 (HSV-1/2), pseudorabies virus (PrV, a swine pathogen widely used as an experimental model), bovine herpesvirus 1 (BoHV-1), and infectious laryngotracheitis virus (ILTV of chickens) — a receptor-binding protein, gD, attaches to cell-surface receptors such as nectin-1 or HVEM (herpesvirus entry mediator) and, according to the prevailing cascade model, signals through gH/gL to trigger gB. Like all viral fusogens, gB is metastable: it is held in a prefusion state on the virion and, upon activation, refolds irreversibly into a stable postfusion hairpin that pulls the viral and cellular membranes together. Unusually for a class III fusogen, gB is not known to be triggered by low pH or by receptor binding alone; its fusogenicity is restrained by its own cytoplasmic domain (CTD), proposed to act as an inhibitory clamp, and by gH/gL, ensuring fusion occurs at the right time and place. The gB ectodomain (the extracellular portion of the protein) comprises five domains (I–V); domain V (DV) lies at the trimer interface close to the viral membrane and continues into the membrane-proximal region (MPR), the transmembrane segment (TMD), and the CTD. Postfusion gB structures have been available for years, and prefusion conformations of HSV-1 gB were later determined using stabilizing mutations, nanobodies (single-domain antibodies), or structure-based design (Vollmer et al. 2020, Science Advances; Roark et al. 2025, Nature Microbiology; Vollmer et al. 2025, Nature), but the metastable prefusion state remains hard to trap and the triggering transition itself has never been directly observed.

In Vallbracht et al. (2021), in vitro evolution of a PrV mutant carrying a C-terminally truncated gB (lacking most of the CTD) yielded a revertant whose gB carries a single substitution, N735S, in a short conserved alpha-helix of the DV ectodomain. This mutation compensates for the missing CTD: gB lacking the CTD but carrying N735S mediates efficient virus-cell and cell-cell fusion without gH/gL — termed autonomous fusion — and the phenotype transfers when homologous substitutions are engineered into BoHV-1 gB (N742S) and ILTV gB (Q684S/N685Q). In HSV-1 gB, by contrast, the homologous substitution (N709S) does not rescue CTD truncation and abolishes fusion in the full-length protein. Independent mutational analysis of the DV arm of HSV-1 gB likewise impaired fusion and delayed entry (Fan et al. 2021), reinforcing DV's regulatory role. Because a single ectodomain point mutation can replace the regulatory inputs of both the CTD and gH/gL, the authors proposed that the DV helix acts as an allosteric switch: the CTD and the DV helical bundle jointly stabilize the prefusion state, and the fusogenic refolding is triggered by interaction with cellular receptor(s) whose identity is unknown. Consistent with a trigger requirement, BoHV-1 gB with the N742S substitution fuses only with bovine cells, implying a species-specific cellular factor, whereas the broad-host-range PrV and the avian ILTV variants may use a more widely expressed receptor or signal. For HSV, several gB-interacting host proteins have been described — paired immunoglobulin-like receptor alpha (PILRα), myelin-associated glycoprotein (MAG), and non-muscle myosin heavy chains IIA/IIB — but none has been demonstrated to trigger gB refolding, and it remains unknown whether gB itself engages a receptor during normal, gH/gL-dependent entry or merely refolds in response to upstream signals from gD and gH/gL (Jambunathan et al. 2021; Wang et al. 2022). BoHV-1, for its part, engages nectin-1 through gD (Yue et al. 2020), leaving the bovine trigger for its autonomous gB mutants unexplained.

Problem Statement

The open problem is the activation mechanism of the conserved herpesvirus fusogen gB: which cellular trigger(s) initiate gB's pre-to-postfusion refolding, and how is trigger engagement allosterically coupled, through the regulatory domain V (DV) helix, to the membrane-proximal region (MPR), transmembrane segment (TMD), and cytoplasmic domain (CTD) of gB?

Building on the autonomous-fusion platform established by in vitro evolution (pseudorabies virus [PrV] gB N735S and its bovine herpesvirus 1 [BoHV-1] N742S and infectious laryngotracheitis virus [ILTV] Q684S/N685Q counterparts), the task addresses four sub-questions of independent standing: each is separately refutable and has its own completion criterion, and an answer may resolve any subset of them without resolving the rest:

(1) Trigger identity for autonomous fusion: identify the bovine-cell-specific trigger that enables BoHV-1 gB lacking most of its CTD but carrying N742S to fuse with bovine but not non-bovine cells, and determine whether the PrV and ILTV autonomous gB variants rely on a broadly expressed receptor or on other cell-specific signals.

(2) gB as a receptor-binding protein in normal entry: determine whether gB itself directly engages host receptors to initiate its conformational change during normal entry that depends on the regulatory gH/gL complex — as the receptor-binding protein gD does via its receptors nectin-1 and HVEM (herpesvirus entry mediator) — and if so, identify the interaction and how receptor binding is coupled to the DV-helix switch.

(3) The structural coupling pathway: determine the pathway by which the DV helix communicates with the MPR, TMD, and CTD during activation and the evidence that establishes it — the trigger-engaged and transitioning states of full-length, membrane-embedded gB that connect CTD clamp release to DV-helix rearrangement and fusogenic refolding.

(4) Genus-specific use of the conserved site: explain mechanistically why the same structurally conserved DV asparagine acts as a switch residue in PrV, BoHV-1, and ILTV gB (conferring autonomous, gH/gL-independent fusion upon substitution) but not in herpes simplex virus type 1 (HSV-1) gB (where N709S abolishes fusion) — that is, which feature of the HSV-1 context (gH/gL partnership, CTD, or DV packing) makes the site mechanistically conserved but not functionally interchangeable across alphaherpesvirus genera.

The authors' proposal that further stabilization of the DV helical bundle could yield more stable prefusion gB for antiviral or vaccine purposes is a motivation and application of this mechanism, not a separate required deliverable.

References

  1. Vallbracht M, Lötzsch H, Klupp BG, Fuchs W, Vollmer B, Grünewald K, Backovic M, Rey FA, Mettenleiter TC. In Vitro Viral Evolution Identifies a Critical Residue in the Alphaherpesvirus Fusion Glycoprotein B Ectodomain That Controls gH/gL-Independent Entry. mBio 12(3):e00557-21, 2021. DOI: 10.1128/mbio.00557-21. https://doi.org/10.1128/mbio.00557-21
  2. Vollmer B, Pražák V, Vasishtan D, Jefferys EE, Hernandez-Duran A, Vallbracht M, Klupp BG, Mettenleiter TC, Backovic M, Rey FA, Topf M, Grünewald K. The prefusion structure of herpes simplex virus glycoprotein B. Science Advances, 2020. DOI: 10.1126/sciadv.abc1726. https://doi.org/10.1126/sciadv.abc1726
  3. Fan Q, Longnecker R, Connolly SA. Herpes Simplex Virus Glycoprotein B Mutations Define Structural Sites in Domain I, the Membrane Proximal Region, and the Cytodomain That Regulate Entry. Journal of Virology, 2021. DOI: 10.1128/jvi.01050-21. https://doi.org/10.1128/jvi.01050-21
  4. Pataki Z, Sanders EK, Heldwein EE. A surface pocket in the cytoplasmic domain of the herpes simplex virus fusogen gB controls membrane fusion. PLoS Pathogens, 2022. DOI: 10.1371/journal.ppat.1010435. https://doi.org/10.1371/journal.ppat.1010435
  5. Zhou M, Vollmer B, Machala E, Chen M, Grünewald K, Arvin AM, Chiu W, Oliver SL. Targeted mutagenesis of the herpesvirus fusogen central helix captures transition states. Nature Communications, 2023. DOI: 10.1038/s41467-023-43011-w. https://doi.org/10.1038/s41467-023-43011-w
  6. Wang C, Liang Q, Sun D, He Y, Jiang J, Guo R, Malla T, Hamrah P, Liu X, Huang Z, Hu K. Nectin-1 and Non-muscle Myosin Heavy Chain-IIB: Major Mediators of Herpes Simplex Virus-1 Entry Into Corneal Nerves. Frontiers in Microbiology, 2022. DOI: 10.3389/fmicb.2022.830699. https://doi.org/10.3389/fmicb.2022.830699
  7. Yue D, Chen Z, Yang F, Ye F, Lin S, He B, Cheng Y, Wang J, Chen Z, Lin X, Yang J, Chen H, Zhang Z, You Y, Sun H, Wen A, Wang L, Zheng Y, Cao Y, Li Y, Lu G. Crystal structure of bovine herpesvirus 1 glycoprotein D bound to nectin-1 reveals the basis for its low-affinity binding to the receptor. Science Advances, 2020. DOI: 10.1126/sciadv.aba5147. https://doi.org/10.1126/sciadv.aba5147
  8. Jambunathan N, Clark CM, Musarrat F, Chouljenko VN, Rudd J, Kousoulas KG. Two Sides to Every Story: Herpes Simplex Type-1 Viral Glycoproteins gB, gD, gH/gL, gK, and Cellular Receptors Function as Key Players in Membrane Fusion. Viruses, 2021. DOI: 10.3390/v13091849. https://doi.org/10.3390/v13091849
  9. Zhong L, Zhang W, Krummenacher C, Chen Y, Zheng Q, Zhao Q, Zeng MS, Xia N, Zeng YX, Xu M, Zhang X. Targeting herpesvirus entry complex and fusogen glycoproteins with prophylactic and therapeutic agents. Trends in Microbiology, 2023. DOI: 10.1016/j.tim.2023.03.001. https://doi.org/10.1016/j.tim.2023.03.001
  10. Sponholtz MR, Byrne PO, Lee AG, Ramamohan AR, Goldsmith JA, McCool RS, Zhou L, Johnson NV, Hsieh CL, Connors M, Karthigeyan KP, Crooks CM, Fuller AS, Campbell JD, Permar SR, Maynard JA, Yu D, Bottomley MJ, McLellan JS. Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation. Proceedings of the National Academy of Sciences USA, 2024. DOI: 10.1073/pnas.2404250121. https://doi.org/10.1073/pnas.2404250121
  11. Vollmer B, Ebel H, Rees R, Nentwig J, Mulvaney T, Schünemann J, Krull J, Topf M, Görlich D, Grünewald K. A nanobody specific to prefusion glycoprotein B neutralizes HSV-1 and HSV-2. Nature, 2025. DOI: 10.1038/s41586-025-09438-5. https://doi.org/10.1038/s41586-025-09438-5
  12. Roark RS, Schaub AJ, Shi W, Wang M, Bahna FA, Becker JE, Biju A, Chong S, Du H, Guo Y, Hong H, Katsamba PS, Mannepalli SM, Olia AS, Ou L, Rubin SK, Sabo Y, Suleiman M, Wells ML, Zhang B, Cheng C, Glasgow A, Ho DD, Huang Y, Pierson TC, Rawi R, Zhou T, Shapiro L, Kwong PD. Prefusion structure, evasion and neutralization of HSV-1 glycoprotein B. Nature Microbiology, 2025. DOI: 10.1038/s41564-025-02153-x. https://doi.org/10.1038/s41564-025-02153-x
  13. Fan Q, Hippler DP, Yang Y, Longnecker R, Connolly SA. Multiple Sites on Glycoprotein H (gH) Functionally Interact with the gB Fusion Protein to Promote Fusion during Herpes Simplex Virus (HSV) Entry. mBio, 2023. DOI: 10.1128/mbio.03368-22. https://doi.org/10.1128/mbio.03368-22
  14. Fan Q, Longnecker R, Connolly SA. Species-specific gB ectodomain interactions and cytoplasmic domain stability regulate herpes simplex virus fusion. mBio, 2026. DOI: 10.1128/mbio.03424-25. https://doi.org/10.1128/mbio.03424-25
  15. Chen X, Kropff B, Li J, Sticht H, Mollik M, Sieger M, Ferrazzi F, Görzer I, Mach M, Thomas M. Functional dissection of HCMV gB's autonomous fusion activity provides insights into how polymorphisms in clinical isolates confer distinct characteristics. mBio, 2026. DOI: 10.1128/mbio.00388-26. https://doi.org/10.1128/mbio.00388-26
  16. Sponholtz MR, Ma D, Byrne PO, Shu Y, Warren C, Thambi N, McCool RS, Slein MD, Johnson NV, Rose WA, Zhang L, Durr E, Wang D, McLellan JS. Structure-based design of soluble prefusion-stabilized herpes simplex virus type 2 glycoprotein B antigens. bioRxiv preprint, 2026. DOI: 10.64898/2026.01.26.701809. https://doi.org/10.64898/2026.01.26.701809
  17. Amlashi P, Kim J, Mendis NN, Wasdin PT, Bass LE, Jordaan G, Abu-Shmais AA, Slein MD, Johnson NV, Bonami RH, Leib DA, Ackerman ME, McLellan JS, Georgiev IS. Prefusion-specific glycoprotein B human antibodies protect against neonatal HSV-2 infection. bioRxiv preprint, 2026. DOI: 10.64898/2026.02.23.707514. https://doi.org/10.64898/2026.02.23.707514