Makio, A. O. | Search bioRxiv

bioRxiv2026

A Prefusion Form of Herpes Simplex Virus 1 gB has a Distinct Antigenic Signature

Herpes simplex virus (HSV) fusion and entry is mediated by a cascade of interactions among gB, gD and gH/gL. The gB homotrimer undergoes a conformational transition from a metastable prefusion state to a more stable postfusion form, driving fusion of the viral envelope and a host cell membrane. An H516P mutation in gB domain III restricts formation of the extended core alpha helix and constrains gB in a prefusion state. Several prefusion gB structures have been determined that contain this mutation. We assessed the antigenic reactivity of gB H516P by quantitative immunodotblot using a panel of gB-reactive monoclonal antibodies All antibodies tested bound to both prefusion (H516P) gB and wild type gB. Antibodies tested to gB domains II, IV and V exhibited differential binding to H516P gB compared to wild type gB. The results suggest that gB H516P has a distinct antigenic profile. The antigenic signature of H516P may be useful as a rapid indicator of prefusion forms of gB. The low pH environment of the cellular endosome is a cell-specific factor for HSV entry and triggers antigenic changes in gB. The step at which low pH impacts gB refolding to execute fusion is not well-understood. The results suggest that gB H516P undergoes wild-type-like conformational changes in gB domains I and V triggered by low pH. We propose that pH acts on an early stage of gB fusion function, prior to extension of the domain III core helix.

microbiology↗

bioRxiv2025

Modulation of specific interactions within a viral fusion protein predicted from machine learning blocks membrane fusion

Enveloped viruses must enter host cells to initiate infections through a fusion process, during which the fusion proteins undergo significant and complex structural changes from pre-fusion to post-fusion conformations. Understanding of the fusion protein conformational stability, rapid and accurate identification of the stabilizing interactions are critically important for inhibiting the infections. Here, we leverage molecular dynamics simulations, novel machine learning models and biological experiments to identify the crucial interactions dictating the structural stability of glycoprotein B (gB), a class III fusion protein. We focused on the interactions between the fusion loops and the membrane proximal region in gB, and a new Q181-R747 polar interaction was identified from our machine learning model as critical in stabilizing the gB pre-fusion conformation. Molecular simulations revealed that mutation of Q181 with proline (Q181P) disrupted the fusion loop secondary structure and reduced gB pre-fusion stability. Experiments were designed to evaluate the impact of the Q181P on fusion. Strikingly, the mutation completely abrogated gB membrane fusion activity. The experiments confirmed the importance of Q181-R747 interaction on fusion, which is consistent with the model predictions. The results deepen our fundamental understanding of the molecular mechanisms of gB during viral fusion, which may lead to novel antiviral interventions. The modeling and experimental framework can be generalized to rapidly identify the critical inter-molecular interactions in other important biological processes.

microbiology↗