Bovine coronavirus (BCoV) is a major pathogen of cattle and the closest known ancestor of the human coronavirus OC43, yet how viral evolution reshapes spike protein structure and function remains poorly understood. Here, we combined comparative genomics, glycoproteomics, cryo-EM, and antigenic characterization to define the structural mechanisms underlying spike evolution across representative BCoV lineages. We identify a previously unrecognized lineage-specific N-glycosylation site in contemporary European viruses and validate its occupancy by glycoproteomics. High-resolution cryo- EM structures reveal that BCoV evolution preserves the overall prefusion architecture of the spike glycoprotein while selectively remodeling key functional regions involved in receptor recognition, conformational dynamics, and antigenicity. Comparative analysis with OC43 demonstrates increased conformational heterogeneity of the receptor-binding loop, whereas the Mebus vaccine strain exhibits enhanced membrane-proximal stalk flexibility despite maintaining thermal stability. Finally, structural modeling together with antibody-binding experiments reveals substantial antigenic remodeling despite >90% spike sequence identity between BCoV and OC43. Together, these findings establish a mechanistic framework for Embecovirus spike evolution and provide structural insights that may inform the development of vaccines based on contemporary circulating strains.
Haneen Tarabih, Jimmy Asiku, Y. Levi-Kalisman et al.· bioRxiv· 0 citations
Hepatitis C virus (HCV) remains a major global health challenge despite the availability of highly effective antiviral therapies, underscoring the need for a broadly protective vaccine. The envelope glycoprotein E2 is the principal target of neutralizing antibodies, yet the full repertoire of vulnerable epitopes and mechanisms of antibody-mediated neutralization remains incompletely understood. Here, we exploited the unique binding properties of camelid nanobodies to probe the antigenic landscape of HCV E2 beyond the immunodominant human antibody response. We isolated a diverse panel of E2-specific nanobodies, including broadly neutralizing antibodies with high-affinity cross-reactivity toward genetically diverse HCV isolates. By combining cross-neutralization assays, competition binding experiments, and high-resolution hydrogen–deuterium exchange mass spectrometry (HDX-MS), we identified three mechanistically distinct classes of neutralizing epitopes. While one class targets the canonical E2 neutralization face, a second class recognizes antigenic region 1 (AR1), independently validating and extending recent evidence that this region represents a functional site of viral vulnerability. These findings demonstrate that broadly neutralizing antibody responses extend beyond the canonical neutralization face and establish a broader framework for understanding HCV neutralization. More broadly, our study illustrates how alternative antibody repertoires can reveal functionally important antigenic surfaces that are underrepresented in conventional human antibody responses, providing new opportunities for the rational design of next-generation HCV vaccine immunogens.
Bacterial protein toxins constitute a vast and largely untapped reservoir of antimicrobial activities with substantial therapeutic and biotechnological potential. However, their intrinsic toxicity frequently prevents stable recombinant expression in bacterial hosts, creating a major bottleneck for biochemical characterization, structural analysis, and development as antimicrobial agents. Here, we present a modular platform for the scalable recombinant production of highly toxic bacterial proteins based on transient intramolecular toxin neutralization. The strategy covalently links each toxin to its cognate immunity protein, promoting neutralization during biosynthesis while permitting recovery of the native toxin through site-specific proteolytic cleavage. Using this approach, we produced multiple previously intractable polymorphic toxin domains that could not be obtained using conventional inducible expression, toxin–immunity co-expression, or bacterial cell-free systems. We further streamlined the production workflow through intracellular protease-mediated cleavage, reducing the purification process from four steps to two and increasing protein recovery. To address cases in which native immunity proteins were insufficient, we incorporated computational protein design to engineer improved toxin-binding partners, enabling production of an additional toxin that remained refractory to the original platform. Purified toxins retained enzymatic activity following denaturation and refolding, confirming recovery of functional proteins and enabling identification of a previously uncharacterized nuclease activity. Together, these findings establish a scalable and adaptable microbial biotechnology platform for the production of intrinsically toxic proteins. The integration of transient intramolecular neutralization with computational engineering provides a route toward systematic production and characterization of toxic proteins for antimicrobial discovery, structural biology, protein engineering, and future biotechnological applications.
R. Fraenkel, Inbar Cahana, Tomer Sivan et al.· bioRxiv· 0 citations
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