Monkeypox virus (MPXV) is a poxvirus endemic to Central and West Africa with high epidemic potential. Pox-viruses enter host cells via a conserved entry-fusion complex (EFC), which mediates viral fusion to the cell membrane. The EFC is a promising therapeutic target, but the absence of structural data has limited the development of fusion-inhibiting treatments. Here, we investigated A16/G9, a subcomplex of the EFC that controls fusion timing. Using cryo-electron microscopy, we showed how A16/G9 interacts with A56/K2, a viral fusion suppressor that prevents superinfection. Immunization with A16/G9 elicited a protective immune response in mice. Using X-ray crystallography, we characterized two neutralizing antibodies and engineered a chimeric antibody that cross-neutralizes several poxviruses more efficiently than 7D11, the most potent antibody targeting the EFC described to date. These findings highlight the potential of A16/G9 as a candidate for subunit vaccines and identify regions of the EFC as targets for antiviral development.

Accepted for publication, to appear October 30, 2025.

Membrane fusion is a fundamental process involved in exocytosis, fertilization, or cell entry by enveloped viruses. Membrane fusion is facilitated by fusion proteins, which are anchored in membranes by helical transmembrane domains (TMDs). Previous studies showed that TMD variations may alter the fusion efficiency, suggesting that TMDs are not merely passive anchors, however the mechanism by which TMDs drive fusion is not well understood. We used high-throughput coarse-grained molecular dynamics simulations and free energy calculations to quantify effects of TMDs on the formation of the first fusion intermediate, that is, of a fusion stalk. We analyzed five physiologically relevant TMDs derived from viral fusion proteins and the SNARE complex embedded in various lipid environments. We find that the addition of TMDs favors stalk formation by typically 10 to 30 kJ/mol in a concentration-dependent manner. Using helices with sequences R₂L_nR₂ (n=6,…, 26), we find that negative hydrophobic mismatch between the TMD and the membrane core strongly promotes fusion. Analysis of the lipid tail order parameters of annular lipids revealed a strong correlation between stalk stabilization and induced lipid disorder. Together, our findings suggest that TMDs actively contribute to membrane fusogenicity by locally perturbing the membrane order.

Photoreceptor proteins initiate, regulate and control fundamental biological processes such as vision, photosynthesis and circadian rhythms. A large photoreceptor subfamily uses vitamin B₁₂ derivatives for light sensing, contrasting with the well-established mode of action of these organometallic derivatives in thermally activated enzymatic reactions. The molecular mechanism of B₁₂ photoreception and how this differs to the thermal pathways remain unknown. Here we provide a detailed spatio-temporal description of photoactivation in the prototypical tetrameric B₁₂ photoreceptor CarH from nanoseconds to seconds by using an integrative approach, combining time- and temperature-resolved structural and spectroscopic methods with quantum chemical calculations. High resolution structural snapshots of key intermediates illustrate how photocleavage of a Co–C bond triggers a pathway of structural changes that propagate throughout CarH from the B₁₂ chromophore, via a previously unknown adduct, to finally cause tetramer dissociation. These unique intermediates, which differentiate CarH from thermally-activated B₁₂ enzymes, steer the photoactivation pathway and act as the molecular bridge between photochemical and photobiological timescales. Our results offer a spatio-temporal understanding of CarH photoactivation and pave the way for designing and optimising B₁₂-dependent photoreceptors for future optogenetic applications.