Structural Syntax of the Vaccinia Virus Entry‐Fusion Complex

The poxvirus family has a unique but uncharacterized mechanism of cell entry involving an 11-chain entry-fusion complex (EFC) anchored to the virus envelope. A computationally confident AlphaFold model for holoEFC contained all 11 chains in single copy and was rational according to crosslinking mass spectrometry (XLMS) and "monolink" (single-ended crosslinker adduct) data. All subunit interfaces, assessed via pDockQ, dG_separated, and dSASA, were computationally authentic, and all 68 ectodomain cysteines fell in pairs within mutual disulfide bonding distance. Two novel features in the model comprised an 11-chain transmembrane (TM) bundle and an atypical hinge-like structure we term the pseudo beta helical connector (PBHC). The holoEFC complex fell into two distinct subregions with apparent mobility between them and the PBHC as a fulcrum. For the subregion 2 hexamer, an assembly pathway could be deduced, and a subregion 2 homotrimer could be modeled with high computational confidence. In the holoEFC model, subunits L1 and F9, typically described as EFC-associated, flank the PBHC core. In modeling involving multiple F9 and L1 chains, L1 could occupy the F9 site in the absence of F9, and forms could be modeled with stacked L1 and/or F9 capping either end of the PBHC core. Fusion inhibitor A26, known to target the A16:G9 subunit pair within EFC, could be modeled as a computationally confident complex with EFC subcomplex A28:H2:A16:G9 in which A26 contacted G9 primarily. Quantitative protein mass spectrometry showed subunits F9 and J5, which clamp one end of the PBHC core, to be ∼10-fold sub-stoichiometric in mature Vaccinia virus preparations.