Uncropped original images of Fig 7B.

Host cell entry of vaccinia virus (a poxvirus) proceeds through multiple steps that involve many viral proteins to mediate cell infection. Upon binding to cells, vaccinia virus membrane fuses with host membranes via a viral entry fusion protein complex comprising 11 proteins: A16, A21, A28, F9, G3, G9, H2, J5, L1, L5 and O3. Despite vaccinia virus having two infectious forms, mature and enveloped, that have different membrane layers, both forms require an identical viral entry fusion complex for membrane fusion. Components of the poxvirus entry fusion complex that have been structurally assessed to date share no known homology with all other type I, II and III viral fusion proteins, and the large number of fusion protein components renders it a unique system to investigate poxvirus-mediated membrane fusion. Here, we determined the NMR structure of a truncated version of vaccinia A28 protein. We also expressed a soluble H2 protein and showed that A28 interacts with H2 protein at a 1:1 ratio in vitro. Furthermore, we performed extensive in vitro alanine mutagenesis to identify A28 protein residues that are critical for H2 binding, entry fusion complex formation, and virus-mediated membrane fusion. Finally, we used molecular dynamic simulations to model full-length A28-H2 subcomplex in membranes. In summary, we characterized vaccinia virus A28 protein and determined residues important in its interaction with H2 protein and membrane components. We also provide a structural model of the A28-H2 protein interaction to illustrate how it forms a 1:1 subcomplex on a modeled membrane.