The Vaccinia virion: Filling the gap between atomic and ultrastructure.

We have investigated the molecular-level structure of the Vaccinia virion in situ by protein-protein chemical crosslinking, identifying 4609 unique-mass crosslink ions at an effective FDR of 0.33%, covering 2534 unique pairs of crosslinked protein positions, 625 of which were inter-protein. The data were statistically non-random and rational in the context of known structures, and showed biological rationality. Crosslink density strongly tracked the individual proteolytic maturation products of p4a and p4b, the two major virion structural proteins, and supported the prediction of transmembrane domains within membrane proteins. A clear sub-network of four virion structural proteins provided structural insights into the virion core wall, and proteins VP8 and A12 formed a strongly-detected crosslinked pair with an apparent structural role. A strongly-detected sub-network of membrane proteins A17, H3, A27 and A26 represented an apparent interface of the early-forming virion envelope with structures added later during virion morphogenesis. Protein H3 seemed to be the central hub not only for this sub-network but also for an attachment protein' sub-network comprising membrane proteins H3, ATI, CAHH(D8), A26, A27 and G9. Crosslinking data lent support to a number of known interactions and interactions within known complexes. Evidence is provided for the membrane targeting of genome telomeres. In covering several orders of magnitude in protein abundance, this study may have come close to the bottom of the protein-protein crosslinkome of an intact organism, namely a complex animal virus. Author summary Vaccinia is one of the most complex virions among the animal viruses, containing 70+ distinct gene products. Although virion ultrastructure has been apparent, at least in outline by electron microscopy since the year 1961 or earlier, its molecular architecture is largely unknown: Vaccinia is resistant to classical structural approaches requiring virus crystallization and moderately resistant to cryoEM. Molecular approaches requiring the maintenance of protein assemblies during virion deconstruction, reconstruction of protein complexes in heterologous or in vitro systems, or internalization of bulky reagents such as antibodies or gold particles may have been already pursued close to exhaustion. Here, protein interfaces within and around the intact virion were identified by virus incubation with bifunctional chemical crosslinkers in situ followed by proteolysis and peptide-level mass spectrometry. This minimally invasive approach revealed the molecular arrangements of structural and membrane protein complexes within the virus, confirming and extending several aspects of virus biology.