Abstract
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.
Highlights
The virion of Vaccinia, the prototypical poxvirus, is one of the largest among the animal viruses
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
Summary
The virion of Vaccinia, the prototypical poxvirus, is one of the largest among the animal viruses. Electron microscopy (EM) and atomic force microscopy (AFM) studies have established clear ultrastructural compartments of the mature virion (MV) [4] including a central, genome-containing ‘core’ that houses a number of virus-encoded enzymes of mRNA transcription and modification, a proteinaceous wall surrounding the core, a pair of ‘lateral body’ structures flanking the core wall, a single lipid bilayer envelope, and an outer protein-rich coat that appears late during maturation. The virion contains between 58 and 73 distinct gene products [5] Some of these have been localized at low resolution on the basis of immunogold EM [6,7,8,9,10], while the compartmental locale of others can be inferred from clearly identifiable transmembrane (TM) domains and other bioinformatics signatures, known function and/or the conditions required for the extraction from the virion. A number of structural proteins of the virion core remain insoluble during virion extraction even in ionic detergent
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