Asymmetric Genome Organization in an RNA Virus Revealed via Graph-Theoretical Analysis of Tomographic Data

March Asymmetric Genome Organization in an RNA Virus Revealed via Graph-Theoretical Analysis of Tomographic Data James A. Geraets 0 1 Eric C. Dykeman 0 1 Peter G. Stockley 0 1 Neil A. Ranson 0 1 Reidun Twarock 0 1 0 1 York Centre for Complex Systems Analysis, University of York, York, United Kingdom, 2 Astbury Centre for Structural Molecular Biology, University of Leeds , Leeds , United Kingdom 1 Editor: Claus O. Wilke, University of Texas at Austin, UNITED STATES Cryo-electron microscopy permits 3-D structures of viral pathogens to be determined in remarkable detail. In particular, the protein containers encapsulating viral genomes have been determined to high resolution using symmetry averaging techniques that exploit the icosahedral architecture seen in many viruses. By contrast, structure determination of asymmetric components remains a challenge, and novel analysis methods are required to reveal such features and characterize their functional roles during infection. Motivated by the important, cooperative roles of viral genomes in the assembly of single-stranded RNA viruses, we have developed a new analysis method that reveals the asymmetric structural organization of viral genomes in proximity to the capsid in such viruses. The method uses geometric constraints on genome organization, formulated based on knowledge of icosahedrally-averaged reconstructions and the roles of the RNA-capsid protein contacts, to analyse cryo-electron tomographic data. We apply this method to the low-resolution tomographic data of a model virus and infer the unique asymmetric organization of its genome in contact with the protein shell of the capsid. This opens unprecedented opportunities to analyse viral genomes, revealing conserved structural features and mechanisms that can be targeted in antiviral drug design. - Funding: This work was funded by the Wellcome Trust [www.wellcome.ac.uk] (090932/Z/09/Z, 094232/ Z/10/Z and 097326/Z/11/Z) and the Engineering and Physical Sciences Research Council [www.epsrc.ac. uk] (EC-2013-019). ECD is supported by a fellowship from the Leverhulme Trust [www.leverhulme.ac.uk] (EC-2013-019). RT is the recipient of a Royal Society Leverhulme Trust Senior Research Fellowship (LT130088), awarded by the Royal Society [www. royalsociety.org]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. their roles in capsid formation. In order to design an additional class of anti-viral drugs that interfere with this process, it is important to understand the asymmetric organization of the genome inside viral capsids. This is currently a challenge, as the averaging techniques used to achieve high resolution structures of the protein containers cannot be used. We present a new approach and demonstrate its predictive power here for a test virus. This paves the way for a better understanding of the functional roles of viral RNAs in virus assembly and their exploitation in anti-viral drug design. Viruses are remarkable examples of symmetry and self-assembly at the nanoscale. The protein containers that encapsulate most viral genomes are formed from just a few different protein building blocks that self-assemble into particles with icosahedral symmetry, and can be described in terms of icosahedral surface lattices [1]. This geometry minimizes the amount of the genome fragment needed to code for the viral capsid, while maximizing its volume/surface area ratio; the principle of genetic economy [2]. Symmetry therefore plays a pivotal role in understanding virus structure. Symmetry averaging techniques have been used to determine viral capsid structures at atomic resolution by X-ray crystallography, and by reconstruction of such structures at medium resolution by cryo-electron microscopy (cryo-EM). However, not all viral components are organized with icosahedral symmetry. Cryo-EM can be used to refine such asymmetric structures provided that they are large enough in mass terms to contribute significantly to the image [3, 4]. However, asymmetric viral components normally contribute too weakly to the images obtained by cryo-EM to allow the refinement of an asymmetric model [5]. Note, in crystals of viral particles, the asymmetric features of the individual viruses usually do not dictate crystal packing contacts, and are therefore averaged out by the lattice. The important functional roles of such viral components in the viral life cycle are therefore difficult to characterize. An example is the single-copy of maturation protein (MP, also called A-protein) in bacteriophage MS2 that is hypothesized to replace a protein dimer in the capsid [6]. It attaches to the bacterial receptor during the infection to facilitate genome extraction. The asymmetric organization of the viral genome inside a capsid is also difficult to reconstruct. Indeed, MS2 is typical in that the high resolution crystal structure lacks density for the *3.7kb genome [7, 8], but cryo-EM reconstructions from both our group and others show extensive density for the RNA [6, 912]. This difference arises because of technical aspects of the ways the EM and X-ray data are collected. We demonstrate here that a better understanding of the asymmetric organization of the viral genome within the capsid can be achieved if specifics about the contacts between capsid protein (CP) and the packaged genome are factored into an analysis of tomographic data. Recently we have shown that a number of positive-sense single-stranded (ss)RNA viruses encode dispersed, degenerate sequence/structure elements within their genomes that bind their cognate coat proteins specifically during assembly, facilitating capsid assembly efficiency [1317]. These packaging signals (PSs) can have dramatic effects on the kinetics and fidelity of virion assembly [18]. There are widespread contacts between genomic RNA and capsid protein in picornaviruses, e.g. rhinovirus [19], and preliminary in vivo experiments for human parechovirus 1 suggest that they function as PSs (ongoing work with collaborators). The requirement for the PSs to contact the coat proteins of the viral capsid at specific positions in the capsid imposes a constraint on the conformation of the genome within each viral particle, that we are exploiting here to analyse tomograms of the packaged genomes. In particular, we exploit knowledge of the PS positions with reference to the icosahedrallyaveraged RNA cages that have been observed in a large number of viruses in proximity to capsid, to formulate constraints on the connections between the PSs. For example, if PSs are located at the vertices of these cages, as in the model system we are considering here, then the RNA organization in proximity to capsid can be modelled as connected paths along the edges of the RNA cage [15, 20]. If the majority of the potential binding sites are (...truncated)