Three-Dimensional Structure of the Enveloped Bacteriophage Φ12: An Incomplete T = 13 Lattice Is Superposed on an Enclosed T = 1 Shell

et al. (2009) Three-Dimensional Structure of the Enveloped Bacteriophage W12: An Incomplete T = 13 Lattice Is Superposed on an Enclosed T = 1 Shell. PLoS ONE 4(9): e6850. doi:10.1371/journal.pone.0006850 Three-Dimensional Structure of the Enveloped Bacteriophage W12: An Incomplete T = 13 Lattice Is Superposed on an Enclosed T = 1 Shell Hui Wei 0 R. Holland Cheng 0 John Berriman 0 William J. Rice 0 David L. Stokes 0 A. Katz 0 David Gene 0 Morgan 0 Paul Gottlieb 0 Robert J. Geraghty, University of Minnesota, United States of America 0 1 Department of Microbiology and Immunology, Sophie Davis School of Biomedical Education, The City College of New York (CCNY) , New York , New York, United States of America, 2 Nanoscience Center, Department of Chemistry, Indiana University , Bloomington , Indiana, United States of America, 3 Department of Cellular and Molecular Biology, University of California at Davis, Davis, California, United States of America, 4 Structural Biology Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine , New York , New York, United States of America, 5 The New York Structural Biology Center , New York , New York, United States of America, 6 Institute for Ultrafast Spectroscopy and Lasers, The City College of New York , New York, New York , United States of America Background: Bacteriophage w12 is a member of the Cystoviridae, a unique group of lipid containing membrane enveloped bacteriophages that infect the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola. The genomes of the virus species contain three double-stranded (dsRNA) segments, and the virus capsid itself is organized in multiple protein shells. The segmented dsRNA genome, the multi-layered arrangement of the capsid and the overall viral replication scheme make the Cystoviridae similar to the Reoviridae. Methodology/Principal Findings: We present structural studies of cystovirus w12 obtained using cryo-electron microscopy and image processing techniques. We have collected images of isolated w12 virions and generated reconstructions of both the entire particles and the polymerase complex (PC). We find that in the nucleocapsid (NC), the w12 P8 protein is organized on an incomplete T = 13 icosahedral lattice where the symmetry axes of the T = 13 layer and the enclosed T = 1 layer of the PC superpose. This is the same general protein-component organization found in w6 NC's but the detailed structure of the entire w12 P8 layer is distinct from that found in the best classified cystovirus species w6. In the reconstruction of the NC, the P8 layer includes protein density surrounding the hexamers of P4 that sit at the 5-fold vertices of the icosahedral lattice. We believe these novel features correspond to dimers of protein P7. Conclusions/Significance: In conclusion, we have determined that the w12 NC surface is composed of an incomplete T = 13 P8 layer forming a net-like configuration. The significance of this finding in regard to cystovirus assembly is that vacancies in the lattice could have the potential to accommodate additional viral proteins that are required for RNA packaging and synthesis. - Funding: PG was supported by grant NIH-RCMI G12RR-03060 from The National Center for Research Resources and the PSC-CUNY Foundation. DGM was supported by the University of California, Davis, Department of Molecular and Cellular Biology and the Nano-Fabrication Center, Department of Chemistry, Indiana University. DLS was supported by grant NIH R01 GM071044 from the National Institutes of Health. The facilities at the New York Structural Biology Center (NYSBC) are supported by grant C000087 from the New York State Foundation for Science, Technology and Innovation (NYSTAR). The funders have 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. . These authors contributed equally to this work. The cystoviruses (w6w14) are a unique group of viruses that infect strains of the plant pathogen Pseudomonas syringae pv phaseolicola. They have been very useful research models for elucidating the replication mechanisms of RNA viruses [1]. In particular, the overall replicative mechanism and the multishell structure of cystoviruses are analogous to members of the Reoviridae family [1]. Both virus families package their mRNA as precursors to the double-stranded RNA (dsRNA) genomic segments. Within the cystovirus family, all species share a very similar genetic organization and encode a comparable set of proteins [2]. In addition, the RNA-directed RNA polymerase (RdRP) is structurally and mechanistically related to the comparable enzyme of the flaviviruses and has been used to study de novo initiation of viral RNA synthesis [35]. A schematic of the structure common to all of the cystovirus species is depicted in Fig. 1 and summarized by Gottlieb [6,7]. Three segments of dsRNA are packaged, replicated, and transcribed within the inner viral shell, the polymerase complex (PC) [8]. The three dsRNA segments are shown in a circular format in the schematic diagram. The PC, as defined in w6, is assembled from four viral proteins (P1, P2, P4, and P7) that are arranged in a dodecahedral conformation [9]. In the work presented here, we find that the w12 PC is composed of proteins P1 and P2, represented by the hexagon in the schematic. The P1 protein is the main structural element of the PC and is responsible for the organization of the dodecahedral assembly in w6, existing as two structurally non-equivalent monomers, named A and B [10]. P2 is an RNA-dependent RNA polymerase [11]. The dsRNA-packaged PC is subsequently covered by a P8 protein shell, which together with P4, P5, and P7, constitute the viral nucleocapsid (NC). The P8 proteins of the NC shell are arranged as trimers organized in a T = 13 icosahedral lattice [10,12]. The P8 shell is shown as the dark circle in the schematic. P4 is a hexameric complex that possesses NTPase activity and is located at the 5-fold symmetry axes of the NC [13,14]. P7 is a dimeric protein and maintains RNA packaging efficiency [15]. All cystoviruses are enveloped by a phospholipid envelope membrane which constitutes the outer virus layer and contains viral encoded proteins P3, P6, P9, P10, and P13 [9,16,17]. Protein P12 is a nonstructural protein that mediates the membrane envelope acquisition that surrounds the viral NC. It is also responsible for inserting the viral proteins into the phospholipid envelope [17,18]. A protein complex composed of membrane proteins P3 and P6 constitute the viral attachment apparatus [17,19,20]. Cryo-electron microscopy (cryoEM) has previously been used to reveal the organization of proteins making up the isolated NC of w6 as well as the docking of the hexameric ATPase at the five-fold vertex of the NC [21]. These studies relied on the icosahedral symmetry innate to (...truncated)