Component tree analysis of cystovirus φ6 nucleocapsid Cryo-EM single particle reconstructions

RESEARCH ARTICLE Component tree analysis of cystovirus φ6 nucleocapsid Cryo-EM single particle reconstructions Lucas M. Oliveira1¤a, Ze Ye1, Al Katz2, Alexandra Alimova3, Hui Wei3¤b, Gabor T. Herman1, Paul Gottlieb3* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Computer Science, Graduate Center of the City University of New York, New York, NY, United States of America, 2 Physics Department, City College of New York, New York, NY, United States of America, 3 City University of New York School of Medicine, City College of New York, New York, NY, United States of America ¤a Current address: Philips Research North America, Cambridge, MA, United States of America ¤b Current address: New York Structural Biology Center, New York, NY, United States of America * Abstract OPEN ACCESS Citation: Oliveira LM, Ye Z, Katz A, Alimova A, Wei H, Herman GT, et al. (2018) Component tree analysis of cystovirus φ6 nucleocapsid Cryo-EM single particle reconstructions. PLoS ONE 13(1): e0188858. https://doi.org/10.1371/journal. pone.0188858 Editor: Raymond Schuch, ContraFect Corp, UNITED STATES Received: July 24, 2017 Accepted: November 14, 2017 Published: January 4, 2018 Copyright: © 2018 Oliveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are available from the World Wide Protein Data Bank (https://www.wwpdb.org/) under the following deposition code: D_1000231282. All relevant software source code (under development) is available from GitHub (https://github.com/matav3/ matav3). Funding: Funding, in part, was provided by grants from the National Institute of General Medical Science Grant SC1-GM092781 to Paul Gottlieb and the National Institutes of Health Research Centers The 3-dimensional structure of the nucleocapsid (NC) of bacteriophage φ6 is described utilizing component tree analysis, a topological and geometric image descriptor. The component trees are derived from density maps of cryo-electron microscopy single particle reconstructions. Analysis determines position and occupancy of structure elements responsible for RNA packaging and transcription. Occupancy of the hexameric nucleotide triphosphorylase (P4) and RNA polymerase (P2) are found to be essentially complete in the NC. The P8 protein lattice likely fixes P4 and P2 in place during maturation. We propose that the viral procapsid (PC) is a dynamic structural intermediate where the P4 and P2 can attach and detach until held in place in mature NCs. During packaging, the PC expands to accommodate the RNA, and P2 translates from its original site near the inner 3-fold axis (20 sites) to the inner 5-fold axis (12 sites) with excess P2 positioned inside the central region of the NC. Introduction Bacteriophage φ6 and its relatives are model systems for virus assembly, genome packaging and dsRNA polymerization. The RNA packaging, replication, transcription mechanism, and overall structure resembles that of reoviruses making the species an excellent model system to study these important pathogens. Of particular interest is the molecular, spatial relationships and overall organization of the RNA packaging and transcription elements found in the procapsid (PC) and mature nucleocapsid (NC). We compare the relative location and occupancy of viral portal proteins in the NC and make comparisons to existing models of the pre-packaged PC. A component tree analysis is employed on single particle reconstructions of the NC to identify and locate the viral elements and to estimate protein occupancy. The φ6 cystovirus species consist of multilayered particles that assemble in a specific order [1, 2]. The initial step in ϕ6 replication is the assembly of an unexpanded procapsid (PC), the PLOS ONE | https://doi.org/10.1371/journal.pone.0188858 January 4, 2018 1 / 16 φ6 component trees in Minority Institutions (NIH/NCRR/RCMI) CCNY/ Grant G12-RR03060 to Paul Gottlieb. Some of this work was performed at the National Resource for Automated Molecular Microscopy (GM103310) at the Simons Electron Microscopy Center (Grant 349247) at the New York Structural Biology Center. Component tree software will be provided upon request. 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. structure responsible for the viral messenger RNA (mRNA) packaging, replication to dsRNA, and the early phase transcription of the mRNA. The PC is composed of four proteins–P1, P2, P4, and P7 [1]. It is built from 120 identical P1 proteins organized into a (triangulation number) T = 1 shell containing 60 non-symmetric dimers where each dimer is composed of subunits A and B of the same 3-dimensional fold [3, 4]. The P1 crystal structure has been determined for viruses ϕ6 and ϕ8 and is a trapezoidal shape that accommodates the significant conformational changes that occur during RNA packaging [3]. Ultimately all three segments are replicated to dsRNA segments that are enclosed into a nucleocapsid (NC), the outer layer of which is a shell composed of a matrix assembled of protein P8 [5–7]. The P8 shell is composed of 200 trimers arranged as a T = 13 lattice that partially covers the filled PC [5, 8, 9]. Recently Sun et al. has shown that Ca+ can induce transition of the P8 trimer from a closed to open conformation during the outer shell assembly [10]. Two states of the P8 trimers were noted as open and closed allowing domain swapping. Of particular interest within the cystovirus field has been the location and precise occupancy number of the protein components that constitute the RNA replicative apparatus. This structure consists of the RNA-directed RNA polymerase (RdRP) P2, the hexameric nucleotide triphosphorylase (NTPase) P4, and the packaging factor P7. The initial position of P2 is at the inner 3-fold axis (20 sites) [11] however, after RNA packaging, P2 translates to the inner 5-fold axis (12 sites) as noted in the related species ϕ12 [12]. The position and function of the P7 packaging factor remains controversial. [13] suggested, based on Cryo-EM studies of the PC, that the P7 density overlaps the P2 density implying mutual exclusion of each protein at an inner 3-fold axis site. However difference maps generated between PC reconstructions and mutants lacking either P2 or P7 strongly suggest that P7 stabilizes P2 at the inner three-fold axis prior to RNA packaging [14]. This implies P2 and P7 can interact and that they are located near each other in the vicinity of the inner 3-fold axis. Single particle reconstructions with imposed symmetry of the PC and NC [5, 9] show the protein locations. However, the imposed symmetry forces the non-symmetric (...truncated)