Protein P7 of the Cystovirus φ6 Is Located at the Three-Fold Axis of the Unexpanded Procapsid

et al. (2012) Protein P7 of the Cystovirus w6 Is Located at the Three-Fold Axis of the Unexpanded Procapsid. PLoS ONE 7(10): e47489. doi:10.1371/journal.pone.0047489 Protein P7 of the Cystovirus w6 Is Located at the Three- Fold Axis of the Unexpanded Procapsid Garrett Katz 0 Hui Wei 0 Alexandra Alimova 0 Al Katz 0 David Gene Morgan 0 Paul Gottlieb 0 Claude Krummenacher, University of Pennsylvania School of Veterinary Medicine, United States of America 0 1 Sophie Davis School of Biomedical Education, City College of New York, New York, New York, United States of America, 2 Department of Physics, City College of New York, New York, New York, United States of America, 3 Chemistry Department, Indiana University , Bloomington, Indiana , United States of America The objective of this study was to determine the location of protein P7, the RNA packaging factor, in the procapsid of the w6 cystovirus. A comparison of cryo-electron microscopy high-resolution single particle reconstructions of the w6 complete unexpanded procapsid, the protein P2-minus procapsid (P2 is the RNA directed RNA-polymerase), and the P7-minus procapsid, show that prior to RNA packaging the P7 protein is located near the three-fold axis of symmetry. Difference maps highlight the precise position of P7 and demonstrate that in P7-minus particles the P2 proteins are less localized with reduced densities at the three-fold axes. We propose that P7 performs the mechanical function of stabilizing P2 on the inner protein P1 shell which ensures that entering viral single-stranded RNA is replicated. - Funding: This work is support in part by the National Institute of General Medical Science - Grant SC1-GM092781 and the Research Centers in Minority Institutions (NIH/NCRR/RCMI) CCNY/Grant G12-RR03060. The electron microscopy facilities at the New York Structural Biology Center are supported by grant C000087 from the New York State Foundation for Science, Technology and Innovation (NYSTAR), and National Institutes of Health grant S10 RR017291. 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. The cystoviridae family of viruses, of which w6 was the first discovered, contain three segments of double stranded RNA. The RNA packaging, replication, transcription mechanism, and overall structure resembles that of the reoviruses making the species an excellent model system for these important pathogens. The initial step in cystoviridae replication is the assembly of a closed and unexpanded procapsid (PC). The PC is responsible for RNA packaging, transcription, and genome replication and is composed of four proteins - P1, P2, P4, and P7 [1,2]. A schematic of w6 is shown in Fig. 1, the hexagon in the center, less RNA, represents the PC. The PC is initially assembled as a dodecahedron with recessed vertices prior to RNA packaging [3]. The 120 copies of P1 form the procapsid shell [4]. During replication, the PC first forms in the unexpanded state, followed by sequential expansion as ssRNA is packaged. Isolated mutants of expanded or unexpanded PC particles can be produced in Escherichia coli [2]. Recently, tomographic reconstructions of cryo-electron micrographs from both unexpanded and expanded PCs showed that all vertices in a individual PC are either in a completely unexpanded or completely expanded state [5]. During RNA packaging, the expansion proceeds in a stepwise mechanism that accommodates the outward pressure of packaged RNA [5]. The packaging of the cystovirus genomic ssRNA into a preformed PC depends on the three portal proteins, P2, P4 and P7. The portals are located at the 12 PC five-fold vertices. These proteins must function in a coordinated mechanism to package the viral RNA segments, replicate them into a double-stranded form and transcribe them [2]. P2 is the RNA-dependent RNA polymerase (RdRP). P4 is the hexameric nucleotide triphosphorylase (NTPase) packaging motor [6]. P4 is assembled on the outer PC surface at the 12 potential RNA portal sites. The six-fold symmetry of P4 forms a mismatch with the five-fold axis of symmetry [7,8]. P7 is assembled into the PC as well, and is required for efficient PC assembly [9], RNA packaging [10,11] and transcription [9]. P7 is the least characterized of the PC proteins. In solution, there is evidence that it forms an elongated dimer [9,12,13]. Poranen et al. observed that an excess concentration of P7 accelerated assembly of P1 in vitro, indicating that P7 may stabilize P1 [13]. The precise position of P7 within the PC, and hence a structural explanation for its importance has not been described [14]. Cryo-EM studies of related cystovirus, w12, indicate that after expansion, P7 surrounds the P4 hexamer on the five-fold axis of symmetry [15]. The crystal structure of the w12 P7 coreproteins was obtained at 1.8 A resolution by x-ray diffraction [16] and together with solution nuclear magnetic resonance (NMR) studies suggest a variety of functional roles for P7. P7 exists as a unique a/b-fold and forms a symmetric homodimer in solution. The C-terminal tail (amino acids 129169) is significantly disordered but on interaction with RNA shows a reduced degree of disorder. The NMR measurements show that the flexible Cterminal tail minimally interacts with the protein core. Of great interest in that work was the suggestion that P7 could play a role in viral RNA recognition. Chemical shifts in the NMR spectrum of the P7 C-terminus (amino acids 159 to 163) were evident in the presence of 5-nucleotide oligoribonucleotides corresponding in sequence to the 59-ends of plus-sense w6 mRNA. The most significant chemical shifts were the negatively charged amino acids Glu135, Glu143, and Asp160. This observation suggests that P7 mechanistically interacts with P2 during RNA packaging and replication. The location of P2 in the expanded PC remains controversial but based upon reconstructions of empty and filled w12 PC, it is suggested that it is directly beneath the central position of the five-fold symmetry axes [15]. Cryo-EM studies have shown that the RNA polymerase of reoviruses are attached to the core shell and overlap the five-fold axis [17]. In the unexpanded w6 PC, P2 is located on the three-fold axis between the inverted five-fold vertices [18]. These studies suggest that P2 in the unpackaged PC is initially at the three-fold axis but during RNA packaging and replication, translates to a position beneath the fivefold symmetry axis. Recent cryo-EM studies by Nemecek et al. [19] suggest that the P2 and P4 sites are randomly incorporated into the PC and no mechanism exists to cause these two proteins to colocalize. Proteins homologous to P7 could exist in reoviruses where, for example, m2 serves as an RNA polymerase cofactor [20]. In this paper we utilized cryo-electron microscopy with single particle reconstruction to (...truncated)