Roles of the Minor Capsid Protein P7 in the Assembly and Replication of Double-Stranded RNA Bacteriophage φ{symbol}6

Institute of Biotechnology, Viikki Biocenter 2, University of Helsinki, Helsinki, Finland.
Journal of Molecular Biology (Impact Factor: 4.33). 10/2008; 383(3):529-38. DOI: 10.1016/j.jmb.2008.08.082
Source: PubMed


The polymerase complexes of double-stranded RNA (dsRNA) viruses are multifunctional RNA processing machineries that carry out viral genome packaging, replication, and transcription. The polymerase complex forms the innermost virion shell and is structurally related in dsRNA viruses infecting a diversity of host organisms. In this study, we analyzed the properties and functions of the minor polymerase complex protein P7 of dsRNA bacteriophage phi6 using terminally truncated P7 polypeptides and an in vitro self-assembly system established for the phi6 polymerase complex. The N-terminally truncated P7 failed to dimerize, whereas C-terminally truncated P7 polypeptides formed functional dimers that were incorporated into the polymerase complex. Nevertheless, the polymerase complex assembly kinetics and stability were altered by the incorporation of the C-terminally truncated P7. Using the in vitro assembly system for phi6 nucleocapsids and subsequent infectivity assays, we confirmed that full-length P7 is necessary for the formation of infectious viral particles. Contrary to previous results, we found that P7 must be incorporated into polymerase complexes during shell assembly.

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    • "Bacteriophage f6, the type member of the Cystoviridae, initially assembles as an RNA-free procapsid with deeply recessed vertices, giving it a dodecahedral morphology (Butcher et al., 1997). The procapsid accommodates the polymerase (P2) and an accessory protein (P7), which has a regulatory function in assembly and RNA packaging (Poranen et al., 2008). P2 is bound to the inner surface of the procapsid (Nemecek et al., 2010; Sen et al., 2008) at sites close to the 3-fold axes that "
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    ABSTRACT: The cystovirus ϕ6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in its icosahedral capsid, and capsids as compartments for transcription and replication. ϕ6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an α-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.
    Structure 07/2013; 21(8). DOI:10.1016/j.str.2013.06.007 · 5.62 Impact Factor
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    • "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, φ12, indicate that after expansion, P7 surrounds the P4 hexamer on the five-fold axis of symmetry [15]. "
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    ABSTRACT: The objective of this study was to determine the location of protein P7, the RNA packaging factor, in the procapsid of the φ6 cystovirus. A comparison of cryo-electron microscopy high-resolution single particle reconstructions of the φ6 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.
    PLoS ONE 10/2012; 7(10):e47489. DOI:10.1371/journal.pone.0047489 · 3.23 Impact Factor
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    ABSTRACT: Bacteriophage phi6 is the type member of the family Cystoviridae and infects Gram-negative Pseudomonas syringae cells. The virion consists of a protein-rich lipid envelope enclosing a nucleocapsid. The nucleocapsid covers the icosahedral polymerase complex that encloses the double-stranded RNA genome. Here, we demonstrate that nucleocapsid surface protein P8 is the single nucleocapsid component interacting with the cytoplasmic membrane. This interaction takes place between P8 and phospholipid. Based on this and previous studies, we propose a model where the periplasmic nucleocapsid interacts with the phospholipid head groups and, when the membrane voltage exceeds the threshold of 110 mV, this interaction drives the nucleocapsid through the cytoplasmic membrane, resulting in an intracellular vesicle containing the nucleocapsid.
    Journal of General Virology 08/2010; 91(Pt 8):2116-20. DOI:10.1099/vir.0.020305-0 · 3.18 Impact Factor
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