Roles of the minor capsid protein P7 in the assembly and replication of double-stranded RNA bacteriophage phi6.
ABSTRACT 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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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; DOI:10.1016/j.str.2013.06.007 · 6.79 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 · 3.53 Impact Factor
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ABSTRACT: Double-stranded RNA (dsRNA) viruses are a diverse group of viruses infecting hosts from bacteria to higher eukaryotes. Among the hosts are humans, domestic animals, and economically important plant species. Fine details of high-resolution virion structures have revealed common structural characteristics unique to these viruses including an internal icosahedral capsid built from 60 asymmetric dimers (120 monomers!) of the major coat protein. Here we focus mainly on the structures and assembly principles of large icosahedral dsRNA viruses belonging to the families of Cystoviridae and Reoviridae. It is obvious that there are a variety of assembly pathways utilized by different viruses starting from similar building blocks and reaching in all cases a similar capsid architecture. This is true even with closely related viruses indicating that the assembly pathway per se is not an indicator of relatedness and is achieved with minor changes in the interacting components.Advances in Experimental Medicine and Biology 01/2012; 726:379-402. DOI:10.1007/978-1-4614-0980-9_17 · 2.01 Impact Factor