title: Probing, by self-assembly, the number of potential binding sites for minor protein subunits in the procapsid of double-stranded RNA bacteriophage Φ6.
abstract: The double-stranded RNA bacteriophage Φ6 is an extensively studied prokaryotic model system for virus assembly. There are established in vitro assembly protocols available for the Φ6 system for obtaining infectious particles from purified protein and RNA constituents. The polymerase complex is a multifunctional nanomachine that replicates, transcribes, and translocates viral RNA molecules in a highly specific manner. The complex is composed of (i) the major structural protein (P1), forming a T=1 icosahedral lattice with two protein subunits in the icosahedral asymmetric unit; (ii) the RNA-dependent RNA polymerase (P2); (iii) the hexameric packaging nucleoside triphosphatase (NTPase) (P4); and (iv) the assembly cofactor (P7). In this study, we analyzed several Φ6 virions and recombinant polymerase complexes to investigate the relative copy numbers of P2, P4, and P7, and we applied saturated concentrations of these proteins in the self-assembly system to probe their maximal numbers of binding sites in the P1 shell. Biochemical quantitation confirmed that the composition of the recombinant particles was similar to that of the virion cores. By including a high concentration of P2 or P7 in the self-assembly reaction mix, we observed that the numbers of these proteins in the resulting particles could be increased beyond those observed in the virion. Our results also suggest a previously unidentified P2-P7 dependency in the assembly reaction. Furthermore, it appeared that P4 must initially be incorporated at each, or a majority, of the 5-fold symmetry positions of the P1 shell for particle assembly. Although required for nucleation, excess P4 resulted in slower assembly kinetics.
authors.affiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Sun
authors.forename: Dennis H
authors.initials: DH
authors.lastname: Bamford
authors.forename: Minna M
authors.initials: MM
authors.lastname: Poranen
mesh_terms: D017126:Bacteriophage phi 6 / Q000378:metabolism*; D001665:Binding Sites; D002213:Capsid / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D018628:Gene Dosage; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D021122:Protein Subunits / Q000235:genetics; D012330:RNA, Double-Stranded / Q000737:chemistry*; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014771:Virion / Q000235:genetics; D019065:Virus Assembly / Q000235:genetics
chemical_list: D010455:Peptides; D021122:Protein Subunits; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011994:Recombinant Proteins