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.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: The double-stranded RNA bacteriophage 6 is an extensively studied prokaryotic model system for virus assembly. There are established in vitro assembly protocols available for the 6 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 6 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.Journal of Virology 08/2012; 86(22):12208-16. · 5.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Bacteriophage 6 is a double-stranded RNA (dsRNA) virus whose genome is packaged sequentially as three single-stranded RNA (ssRNA) segments into an icosahedral procapsid which serves as a compartment for genome replication and transcription. The procapsid shell consists of 60 copies each of P1(A) and P1(B), two nonequivalent conformers of the P1 protein. Hexamers of the packaging ATPase P4 are mounted over the 5-fold vertices, and monomers of the RNA-dependent RNA polymerase (P2) attach to the inner surface, near the 3-fold axes. A fourth protein, P7, is needed for packaging and also promotes assembly. We used cryo-electron microscopy to localize P7 by difference mapping of procapsids with different protein compositions. We found that P7 resides on the interior surface of the P1 shell and appears to be monomeric. Its binding sites are arranged around the 3-fold axes, straddling the interface between two P1(A) subunits. Thus, P7 may promote assembly by stabilizing an initiation complex. Only about 20% of the 60 P7 binding sites were occupied in our preparations. P7 density overlaps P2 density similarly mapped, implying mutual occlusion. The known structure of the 12 homolog fits snugly into the P7 density. Both termini-which have been implicated in RNA binding-are oriented toward the adjacent 5-fold vertex, the entry pathway of ssRNA segments. Thus, P7 may promote packaging either by interacting directly with incoming RNA or by modulating the structure of the translocation pore.Journal of Virology 08/2012; 86(21):11616-24. · 5.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Enveloped double-stranded RNA (dsRNA) bacterial virus Pseudomonas phage ϕ6 has been developed into an advanced assembly system where purified virion proteins and genome segments self-assemble into infectious viral particles, inferring the assembly pathway. The most intriguing step is the membrane assembly occurring inside the bacterial cell. Here, we demonstrate that the middle virion shell, made of protein 8, associates with the expanded viral core particle and the virus-specific membrane vesicle.Journal of Virology 02/2012; 86(9):5376-9. · 5.08 Impact Factor