Initial Location of the RNA-dependent RNA Polymerase in the Bacteriophage Φ6 Procapsid Determined by Cryo-electron Microscopy

Abstract

The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and transcription. In bacteriophage ⌽6, this inner shell is first assembled as an icosahedral procapsid with recessed 5-fold vertices that subsequently undergoes major structural changes during maturation. The tripartite genome is packaged as singlestranded RNA molecules via channels on the 5-fold vertices, and transcripts probably exit the mature capsid by the same route. The RdRP (protein P2) is assembled within the procapsid, and it was thought that it should be located on the 5-fold axes near the RNA entry and exit channels. To determine the initial location of the RdRP inside the procapsid of bacteriophage ⌽6, we performed cryo-electron microscopy of wild type and mutant procapsids and complemented these data with biochemical determinations of copy numbers. We observe ring-like densities on the 3-fold axes that are strong in a mutant that has ϳ10 copies of P2 per particle; faint in wild type, reflecting the lower copy number of ϳ3; and completely absent in a P2-null mutant. The dimensions and shapes of these densities match those of the known crystal structure of the P2 monomer. We propose that, during maturation, the P2 molecules rotate to occupy positions closer to adjacent 5-fold vertices where they conduct replication and transcription.

Probing, by Self-Assembly, the Number of Potential Binding Sites for Minor Protein Subunits in the Procapsid of Double-Stranded RNA Bacteriophage φ6

“…Early biochemical studies based on quantification of individual proteins by use of radioactively labeled virus proposed that there are 100, 14, 111, and 92 copies of P1, P2, P4, and P7, respectively, in the virions of 6 (13). Cryo-electron microscopy data have located minor protein components at the 5-fold (P4) or 3-fold (P2 and P7) axis of icosahedral symmetry (14,35,46). Here we describe a Coomassie brilliant blue staining-based biochemical quantification method for measurement of 6 PC protein components (see Fig.…”

“…P4 hexamers are located at the icosahedral 5-fold symmetry positions on the surface of the P1 shell. P2 and P7 are located in the interior of the capsid, near the icosahedral 3-fold symmetry axes (34,35,46). The theoretical maximal copy numbers for P2, P4, and P7 are 20, 72, and 60, respectively.…”

“…Protein P2 has been suggested to reside close to the 3-fold symmetry axes of the 6 PC, with a proposed 20 binding sites per particle, although no PC with a full occupancy of P2 has been detected (34,46). We probed the number of potential P2 binding sites by titrating the amount of P2 in the in vitro assembly reaction mix for 6 PCs.…”

“…In dsRNA viruses of the family Reoviridae, such as orthoreovirus (52), rotavirus (29), rice dwarf virus (33), aquareovirus (11), and gram carp reovirus (10), the polymerase subunit localizes to the interior of the Tϭ1 shell, close to 5-fold axes of icosahedral symmetry, resulting in a maximal number of 12 polymerase subunits per particle. However, a recent cryo-electron tomography study of recombinant 6 PCs suggests that the polymerase (P2) resides near the 3-fold axes in the PC interior, with 20 potential binding sites per particle (46). Nemecek et al (34) observed that P2 is assembled randomly at the proposed binding locations, with a mean copy number of eight subunits per recombinant PC.…”

“…The existing information regarding the stoichiometry, location, and occupancy of PC proteins was obtained primarily from electron microscopy studies (20,21,34,46). The recent observation of the P2 location near the 3-fold symmetry axes of the PC, as well as the apparently low occupancy of P2 at the 20 potential binding locations of the recombinant PC particle (34,46), encouraged us to systematically analyze the average copy numbers of proteins P2, P4, and P7 in several recombinant PC and 6 virion preparations. We used the in vitro self-assembly system for 6 PCs to probe the number of maximal binding sites for P2 and P7 in the 6 PC structure.…”