Packaging Accessory Protein P7 and Polymerase P2 Have Mutually Occluding Binding Sites inside the Bacteriophage ϕ6 Procapsid

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.

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

“…5D), whereas approximately 36 or 40 copies were detected in recombinant PCs or 6 virions, respectively ( Table 1). The observed 59 P7 subunits could occupy almost all of the theoretical 60 binding sites in the PC (three subunits at each of the 20 3-fold symmetry axes of the PC) that were also indicated in a recent cryo-electron microscopy study (35). Apparently, the association of P7 with other PC components could stabilize the trimeric state detected for cross-linked, purified P7 (see Fig.…”

“…The amounts of P2 and P7 in the PCs were significantly diminished if these proteins were added to preassembled particles (Fig. 7), which is in accordance with their internal location (34,35).…”

“…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 P7 is an assembly cofactor that accelerates PC assembly and is essential for the formation of infectious virions (42,43). P7 forms elongated dimers in solution (23); however, recent structural studies indicate that P7 subunits are arranged as monomers around the 3-fold axes of empty PCs (35). Thus, the multimeric state of P7 has not been established.…”