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. 2012 Nov;86(21):11616-24.
doi: 10.1128/JVI.01347-12. Epub 2012 Aug 15.

Packaging accessory protein P7 and polymerase P2 have mutually occluding binding sites inside the bacteriophage 6 procapsid

Daniel Nemecek  1 , Jian Qiao, Leonard Mindich, Alasdair C Steven, J Bernard Heymann
Affiliations

Packaging accessory protein P7 and polymerase P2 have mutually occluding binding sites inside the bacteriophage 6 procapsid

Daniel Nemecek et al. J Virol. 2012 Nov.

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.

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Figures

Fig 1
Fig 1
(A to D) Cryo-electron micrographs of P1247 (A), P124 (B), P147 (C), and P14 (D) procapsids taken with an ∼2.1-μm defocus. Bars, 500 Å. The procapsids are randomly oriented. As a result, the images show markedly varying projections of the icosahedral shell with its deeply inverted vertices. (E) SDS-PAGE gel containing P1247 (lane A), P124 (lane B), P147 (lane C), and P14 (lane D) procapsids. The P124 sample also contained a minor contaminant of ∼35 kDa (X). (F) Densitometry profiles (cyan lines) obtained from the SDS-PAGE gels shown in panel E were fitted with asymmetric Gaussian curves (red lines). Peaks of the P1, P2, P4, and P7 subunits are indicated in the sample of P1247 procapsids (profile A).
Fig 2
Fig 2
(A) Segmentation of P1 subunits in the procapsid shell. The P1A subunits (teal) form inverted pentamers located at the 5-fold axes. The P1B subunits (orange) connect neighboring 3-fold vertices via 2-fold axes. One P1A subunit and one P1B subunit are depicted in green and yellow, respectively. (B) Fit of the segmented P1A (green) and P1B (yellow) subunits from the procapsid into the mature shell of the RNA-packaged capsid (gray) (9) (EMDataBank [EMDB] ID 1206). The unassigned densities in the previously described segmentation (12), near the 5-fold and 3-fold axes, are included in the P1A and P1B subunits, respectively (black arrows). (C) Overlay of the segmented P1A (green mesh) and P1B (yellow surface) subunits as viewed from the procapsid exterior (left) and interior (right). Most of the rod-like densities (putative α-helices) are superimposable, with only a few at the edges having relative shifts (black arrows).
Fig 3
Fig 3
(A to D) Slices through icosahedral reconstructions of ϕ6 procapsids filtered to 8-Å resolution and viewed along the 5-fold icosahedral axis ∼73 Å from the procapsid center. Bar, 100 Å. The P4 density is indicated in the map of the P14 procapsid (white arrow). (E to H) Difference maps calculated between P1247 and P147 procapsids or P124 and P14 procapsids show locations of P2, while the difference maps calculated between P1247 and P124 procapsids or P147 and P14 procapsids show locations of P7. (I to K) Corresponding slices through models of P2 and P7 in the procapsid, determined by fitting the P2 structure (PDB ID 1UVJ) and the P7 homology model into the difference maps (see the text). The densities are shown for P2 (I) and P7 (K) subunits. An overlay of the P2 (black contour) and P7 (gray density) locations is shown in panel J.
Fig 4
Fig 4
Overlap of P2 and P7 as shown by difference density maps. (A) Inside view of the procapsid along the 3-fold axis, showing one P2 subunit (yellow mesh) and three neighboring P7 subunits (red, blue, and green). P2 significantly overlaps all three P7 subunits (white arrows). (B) Large overlap between P2 and P7 (white arrows), as seen in a side view.
Fig 5
Fig 5
(A) In the icosahedrally averaged map, the P7 densities (yellow mesh) appear as triplets surrounding the 3-fold axis between the 5-fold vertices of the procapsid shell (gray). One of the three P7 densities is shown in blue. (B) Radial shells of the P1247-P124 difference map at radii of 86, 106, 113, and 120 Å. The P7 density (dark) is organized around the 3-fold axes. (C) Due to the small amounts of P7 in the procapsid and to partial overlap of the P7 and P2 binding sites, most locations at the 3-fold axes are occupied by only one P7 (yellow) or one P2 (magenta) molecule.
Fig 6
Fig 6
Central slices through P124 (A) and P1247 (B) procapsids filtered to 15-Å resolution and oriented along the 2-fold icosahedral axis. The P124 procapsid exhibits additional density under the 5-fold vertex (arrow). Bars, 100 Å. (C) Inside view of the 5-fold vertex of the P1247 (gray mesh) and P124 (yellow) procapsids filtered to 8-Å resolution and depicted at a 3-sigma threshold. The P124 procapsid shows decreased density in the short helices at the tip of P1A subunits at the 5-fold vertex (arrow). The additional density in the P124 procapsid (orange) has a similar size and shape to those of the short helix and may represent its alternative conformation in the absence of P7. Bar, 10 Å.
Fig 7
Fig 7
Orientation of P7 in the procapsid. (A) Crystal structure of ϕ12 P7 (blue ribbon) (PDB ID 2Q82) (6) fitted into the P7 density map (yellow) from the P1247-P124 difference map. The fitting, which involved a global search over all orientations, is described in Materials and Methods. (B) In this setting, the C- and N-terminal helices of P7 both lie close to the 5-fold vertex of the procapsid. Both P7 termini were implicated in RNA binding and could thus interact with packaged RNA in this orientation. Additionally, the EM map rendered at a lower threshold shows an unassigned density near the vertex (arrow) that may correspond to a part of the C-terminal region of P7 that is missing from the crystal structure.
Fig 8
Fig 8
Interaction of P7 with P1 subunits in the procapsid. (A) Interior view of the 5-fold vertex (marked by a pentagon) in the procapsid. P7 (orange mesh) is attached to an interface that comprises two P1A subunits (green and blue). A hypothetical attachment of P7 (red mesh) at the corresponding site between P1B (yellow) and P1A (green) subunits produces a clash between P7 and the adjacent P1A subunit (blue). (B) Densities of bound P7 (orange) and two P1A (green and blue) subunits are shown at a high-density threshold. The binding interface is defined by the distance between centers of neighboring α-helices, which falls within 10 to 15 Å. The interface comprises a long α-helix (a) of the blue P1A subunit that points toward the 5-fold vertex. (C) The hypothetical binding interface between P7 (red) and the P1B (yellow) and P1A (green) subunits does not comprise the same α-helix (a) of P1A as that in panel B, but another α-helix (b) on the opposite site of P1A.
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