Structural changes in a marine podovirus associated with release of its genome into Prochlorococcus

Abstract

Podovirus P-SSP7 infects Prochlorococcus marinus, the most abundant oceanic photosynthetic microorganism. Single-particle cryo-electron microscopy yields icosahedral and asymmetrical structures of infectious P-SSP7 with 4.6-A and 9-A resolution, respectively. The asymmetric reconstruction reveals how symmetry mismatches are accommodated among five of the gene products at the portal vertex. Reconstructions of infectious and empty particles show a conformational change of the 'valve' density in the nozzle, an orientation difference in the tail fibers, a disordering of the C terminus of the portal protein and the disappearance of the core proteins. In addition, cryo-electron tomography of P-SSP7 infecting Prochlorococcus showed the same tail-fiber conformation as that in empty particles. Our observations suggest a mechanism whereby, upon binding to the host cell, the tail fibers induce a cascade of structural alterations of the portal vertex complex that triggers DNA release.

Figure 1. Image and reconstruction of P-SSP7 at 4.6 Å resolution

(a) 300kV image of P-SSP7 embedded in vitreous ice. (b) Segmented density of the shell protein subunit (gp10) and its Cα model from the 4.6 Å icosahedral reconstruction. (c) Subunit interactions within and across capsomeres. Four subunits are shown; the upper two belong to one pseudo-6 fold capsomere, the bottom two belong to another capsomere. The view is from the outside of the capsid looking almost down the 2-fold symmetry axis. (d) A zoomed-in view, but viewed from inside the capsid, with the corresponding density around the F loop and domain P. The densities (annotated in yellow) near the 2-fold axis indicate interactions between Lys61 (in blue) and Glu120 (in red) from two capsomeres.

Figure 2. Asymmetric reconstructions of P-SSP7

(a) 9.2 Å resolution map of the full phage. (b) Central slice of the full phage. (c) Fiber trimer viewed from its proximal end. (d) A cutaway zoomed-in view of the adaptor, nozzle and tail fibers. In (c) and (d), the full phage map was low-pass filtered to the same resolution as the empty phage map to facilitate comparison between the structures. (e) 24 Å resolution asymmetric reconstruction of the empty phages. (f) Central slice of the empty phage. (g) Cutaway view for the empty phage equivalent to the view in (d). All phage protein components are annotated and colored differently. Two special features (valve density and DNA strand separation) are observed in the slice from the full phage (b). There are substantial conformational changes in the nozzle, tail fibers, portal and core proteins between the full and empty particles. The yellow dashed box in (d) shows that a fiber interacts with both nozzle and adaptor in full phage, but the links between fiber and adaptor are broken in empty phage (g). The full phage has strong density at and around the valve (colored in orange and red), but the density of empty phage in the corresponding location is markedly reduced and there is no density at the valve.

Figure 3. Symmetry mismatch at the portal vertex

(a) Interface between 12 copies of portal (greenish) and 10 copies of gp10 models (purple and cyan). The purple and cyan molecules represent two subunits from each of the 5 surrounding hexons. The gray density segmented from the asymmetric reconstruction is the interfacial density between capsid and portal. (b) Schematic diagram to illustrate the 4 apparent types of interactions in our density map: F loop interaction (2, 4, 7, 9, 12), hook of domain P interaction (1, 3, 6, 8), N terminus of long helix interaction (10) and no interaction (5, 11). (c) 12 copies of adaptor proteins in alternating conformations. (d) Interface between the portal (red and black footprints – black indicates one subunit) and the adaptor. One subunit of the portal interacts with two neighboring adaptor subunits. (e) Interface between the nozzle (red and black footprints, black for 1 subunit) and the adaptor. One nozzle subunit interacts with 4 contiguous adaptor subunits and also interacts with a tail fiber. The nozzle footprint outlines coverage around the center of the nozzle and indicates the presence of the “valve”. Figure 3a, 3b and 3e are viewed from the outside along the portal vertex complex axis, while Figure 3c and 3d are viewed from the inside of the capsid.

Figure 4. Cryo-ET of P-SSP7 infecting MED-4 cell

(a) Slice of a tomogram showing three states of P-SSP7 in its native environment: infecting phages (all dark particles with one annotated by a yellow arrow); post-infection (empty) phages still attached to the cell (white arrow); and unattached phages. The attachment of the phages to the host cell can only be ascertained from the visualization of the 3-D tomogram, but not in a 2-D slice as exemplified here. (b) An average of 26 infecting phage subtomograms with the portal vertex oriented normal to the cell surface. The phage tail fibers are extended horizontally. (c) Central slice of the averaged subtomograms of infecting phages. (d) Schematic cartoon of P-SSP7 phage infecting the host cell (the cell membranes are represented by the double layer at the bottom).