A stargate mechanism of Microviridae genome delivery unveiled by cryogenic electron tomography

Single-stranded DNA bacteriophages of the Microviridae family are major components of the global virosphere. Microviruses are highly abundant in aquatic ecosystems and are prominent members of the mammalian gut microbiome, where their diversity has been linked to various chronic health disorders. Despite the clear importance of microviruses, little is known about the molecular mechanism of host infection. Here, we have characterized an exceptionally large microvirus, Ebor, and provide crucial insights into long-standing mechanistic questions. Cryogenic electron microscopy of Ebor revealed a capsid with trimeric protrusions that recognise lipopolysaccharides on the host surface. Cryogenic electron tomography of the host cell colonized with virus particles demonstrated that the virus initially attaches to the cell via five such protrusions, located at the corners of a single pentamer. This interaction triggers a stargate mechanism of capsid opening along the 5-fold symmetry axis, enabling delivery of the virus genome. Despite variations in specific virus-host interactions among different Microviridae family viruses, structural data indicate that the stargate mechanism of infection is universally employed by all members of the family. Startlingly, our data reveal a mechanistic link for the opening of relatively small capsids made out of a single jelly-roll fold with the structurally unrelated giant viruses.

Ebor variant R120 purified using ion exchange chromatography (b).The particles formed aggregates (green arrows) and the ratio of empty and native particles was close to 1:1.Ebor variant R120 purified using CsCl ultracentrifugation (c), all observed particles were native.Ebor variant S120 purified using sucrose ultracentrifugation (d), all observed particles were empty, the contaminants are likely ejected genome molecules.Ebor variant S120 purified using CsCl ultracentrifugation (e), the particles appear to be releasing their genome.Images were collected at 200 kV using a Glacios TEM equipped with Falcon 4 camera, with the total exposure dose of ~3 e -/Å 2 in case of cells and 50 e -/Å 2 in case of purified viruses.

Supp Figure S3 :
Additional structural analysis of the native virion of Ebor.a) Diameters of individual Microviridae phages.Central Z slices of the capsids are shown, the densities of phiX174 and SpV4 were generated by ChimeraX molmap command (2) of PDB bioassemblies 2BPA_A and 1KVP_A respectively.For EC6098, a segmentation of EMD-27397 was used, masking out the central virion density for clarity.b) Ribbon diagrams of the major capsid protein of phage EC6098, PDB code 8DES_A.Regions of interest are highlighted in colour and delimiting residues numbers are shown.c) Map of Ebor virion releasing the genome in vitro reconstructed by single particle analysis of variant S120 virions purified using CsCl gradient.d-e) electrostatic potential of Ebor protrusion (d) and penton (e) estimated according to Adaptive Poisson-Boltzmann Solver (3).The AlphaFold2-predicted (4) protrusion loops were connected into a single model with respective subunits of major capsid protein in Coot (5) with the axis of symmetry highlighted (magenta triangle).The colour coding corresponds to the electrostatic potential values of the protein surface calculated at T=298.15K and pH=7.0.Supp Figure S4: Additional cryo-EM images of Ebor.a) The same virus stock as used in the experiments shown in the Figure 3d-f) was imaged after 5 min of incubation with the host strain SB1003 to which 5 mM CaCl2 was added.The effect of the calcium addition on the plaque size is shown in left.b-e) Images of Ebor particles purified using different methods.
Figure S5: EMAN2 pipeline (6) applied for the subtomogram reconstruction of the dataset of phage Ebor attached to cells.The colour bar indicates the distance from the centre of the capsid.Different iterations of the refinement algorithm are labelled according EMAN2 conventions: p, 3D particle orientation; t, 2D subtilt translation; r, subtilt translation and rotation; d, subtilt defocus refinement.Supp Figure S6: EMAN2 pipeline (6) applied for the subtomogram reconstruction of the dataset of phage Ebor attached to OMVs.The colour bar indicates the distance from the centre of the capsid.Different iterations of the refinement algorithm are labelled according EMAN2 conventions: p, 3D particle orientation; t, 2D subtilt translation; r, subtilt translation and rotation; d, subtilt defocus refinement.Supp Figure S7: RELION pipeline (7-9) applied for the single particle reconstruction of the dataset of phage Ebor attached to cells.The colour bar indicates the distance from the centre of the capsid.Supp Figure S8: Single particle analysis of Ebor attached to cells.a-c) C5 symmetry maps of the attached particles classified into class 1 (a), 2 (b) and 3 (c) reconstructed by single particle analysis (SPA).Side view, top view and central slice through y axis are shown from top to bottom, with a central slice through y axis of unfiltered maps reconstructed by subtomogram averaging (STA) shown on the very bottom for comparison.The colour bar indicates the distance from the centre of the capsid.The arrows point at the density of an internal cavity formed above the interacting penton.d) Fitting of the major capsid protein model shown as ribbon diagrams into the interacting penton of classes 1 and 2 reconstructed by SPA s and class 2 reconstructed by STA.A similar pore was identified in class 2 reconstructed by both methods, with detached density present right on the 5-fold axis.e) Fitting of the major capsid protein in class 3 map reconstrcuted from SPA shows the density connecting the capsid with the membrane corresponds to the position of EF bulge.Close up of the region is shown in the bottom rectangle.Individual residues are shown as sticks with R120 and L124 highlighted.

Table S2 :
Raw data related to the Ebor inhibition assay.
*at the recommended contour level = 0.1