Transmission electron microscopy images of negative stained virus-like particles isolated from Finnish samples

A) Left, diamond-shaped virions found in a Recirculating Aquaculture System (RAS) tank sample and in experimental aquarium samples. Right, enlarged view showing the virion interior with at least three attachment points to the external capsid. B) Left, virions with loosely structured capsids found in a RAS tank sample and in experimental aquarium samples. Right, detailed view of one virion of this morphotype. C) Left, round morphotype found in an experimental aquarium sample; right, enlarged view of a spherical-shaped virion. D) Left, clusters of full and empty Jyvaskylavirus virions isolated from a composting soil sample. Center, an empty capsid showing the double-layered architecture of Jyvaskylavirus; C marks the capsid, and IM marks the putative internal membrane. Right, a fully packaged Jyvaskylavirus virion.

Jyvaskylavirus genomic data

A) Representative map of Jyvaskylavirus genome features. The G-C content, G-C skew, and ORFs distribution throughout the DNA sequence are coded by different ring colors as indicated in the color legend above. The outer blue ring represents the forward strand (positive sense) whereas the inner blue ring represents the reverse strand (negative sense). This illustrative genome map was constructed using CGview server (Grant et al 2008). B) Number of Jyvaskylavirus proteins according to the function predicted during genome annotation; n= 388. C) Maximum-likelihood phylogenetic tree based on DNA polymerase family B amino acid sequences from different nucleocytoviruses. The Jyvaskylavirus sequence is indicated by a blue circle. The alignment was performed with MUSCLE and the maximum-likelihood tree was reconstructed using IQtree software using ultrafast bootstrap (1000 replicates). The best-fit model selected using ModelFinder (implemented in IQtree) was VT+F+R5. Scale bar indicates the number of substitutions per site.

Helium ion microscopy images of Jyvaskylavirus attachment to A. castellanii cells

A) A. castellanii cells with spined structures (acanthopodia); elongated cell at the center. B) Details of a cell containing several viral particles on its surface (white arrowheads mark virions). C) Icosahedrally shaped virions near cell surface invaginations appearing as craters; inset, details of a virion likely starting the infection process through endocytosis. D) Centre, one cell containing viruses on its surface (white arrowhead) near a burst cell displayed with its ruptured content. Right inset, details of the burst cell content, showing several vesicles (yellow asterisks) and viruses (white arrowheads). Left inset, clusters of virions inside extracellular vesicles indicated by white arrows and individual virions by white arrowheads.

Transmission electron microscopy images of thin sections of A. castellanii cells infected by Jyvaskylavirus

A) Infected cell containing viruses spread over its cytoplasm marked by C (green) and with intracellular vesicles filled with viruses indicated by white arrows. The nucleus, whose boundary is highlighted in semi-transparent cyan, is indicated by the letter N (cyan). B) One infected cell with a large viral factory (VF, red) in its cytoplasm. C) View of an intracellular vesicle with icosahedral genome-filled virions as marked by a white arrowhead; membrane related structures nearby the vesicle interior are marked by dark-yellow triangles. NM (light cyan) and M (yellow) mark the nuclear membrane and the mitochondria, respectively. D) Enlarged view of the region marked by the black rectangle in C) showing possible membrane-related structure juxtaposed to or detached from the vesicle interior; black triangles possibly indicated a forming membrane vesicle. E) Details of virions in different stages of maturation inside the viral factory; DNA-full particles in white arrowheads, empty particles in blue arrowheads. F) Putative stages of virion assembly (indicated by the black arrows) as derived from the inspection of distinct particles in E and other cellular sections. The white elliptical line highlights a capsid aperture, while the red asterisk indicates an assembling capsid; in the remaining virion images, the capsid appears more assembled.

Jyvaskylavirus cryo-EM reconstruction.

A) Left, a slab (10 central sections) of the isosurface (low-pass filtered to 15 Å) showing the interior of the virion colour-coded by radius as from key. Right, isosurface of half the virion with black arrows indicating the triangulation indices h=7, k=13 (T = 309). B) Representation of the virion using trisymmetron geometry, with each trisymmetron color-coded differently, and a pentasymmetron marked by a pentagonal black line. C) A schematic of a virus facet with a trisymmetron marked with a white triangle with three icosahedral asymmetric units (IAU), one of which is marked by a thick black line. The pseudo-hexameric morphology displayed by a capsomer is represented by a hexagon coloured in light blue, while the true trimeric state of the MCP is depicted as a yellow triangle. To build the IAU (excluding the penton protein), 51 pseudo-hexameric capsomers and one-third of the capsomer located at the 3-fold symmetry axis are required, resulting in a total of 154 major capsid proteins (MCP) forming the IAU. The inset shows a cut-through of the density along the three-fold axis of a capsomer. D) Alphafold prediction of the MCP ORF184 shows, with very high confidence, that the fold adopted by the ORF is a vertical double jellyroll. E) Trimeric model of the capsomers rigid-body fitted into the original cryo-EM density and rendered in ChimeraX; left, viewed along the trimer fold axis and on the right, viewed orthogonally to it. The three copies of the MCP are represented in cartoon and coloured in green, light magenta, and light blue, while the corresponding density is shown in white transparent surface.

Jyvaskylavirus penton and capsomer-cap proteins

A) Cut-through view of the virion cryo-EM density (binned twice from original size) at the 5-fold axis rendered in white in ChimeraX, with regions corresponding to different proteins forming the capsid outlined in black. B) Top, a cartoon representation of the predicted model of the penton protein, with strands labeled BIDG/CHEF and color-coded by confidence level. Center and bottom, penton complex fitted into the density, shown from the top and as a cut-through, respectively. C) Top, atomic model of the trimeric cap in pink cartoon, composed of three copies of ORF121 with a β-barrel fold inserted on top of the pseudo-hexameric capsomer, shown in white cartoon. Bottom, cut-through view of the cap model fitted into the original density, Gaussian filtered and rendered as semi-transparent grey in ChimeraX.

Jyvaskylavirus pentasymmetron protein components and trisymmetron facet glueing protein

A) Top left, view of the cryo-EM density of Jyvaskylavirus (binned four times from the original size and Gaussian filtered) as seen from within the virion along the five-fold icosahedral symmetry axis. The white pentameric line marks the pentasymmetron region from below, while the white circle highlights the density corresponding to some identified proteins. Further densities corresponding to cementing, zippering and lattice scaffolding proteins are also visible, with one of them coloured in dodger-blue (see also panel B). The enlarged inset (top right and below) shows the spatial organization of the four identified ORFs and modelled using AlphaFold3, represented as cartoon tubes and coloured as per the legend. The penton proteins are coloured light brown, while peripentonal capsomers and capsomer-cap proteins are shown as space-filled atoms and coloured as slate-blue and pink, respectively. Left bottom, the different protein components fitted into the original 6.3 Å resolution Jyvaskylavirus cryo-EM density (white semi-transparent) binned to 2.68 Å/pix and rendered in ChimeraX; MCPs and capsomer-cap proteins have been omitted for clarity. B) Left, view of the cryo-EM density of Jyvaskylavirus, as shown in the top left panel of A), but along the three-fold icosahedral symmetry axis. A cementing protein, colored red, runs through the capsomers; however, the corresponding ORF has not been identified. Densities colored dodger-blue, which glue the capsomers across two trisymmetrons, correspond to ORF119, as shown in the large inset on the right. In this inset, dimers of ORF119 are depicted as dodger-blue cartoons fitted into the original cryo-EM density, Gaussian filtered and rendered as semi-transparent grey in ChimeraX. At the bottom right, a 90-degree view shows the spatial arrangement between two capsomers (navy blue), with the cap protein ORF121 (pink cartoon) positioned on top, and ORF119 fitted into the density at the base of the adjacent MCPs.