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. 2022 Dec 3;13(1):7478.
doi: 10.1038/s41467-022-35123-6.

Cryo-EM structure of ssDNA bacteriophage ΦCjT23 provides insight into early virus evolution

Nejc Kejzar #  1   2 , Elina Laanto #  3   4 , Ilona Rissanen #  1 , Vahid Abrishami  1 , Muniyandi Selvaraj  1   5 , Sylvain Moineau  6 , Janne Ravantti  3 , Lotta-Riina Sundberg  7 , Juha T Huiskonen  8
Affiliations

Cryo-EM structure of ssDNA bacteriophage ΦCjT23 provides insight into early virus evolution

Nejc Kejzar et al. Nat Commun. .

Abstract

The origin of viruses remains an open question. While lack of detectable sequence similarity hampers the analysis of distantly related viruses, structural biology investigations of conserved capsid protein structures facilitate the study of distant evolutionary relationships. Here we characterize the lipid-containing ssDNA temperate bacteriophage ΦCjT23, which infects Flavobacterium sp. (Bacteroidetes). We report ΦCjT23-like sequences in the genome of strains belonging to several Flavobacterium species. The virion structure determined by cryogenic electron microscopy reveals similarities to members of the viral kingdom Bamfordvirae that currently consists solely of dsDNA viruses with a major capsid protein composed of two upright β-sandwiches. The minimalistic structure of ΦCjT23 suggests that this phage serves as a model for the last common ancestor between ssDNA and dsDNA viruses in the Bamfordvirae. Both ΦCjT23 and the related phage FLiP infect Flavobacterium species found in several environments, suggesting that these types of viruses have a global distribution and a shared evolutionary origin. Detailed comparisons to related, more complex viruses not only expand our knowledge about this group of viruses but also provide a rare glimpse into early virus evolution.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Genome of a temperate phage phiCjT23 and related prophages found in Flavobacterium genomes.
a ΦCjT23 genome is a 7642-nt long circular single-stranded DNA molecule with 15 predicted open-reading frames (ORFs). The annotated replication initiation protein is indicated in light blue and the two identified structural proteins, major capsid protein (MCP, gp5) and spike protein (gp13), are in pink. Hypothetical proteins are indicated in dark blue. b Examples of putative prophages found in Flavobacterium genomes. The lengths of the sequences range between ~6000 and 8000 bp. Shared genome synteny between the identified prophages included replication initiation protein near the start of the sequence, followed by a gene encoding a major capsid protein MCP (sequence similarity shared between the prophages). c Phages and bacterial dif-motifs were identified from the non-coding regions surrounding putative prophages implying XerC/XerD dependent integration. The 28-nt long dif locus (including six extra surrounding nucleotides) with XerC and XerD-binding sites, as well as the central hexanucleotide, are shown.
Fig. 2
Fig. 2. Structure of the ΦCjT23 bacteriophage capsid and major capsid protein trimer.
a Surface rendering of the ΦCjT23 virion cryo-EM map at 4.1 Å resolution. The four different types of major capsid protein trimers are colored yellow (1), green (2), red (3) and blue (4). The penton base (P) is colored cyan with the remaining structural components colored gray. On top, a gray transparent surface, calculated from the cryo-EM map filtered to 12 Å resolution, is shown. This lower-resolution surface reveals disordered density attributed to flexible spikes which originate from the penton bases. A part of the density is removed, to reveal the lipid bilayer (M), DNA, and putative minor protein components (circles) bridging MCPs to the lipid bilayer. b A close-up of the high-resolution surface from the area indicated in a and rotated to face the viewer. The icosahedral axes of symmetry are marked with a pentagon (five-fold axis), triangle (three-fold axis), and diamond (two-fold axis). Type 1 trimers are peripentonal, circling the five-folds. Type 2 trimers are adjacent to the two folds. Type 3 trimers locate on the three-folds. Type 4 trimers are farthest from any symmetry axes. c Model for one trimer (type 3) is shown as a ribbon. The three different subunits are shown in different shades. d The view in c is shown after rotating 90° around the horizontal axis as indicated. Only the frontmost monomer is shown. The two β-sandwich domains (V1 and V2) are labeled, in addition to the protein termini. e Closeup of the area indicated in c show how the two upright loops (FG1 and FG2) lock the monomer with the V2 domains (V2’ and V2”) of its neighbors (shown as surfaces).
Fig. 3
Fig. 3. Structural phylogeny of major capsid proteins with double jelly-roll fold.
The two different parts of the phylogeny, corresponding to viruses harboring either an ssDNA (blue) or dsDNA (light blue) genome, are shown in different colors. A generalized common core of the fold determined by the Homologous Structure Finder software and used in the comparisons is shown as a Cα-trace in the inset. The T-number for the corresponding icosahedral capsid, when applicable, is given.
Fig. 4
Fig. 4. ΦCjT23 vertex.
a A composite cryo-EM map of ΦCjT23 bacteriophage capsid combining localized reconstructions of the four MCP trimers (yellow, green, red, and blue), penton base (cyan), and spike (gray). b A close-up of one vertex seen from the top of the area indicated in a. c The same view as in b, rotated by 90° around the axis indicated. The dimensions of the spike, corresponding to the external domains of spike protein P13, are indicated. Note the discontinuation in the density (arrowhead). d A close-up of the area indicated in c. The model of the penton base part of the spike is shown in ribbon (cyan). The ordered part of the map ends around the area occupied by G92. Putative linker regions spanning the 1-nm gap between the penton base and the external part of the spike (gray) are illustrated with dashed lines. The sequence of the putative linker is given. e A model of the pentamer formed by the penton base domain seen along a 5-fold axis of symmetry. Different monomers are colored with different shades of cyan. The positions of the N-terminus, C-terminal G92, αhelix at the monomer–monomer interface (α) and loop (L) closing the oculus in the middle of the pentamer are labeled. f The monomer is colored from blue (N-terminus) to red (C-terminal G92) and marked with an asterisk in e is shown rotated by 90° around the axis indicated. The β-strands are numbered 1–6. Strands 2-1-4 make a β-sheet. The α-helix, residing in the region connecting strands 5 and 6, is labeled.
Fig. 5
Fig. 5. Mapping of ΦCjT23-like prophage sequence variation.
a Sequence conservation (score –2.12–2.26) determined by multiple sequence alignment of ΦCjT23-like prophage sequences (N = 21) and ΦCjT23 major capsid protein (MCP) mapped per residue on the ΦCjT23 MCP structure. b The same view as in a with residues surrounding insertions highlighted in pink. c Sequence conservation (score –1.84–2.36) determined by multiple sequence alignment of ΦCjT23-like prophage sequences (N = 8) and ΦCjT23 spike protein N-terminal domain. d The same view as in c with residues surrounding insertions highlighted.
Fig. 6
Fig. 6. Icosahedral virus capsid architectures.
A schematic diagram is shown for different types of icosahedrally symmetric capsids built of 12 pentagons (gray) and N = 60×(T–1)/6 hexagons (white). The triangulation (T) number of each capsid is defined by two lattice indexes h and k as T = h2 + h × k + k2. In the members of the PRD1–adenovirus lineage (Bamfordvirae), the allowed hexagon positions are occupied by pseudohexameric trimers, hence the T-number is indicated being pseudo (p). Different capsid types are classified in classes 1, 2, and 3 (ref. 32). Only the first members of the series (…) are shown in each class. The lattice axes are illustrated for one capsid in each class with arrows. Hexagons that are located on the icosahedral two-fold axes of symmetry, and that thus cannot be occupied by a pseudohexameric, trimeric major capsid protein (MCP), are colored in red (corresponding capsids are labeled as “not allowed”). For each capsid, the value of hexamer complexity (Ch) is given. Some names of the characterized viruses with a certain capsid type (pT = 21, 25, 27) are given. Capsid geometries with no characterized representatives are labeled as unobserved. Note that the lattice in class 2 capsids is handed and only the right-handed (dextro) organization, which is observed in PM2, FLiP, and ΦCjT23, is shown. Allowed capsid geometries (pT = 3, pT = 9, pT = 21 and pT = 27) where a hexagon occupies the three-fold axes of symmetry are indicated by coloring one such hexagon blue. D and V denote the relative diameter and volume, respectively, relative to the pT = 21 shell.
Fig. 7
Fig. 7. Models for capsid assembly and spike swapping.
a A hypothetical model for the assembly of ΦCjT23 and PM2-like viruses with T = 21 capsids. First, a membrane vesicle wrapping around the genome assembles to create a lipid core (1). Major capsid protein (MCP) trimers attach to the lipid core via minor membrane proteins (2). More MCPs can be incorporated in a symmetrical fashion via MCP–MCP interactions (3,4) until a nearly closed shell is formed (5). Finally, spikes are incorporated into the capsid (6). b The genome region encoding for the spikes is depicted in gray. Genetic changes (insertions and deletions) resulting in swapping first the complete spike (such as those observed in ΦCjT23 and PM2) with a penton base and then additional spike proteins being incorporated (such as those observed in FLiP and adenovirus) are depicted.
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