Evolution and diversity of the Microviridae viral family through a collection of 81 new complete genomes assembled from virome reads

Abstract

Recent studies suggest that members of the Microviridae (a family of ssDNA bacteriophages) might play an important role in a broad spectrum of environments, as they were found in great number among the viral fraction from seawater and human gut samples. 24 completely sequenced Microviridae have been described so far, divided into three distinct groups named Microvirus, Gokushovirinae and Alpavirinae, this last group being only composed of prophages. In this study, we present the analysis of 81 new complete Microviridae genomes, assembled from viral metagenomes originating from various ecosystems. The phylogenetic analysis of the core genes highlights the existence of four groups, confirming the three sub-families described so far and exhibiting a new group, named Pichovirinae. The genomic organizations of these viruses are strikingly coherent with their phylogeny, the Pichovirinae being the only group of this family with a different organization of the three core genes. Analysis of the structure of the major capsid protein reveals the presence of mushroom-like insertions conserved within all the groups except for the microviruses. In addition, a peptidase gene was found in 10 Microviridae and its analysis indicates a horizontal gene transfer that occurred several times between these viruses and their bacterial hosts. This is the first report of such gene transfer in Microviridae. Finally, searches against viral metagenomes revealed the presence of highly similar sequences in a variety of biomes indicating that Microviridae probably have both an important role in these ecosystems and an ancient origin.

Figure 1. Phylogenetic tree drawn from the major capsid protein multiple alignment.

Linearized genomes are represented for each virus. The open reading frames in each genome are color-coded following the nomenclature used for Chlamydia phage genomes (i.e.VP1 : major capsid protein, VP2 : DNA pilot protein, VP3 : internal scaffolding protein, VP4 : genome replication initiation protein, and VP5 : DNA binding protein). Striped-colored genes encode proteins possessing features characteristic of VP2 proteins, but displaying no significant sequence similarity, as assessed by BLAST. The four Microviridae subgroups are highlighted on the tree. Bootstrap scores greater than 80 are marked with gray dots.

Figure 2. Major capsid protein (MCP) variation within the Microviridae.

(A) Three-dimensional model of the SpV4 virion (PDB ID:1KVP). Three capsomers donating long insertion loops to form the ‘mushroom-like’ protrusions at the three-fold axes of symmetry of the icosahedral capsid are highlighted in blue, green, and red. (B) A boxplot illustrating the variation of MCP sizes between the four subgroups of the Microviridae. (C) Three-dimensional models of the MCPs from viruses representing the four subgroups of the Microviridae: Microvirus (ΦX174 protein F; PDB ID:1CD3), Gokushovirinae (SpV4 VP1, PDB ID:1KVP), Alpavirinae (Prevotella bucalis prophage BMV5 protein VP1; GI:282877220), Pichovirinae (Pavin_279 protein VP1). The insertions within the VP1 proteins of gokusho-, alpa- and pichoviruses relative to the F protein of ΦX174 are highlighted in green.

Figure 3. Genomic context and phylogenetic analysis of the Peptidase M15 Microviridae sequences.

(A) Organization of the Peptidase M15_3 region in the 11 newly assembled Microviridae genomes. The regions encompassing homologous peptidase genes in three bacterial and two phages (noted with a black circle) genomes are also shown. P2 GpR stands for the P2 phage tail completion protein. (B) Maximum-likelihood tree computed from the multiple alignment of peptidase M15 sequences of the Microvidae and their closest homologues in viral and bacterial genomes. Bootstrap support values are indicated on each node. A fully expanded view of this tree is available as Fig. S9.

Figure 4. Abundance and distribution of VP1-like sequences in the environment.

The number of viromes and the origin of the samples used for virome preparation are indicated. VP1 sequences were affiliated by best BLAST hit against a database including VP1 sequences from both the previously published Microviridae genomes and the complete genomes assembled in this study.