The DNA methylation landscape of giant viruses

Abstract

DNA methylation is an important epigenetic mark that contributes to various regulations in all domains of life. Giant viruses are widespread dsDNA viruses with gene contents overlapping the cellular world that also encode DNA methyltransferases. Yet, virtually nothing is known about the methylation of their DNA. Here, we use single-molecule real-time sequencing to study the complete methylome of a large spectrum of giant viruses. We show that DNA methylation is widespread, affecting 2/3 of the tested families, although unevenly distributed. We also identify the corresponding viral methyltransferases and show that they are subject to intricate gene transfers between bacteria, viruses and their eukaryotic host. Most methyltransferases are conserved, functional and under purifying selection, suggesting that they increase the viruses' fitness. Some virally encoded methyltransferases are also paired with restriction endonucleases forming Restriction-Modification systems. Our data suggest that giant viruses' methyltransferases are involved in diverse forms of virus-pathogens interactions during coinfections.

Fig. 1. Encoded MTases and targeted methylated motifs in the giant viruses’ genomes.

The encoded DNA MTases of each virus are shown along with the number of occurrences of the predicted targets (if any) on both strands of the cognate genomic sequence. Modified nucleotides within the motifs are underlined. Red circles indicate methylated motifs experimentally verified from SMRT data (filled circles) or with unidentified predicted methylation (empty circles). Likewise, predicted (filled circle) and not predicted (empty circle) targets based on sequence homology of the encoded MTase are shown using blue circles. Bar graphs correspond to the median IPDr profiles of the motifs (gray region) and the surrounding 20 nucleotides on each side. Each bar displays the median IPDr value and a 95% confidence interval (error bars) based on 1000 bootstraps. These statistics are derived from the number of occurrences (n) of the motifs in each genome. Red bars correspond to positions with significantly high IPDr values. Individual data points are displayed for viruses with n ≤ 10.

Fig. 2. Presence/absence of R-M systems in the Marseilleviridae family.

Phylogeny of the Marseilleviridae completely sequenced viruses with the following GenBank accessions: melbournevirus (KM275475.1), cannes 8 virus (KF261120.1), marseillevirus (GU071086.1), marseillevirus shanghai 1 (MG827395.1), tokyovirus A1 (AP017398.1), kurlavirus (KY073338.1), noumeavirus (KX066233.1), port-Miou virus (KT428292.1), lausannevirus (HQ113105.1), brazilian marseillevirus (KT752522.1), insectomimevirus (KF527888.1), tunisvirus (KF483846.1) and golden marseillevirus (KT835053.1). The phylogeny was based on protein sequence alignments of the 115 strictly conserved single copy orthologues (see Supplementary Data 1). The tree was calculated using the best model of each partitioned alignment as determined by IQtree. Bootstrap values were computed using the UFBoot method. The red and blue filled circles highlight the presence of encoded marseilleviruses R-M systems MTases and REases respectively. The empty circles highlight the absence of the MTase (in red) and the REase (in blue). The arrow points to the most parsimonious acquisition of a R-M system in the Marseilleviridae family.

Fig. 3. Host and marseilleviruses DNA protection against GATC-targeting REases.

Agarose gel electrophoresis analysis of A. castellanii, melbournevirus and noumeavirus DNA digested with GATC-targeting restriction enzymes. Restriction patterns using DpnI and DpnII enzymes are presented with control DNA. DpnI cleaves DNA at GATC sites containing N⁶-methyl-adenines and DpnII at GATC sites containing unmethylated adenines. This experiment was repeated twice with similar results.

Fig. 4. Expression timing of the melbournevirus R-M system MTase and REase.

Shown are the RT-PCRs of the transcripts corresponding to the mel_015 and mel_016 genes during a melbournevirus infection. Times (post infection) are listed on the top of the figure. NI corresponds to non-infected. This experiment was repeated twice with similar results.

Fig. 5. Phylogenetic tree of the giant viruses MTases.

Phylogenetic tree of the giant viruses’ MTases along with prokaryotic and eukaryotic homologs. The blue triangles mark viral genes, the red ones eukaryotic genes and the unmarked genes are prokaryotic. The tree was computed using the LG + R6 model from a multiple alignment of 678 informative sites. Bootstrap values were computed using the UFBoot method from IQtree. All branches with support value > 80 are highlighted using purple circles. The GenBank accessions and taxonomic assignations extracted from GenBank entries are shown. The tree was rooted using the midpoint rooting method. The tree was split into five subgroups highlighted using different colors (blue, orange, red, purple, and green).