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 viruspathogens interactions during coinfections.

Giant viruses are abundant in aquatic environments and ecologically important through the metabolic reprogramming of their hosts. Less is known about giant viruses from soil even though two of them, belonging to two different viral families, were reactivated from 30,000-y-old permafrost samples. This suggests an untapped diversity of Nucleocytoviricota in this environment. Through permafrost metagenomics we reveal a unique diversity pattern and a high heterogeneity in the abundance of giant viruses, representing up to 12% of the sum of sequence coverage in one sample. Pithoviridae and Orpheoviridae-like viruses were the most important contributors. A complete 1.6 Mb Pithoviridae-like circular genome was also assembled from a 42,000-y-old sample. The annotation of the permafrost viral sequences revealed a patchwork of predicted functions amidst a larger reservoir of genes of unknown functions. Finally, the phylogenetic reconstructions not only revealed gene transfers between cells and viruses, but also between viruses from different families.

Marseilleviridae members are large dsDNA viruses with icosahedral particles 250 nm in diameter infecting Acanthamoeba. Their 340 to 390 kb genomes encode 450 to 550 protein-coding genes. Since the discovery of marseillevirus (the prototype of the family) in 2009, several strains were isolated from various locations, among which 13 are now fully sequenced. This allows the organization of their genomes to be deciphered through comparative genomics. Here, we first experimentally demonstrate that the Marseilleviridae genomes are circular. We then acknowledge a strong bias in sequence conservation, revealing two distinct genomic regions. One gathers most Marseilleviridae paralogs and has undergone genomic rearrangements, while the other, enriched in core genes, exhibits the opposite pattern. Most of the genes whose protein products compose the viral particles are located in the conserved region. They are also strongly biased toward a late gene expression pattern. We finally discuss the potential advantages of Marseilleviridae having a circular genome, and the possible link between the biased distribution of their genes and the transcription as well as DNA replication mechanisms that remain to be characterized.