Recent discoveries of new large DNA viruses reveal high diversity in their morphologies, genetic repertoires, and replication strategies. Here, we report the novel features of medusavirus, a large DNA virus newly isolated from hot spring water in Japan. Medusavirus, with a diameter of 260 nm, shows a T=277 icosahedral capsid with unique spherical-headed spikes on its surface. It has a 381-kb genome encoding 461 putative proteins, 86 of which have their closest homologs in Acanthamoeba, whereas 279 (61%) are orphan genes. The virus lacks the genes encoding DNA topoisomerase II and RNA polymerase, showing that DNA replication takes place in the host nucleus, whereas the progeny virions are assembled in the cytoplasm. Furthermore, the medusavirus genome harbored genes for all five types of histones (H1, H2A, H2B, H3, and H4) and one DNA polymerase, which are phylogenetically placed at the root of the eukaryotic clades. In contrast, the host amoeba encoded many medusavirus homologs, including the major capsid protein. These facts strongly suggested that amoebae are indeed the most promising natural hosts of medusavirus, and that lateral gene transfers have taken place repeatedly and bidirectionally between the virus and its host since the early stage of their coevolution. Medusavirus reflects the traces of direct evolutionary interactions between the virus and eukaryotic hosts, which may be caused by sharing the DNA replication compartment and by evolutionarily long lasting virus-host relationships. Based on its unique morphological characteristics and phylogenomic relationships with other known large DNA viruses, we propose that medusavirus represents a new family, MedusaviridaeIMPORTANCE We have isolated a new nucleocytoplasmic large DNA virus (NCLDV) from hot spring water in Japan, named medusavirus. This new NCLDV is phylogenetically placed at the root of the eukaryotic clades based on the phylogenies of several key genes, including that encoding DNA polymerase, and its genome surprisingly encodes the full set of histone homologs. Furthermore, its laboratory host, Acanthamoeba castellanii, encodes many medusavirus homologs in its genome, including the major capsid protein, suggesting that the amoeba is the genuine natural host from ancient times of this newly described virus and that lateral gene transfers have repeatedly occurred between the virus and amoeba. These results suggest that medusavirus is a unique NCLDV preserving ancient footprints of evolutionary interactions with its hosts, thus providing clues to elucidate the evolution of NCLDVs, eukaryotes, and virus-host interaction. Based on the dissimilarities with other known NCLDVs, we propose that medusavirus represents a new viral family, Medusaviridae.

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