With DNA genomes reaching 2.5 Mb packed in particles of bacterium-like shape and dimension, the first two Acanthamoeba-infecting pandoraviruses remained up to now the most complex viruses since their discovery in 2013. Our isolation of three new strains from distant locations and environments is now used to perform the first comparative genomics analysis of the emerging worldwide-distributed Pandoraviridae family. Thorough annotation of the genomes combining transcriptomic, proteomic, and bioinformatic analyses reveals many non-coding transcripts and significantly reduces the former set of predicted protein-coding genes. Here we show that the pandoraviruses exhibit an open pan-genome, the enormous size of which is not adequately explained by gene duplications or horizontal transfers. As most of the strain-specific genes have no extant homolog and exhibit statistical features comparable to intergenic regions, we suggest that de novo gene creation could contribute to the evolution of the giant pandoravirus genomes.

Acanthamoeba are infected by a remarkable diversity of large dsDNA viruses, the infectious cycles of which have been characterized using genomics, transcriptomics and electron microscopy. Given their gene content and the persistence of the host nucleus throughout their infectious cycle, the Marseilleviridae were initially assumed to fully replicate in the cytoplasm. Unexpectedly, we find that their virions do not incorporate the virus-encoded transcription machinery, making their replication nucleus-dependent. However, instead of delivering their DNA to the nucleus, the Marseilleviridae initiate their replication by transiently recruiting the nuclear transcription machinery to their cytoplasmic viral factory. The nucleus recovers its integrity after becoming leaky at an early stage. This work highlights the importance of virion proteomic analyses to complement genome sequencing in the elucidation of the replication scheme and evolution of large dsDNA viruses.

With DNA genomes up to 2.5 Mb packed in particles of bacterium-like shape and dimension, the first two Acanthamoeba-infecting Pandoraviruses remained the most spectacular viruses since their description in 2013. Our isolation of three new strains from distant locations and environments allowed us to perform the first comparative genomics analysis of the emerging worldwide-distributed Pandoraviridae family. Thorough annotation of the genomes combining transcriptomic, proteomic, and bioinformatic analyses, led to the discovery of many non-coding transcripts while significantly reducing the former set of predicted protein-coding genes. We found that the Pandoraviridae exhibit an open pan genome, the enormous size of which is not adequately explained by gene duplications or horizontal transfers. As most of the strain specific genes have no extant homolog and exhibit statistical features comparable to intergenic regions, we suggests that de novo gene creation is a strong component in the evolution of the giant Pandoravirus genomes.