Pandoravirus Celtis Illustrates the Microevolution Processes at Work in the Giant Pandoraviridae Genomes Matthieu Legendre, Jean-Marie Alempic, Nadège Philippe, Audrey Lartigue, Sandra Jeudy, Olivier Poirot, Ngan Thi Ta, Sébastien Nin, Yohann Couté, Chantal Abergel, et al. To cite this version: Matthieu Legendre, Jean-Marie Alempic, Nadège Philippe, Audrey Lartigue, Sandra Jeudy, et al.. Pandoravirus Celtis Illustrates the Microevolution Processes at Work in the Giant Pandoraviridae Genomes. Frontiers in Microbiology, Frontiers Media, 2019, 10, pp.430. 10.3389/fmicb.2019.00430. hal-02135607 HAL Id: hal-02135607 https://hal.archives-ouvertes.fr/hal-02135607 Submitted on 5 Nov 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Distributed under a Creative Commons Attribution| 4.0 International License fmicb-10-00430 March 6, 2019 Time: 17:27 # 1 ORIGINAL RESEARCH published: 08 March 2019 doi: 10.3389/fmicb.2019.00430 Pandoravirus Celtis Illustrates the Microevolution Processes at Work in the Giant Pandoraviridae Genomes Matthieu Legendre1, Jean-Marie Alempic1, Nadège Philippe1, Audrey Lartigue1, Sandra Jeudy1, Olivier Poirot1, Ngan Thi Ta1, Sébastien Nin1, Yohann Couté2, Chantal Abergel1* and Jean-Michel Claverie1* 1 Aix Marseille Univ, CNRS, IGS, Structural and Genomic Information Laboratory (UMR7256), Mediterranean Institute of Microbiology (FR3479), Marseille, France, 2 Inserm, BIG-BGE, CEA, Université Grenoble Alpes, Grenoble, France With genomes of up to 2.7 Mb propagated in mm-long oblong particles and initially predicted to encode more than 2000 proteins, members of the Pandoraviridae family display the most extreme features of the known viral world. The mere existence of such giant viruses raises fundamental questions about their origin and the processes governing their evolution. A previous analysis of six newly available isolates, independently confirmed by a study including three others, established that the Pandoraviridae pan-genome is open, meaning that each new strain exhibits protein- Edited by: Ricardo Flores, coding genes not previously identified in other family members. With an average Instituto de Biología Molecular y increment of about 60 proteins, the gene repertoire shows no sign of reaching a Celular de Plantas (IBMCP), Spain limit and remains largely coding for proteins without recognizable homologs in other Reviewed by: viruses or cells (ORFans). To explain these results, we proposed that most new protein- Amparo Latorre, University of Valencia, Spain coding genes were created de novo, from pre-existing non-coding regions of the GCC Victor Krylov, rich pandoravirus genomes. The comparison of the gene content of a new isolate, I. I. Mechnikov Research Institute for Vaccines and Sera (RAS), Russia pandoravirus celtis, closely related (96% identical genome) to the previously described *Correspondence: p. quercus is now used to test this hypothesis by studying genomic changes in a Chantal Abergel microevolution range. Our results confirm that the differences between these two similar [email protected] gene contents mostly consist of protein-coding genes without known homologs, with Jean-Michel Claverie [email protected] statistical signatures close to that of intergenic regions. These newborn proteins are under slight negative selection, perhaps to maintain stable folds and prevent protein Specialty section: This article was submitted to aggregation pending the eventual emergence of fitness-increasing functions. Our study Virology, also unraveled several insertion events mediated by a transposase of the hAT family, a section of the journal 3 copies of which are found in p. celtis and are presumably active. Members of the Frontiers in Microbiology Pandoraviridae are presently the first viruses known to encode this type of transposase. Received: 18 December 2018 Accepted: 19 February 2019 Keywords: de novo gene creation, comparative genomics, Acanthamoeba, giant viruses, soil viruses, hAT Published: 08 March 2019 transposase Citation: Legendre M, Alempic J-M, Philippe N, Lartigue A, Jeudy S, INTRODUCTION Poirot O, Ta NT, Nin S, Couté Y, Abergel C and Claverie J-M (2019) The Pandoraviridae is a proposed family of giant dsDNA viruses - not yet registered by the Pandoravirus Celtis Illustrates International Committee on Taxonomy of Viruses (ICTV) - multiplying in various species of the Microevolution Processes at Work Acanthamoeba through a lytic infectious cycle. Their linear genomes, flanked by large terminal in the Giant Pandoraviridae Genomes. Front. Microbiol. 10:430. repeats, range from 1.9 to 2.7 Mb in size, and are propagated in elongated oblong particles doi: 10.3389/fmicb.2019.00430 approximately 1.2 mm long and 0.6 mm in diameter (Supplementary Figure S1). The prototype Frontiers in Microbiology| www.frontiersin.org 1 March 2019| Volume 10| Article 430 fmicb-10-00430 March 6, 2019 Time: 17:27 # 2 Legendre et al. Evolutionary Mechanisms in Pandoraviridae strain (and the one with the largest known genome) is cloned, mass-produced and purified as previously described pandoravirus salinus (p. salinus), isolated from shallow (Philippe et al., 2013). marine sediments off the coast of central Chile (Philippe et al., 2013). Other members were soon after isolated from Genome and Transcriptome Sequencing, worldwide locations. Complete genome sequences have been Annotation determined for p. dulcis (Melbourne, Australia) (Philippe et al., The p. celtis genome was fully assembled from one PacBio SMRT 2013), p. inopinatum (Germany) (Antwerpen et al., 2015), cell sequence data with the HGAP 4 assembler (Chin et al., 2013) p. macleodensis (Melbourne, Australia), p. neocaledonia (New from the SMRT link package version 5.0.1.9585 with default Caledonia), and p. quercus (France) (Legendre et al., 2018), parameters and the “aggressive” option = true. Genome polishing and three isolates from Brazil (p. braziliensis, p. pampulha, and was finally performed using the SMRT package. Stringent gene p. massiliensis) (Aherfi et al., 2018). A standard phylogenetic annotation was performed as previously described (Legendre analysis of the above strains suggested that the Pandoraviridae et al., 2018). Briefly, data from proteomic characterization family consists of two separate clades (Claverie et al., 2018; of the purified virions were combined with stranded RNA- Figure 1). The average proportion of identical amino acids seq transcriptomic data, as well as protein homology among between pandoravirus orthologs within each clade is above previously characterized pandoraviruses. The transcriptomic 70% while it is below 55% between members of the A and B data were generated from cells collected every hour over an clades. Following a stringent reannotation of the predicted infectious cycle of 15 h. They were pooled and RNAs were protein-coding genes using transcriptomic and proteomic data, extracted prior poly(A)C enrichment. The RNA were then sent our comparative genomic analysis reached the main following for sequencing. Stranded RNA-seq reads were then used to conclusions (Legendre et al., 2018): accurately annotate protein-coding as well as non-coding RNA (1) The uniquely large proportion of predicted proteins genes. A threshold of gene expression (median read coverage > 5 without homologs outside of the Pandoraviridae over the whole transcript) larger than the lowest one associated (ORFans) is real and not due to bioinformatic errors to proteins detected in proteomic analyses was required to induced by the above-average GCC content (>60%) of validate all predicted genes (including novel genes) (Table 1). pandoravirus genomes; Genomic regions exhibiting similar expression levels but did (2) the Pandoraviridae pan genome appears “open” not encompass predicted proteins or did overlap with protein- (i.e., unbounded); coding genes expressed from opposite strands were annotated as (3) as most of the genes are unique to each strain are “non-coding RNA” (ncRNA) after assembly by Trinity (Grabherr ORFans, they were not horizontally acquired from other et al., 2011). When only genomic data were available, namely (known) organisms; for p. pampulha, p. massiliensis, and p. braziliensis (Aherfi (4) they are neither predominantly the result of et al., 2018), we annotated protein-coding genes using ab initio gene duplications. prediction coupled with sequence conservation information as previously described (Legendre et al., 2018). The scenario of de novo and in situ gene creation, supported by Gene clustering was performed on all available the analysis of their sequence statistical signatures, thus became pandoraviruses’ protein sequences using Orthofinder (Emms our preferred explanation for the origin of strain specific genes. and Kelly, 2015) with defaults parameters except for the “msa” In the present study, we take advantage of the high similarity option for the gene tree inference method. (96.7% DNA sequence identity) between p. quercus and a newly Functional annotation of protein-coding genes was performed characterized isolate,