PT - JOURNAL ARTICLE AU - Sarah Aherfi AU - Djamal Brahim Belhaouari AU - Lucile Pinault AU - Jean-Pierre Baudoin AU - Philippe Decloquement AU - Jonatas Abrahao AU - Philippe Colson AU - Anthony Levasseur AU - David C. Lamb AU - Eric Chabriere AU - Didier Raoult AU - Bernard La Scola TI - Tricarboxylic acid cycle and proton gradient in <em>Pandoravirus massiliensis</em>: Is it still a virus? AID - 10.1101/2020.09.21.306415 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.09.21.306415 4099 - http://biorxiv.org/content/early/2020/09/21/2020.09.21.306415.short 4100 - http://biorxiv.org/content/early/2020/09/21/2020.09.21.306415.full AB - Since the discovery of Acanthamoeba polyphaga Mimivirus, the first giant virus of amoeba, the historical hallmarks defining a virus have been challenged. Giant virion sizes can reach up to 2.3 µm, making them visible by optical microscopy. They have large genomes of up to 2.5 Mb that encode proteins involved in the translation apparatus. Herein, we investigated possible energy production in Pandoravirus massiliensis, the largest of our giant virus collection. MitoTracker and TMRM mitochondrial membrane markers allowed for the detection of a membrane potential in virions that could be abolished by the use of the depolarizing agent CCCP. An attempt to identify enzymes involved in energy metabolism revealed that 8 predicted proteins of P. massiliensis exhibited low sequence identities with defined proteins involved in the universal tricarboxylic acid cycle (acetyl Co-A synthase; citrate synthase; aconitase; isocitrate dehydrogenase; α-ketoglutarate decarboxylase; succinate dehydrogenase; fumarase). All 8 viral predicted ORFs were transcribed together during viral replication, mainly at the end of the replication cycle. Two of these proteins were detected in mature viral particles by proteomics. The product of the ORF132, a predicted protein of P. massiliensis, cloned and expressed in Escherichia coli, provided a functional isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle, which converts isocitrate to α-ketoglutarate. We observed that membrane potential was enhanced by low concentrations of Acetyl-CoA, a regulator of the tricarboxylic acid cycle. Our findings show for the first time that energy production can occur in viruses, namely, pandoraviruses, and the involved enzymes are related to tricarboxylic acid cycle enzymes. The presence of a proton gradient in P. massiliensis coupled with the observation of genes of the tricarboxylic acid cycle make this virus a form a life for which it is legitimate to question ‘what is a virus?’.Competing Interest StatementThis work was supported by the French Government under the "Investments for the Future" program managed by the National Agency for Research (ANR) Mediterranee-Infection 10-IAHU-03. It was also supported by Region PACA and European funding FEDER PRIMMI.