The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question “what is a virus?”.

棘阿米巴拟菌病毒（ Acanthamoeba polyphaga Mimivirus）是第一个分离的阿米巴巨型病毒，它的发现挑战了定义病毒的历史标志。已知巨型病毒体尺寸可达 2.3 µm，通过光学显微镜即可观察到。它们的基因组大小高达 2.5 Mb，可以编码参与翻译装置的蛋白质。我们研究了马西潘多拉病毒可能产生的能量。线粒体膜标记允许检测纯化病毒体中的膜电位，并且三羧酸循环的调节剂增强了这一点，但通过使用去极化剂消除了这一点。采用生物信息学来鉴定参与病毒粒子质子梯度生成的酶，该方法揭示了八种假定的马西里巴氏菌蛋白质与参与通用三羧酸循环的已知细胞酶表现出较低的序列同一性。此外，所有八个病毒基因在复制过程中都被转录。这些基因之一的产物 ORF132 在大肠杆菌中被克隆和表达，并显示出其作为异柠檬酸脱氢酶（三羧酸循环的关键酶）的功能。我们的研究结果首次表明潘多拉病毒中存在膜电位，这可能与三羧酸循环有关。 P. Massiliensis中质子梯度的存在使这种病毒成为一种生命形式，因此可以合理地提出“什么是病毒？”的问题。