The discovery of Mimivirus in 2003 challenged the established definition of viruses due to its large size and genome. Pandoraviruses, with even larger genomes (up to 2.5 Mb) and unique characteristics like a lack of capsid protein genes, further blurred the lines. Giant viruses have also exhibited features previously thought exclusive to cellular life, including virophage infection, a CRISPR-Cas-like defense mechanism (MIMIVIRE), and components of the protein translation apparatus. Furthermore, some giant viruses possess genes for metabolic pathways such as fermentation, sphingolipid biosynthesis, and nitrogen metabolism. However, no previous evidence existed suggesting that these gene products were used by the viruses themselves for their metabolic needs. This study sought to investigate another hallmark of independent life: energy production, specifically in Pandoravirus massiliensis, a giant virus with a high proportion of ORFan genes (no homologs in databases), offering a unique system for discovering novel gene functions. Energy generation is often associated with proton gradients; thus, the researchers looked for such gradients in P. massiliensis, hypothesizing that the presence of a proton gradient in this virus would strongly suggest that the classification of this virus should be re-evaluated.

Giant viruses have challenged the traditional understanding of viruses, pushing the boundaries of the definition. The discovery of Mimivirus, with its large size and genome, initiated this paradigm shift. Subsequent discoveries of Pandoraviruses, with even larger genomes and unique characteristics such as the absence of genes encoding for capsid proteins, further questioned the classical viral definition. The identification of associated virophages, a functioning defense mechanism (MIMIVIRE) similar to the CRISPR-Cas system in bacteria, and components of the protein translation apparatus in some giant viruses like Tupanvirus and Klosneuvirus, have added to the complexity. The presence of genes involved in metabolic pathways, like fermentation, sphingolipid biosynthesis, and nitrogen metabolism, in many giant viruses, fueled the debate further. However, prior to this research, no conclusive evidence suggested that giant viruses themselves utilized these gene products for their own metabolic needs. This provided the impetus for exploring the possibility of energy production within giant viruses.

This study used a multifaceted approach to investigate the energy production capabilities of Pandoravirus massiliensis. First, they developed mouse polyclonal antibodies specific to P. massiliensis to aid in the visualization and differentiation of viral structures from amoeba components. Second, the presence of a membrane electrochemical gradient in P. massiliensis virions was assessed using two fluorescent dyes: Mitotracker Red 633 and tetramethyl rhodamine methyl ester (TMRM). These dyes are selectively sequestered in hyperpolarized inner cell membranes, their uptake depending on the mitochondrial membrane potential. The experiments were conducted with both amoeba infected with P. massiliensis and purified virions. A negative control (cowpox virus) and a positive control (Staphylococcus aureus) were used. The effect of the uncoupling agent chloridyl cyanide-methoxyphenylhydrazone (CCCP), which disrupts the proton gradient, was assessed on the TMRM staining of the virions. Third, bioinformatics analyses were performed on the P. massiliensis genome using BLASTp against the GenBank nr database and delta-BLAST against the conserved domain database (CDD) to identify potential genes involved in energy metabolism, focusing on the tricarboxylic acid (TCA) cycle. Phylogenetic analyses were conducted to determine the evolutionary origin of identified genes. RNA sequencing was utilized to assess the transcription of the candidate genes during the viral replication cycle. Finally, the genes encoding suspected TCA enzymes were cloned, expressed in E. coli, and purified. Enzymatic activity assays were then conducted to confirm their functionality. Statistical analyses were performed to analyze the results.

The study revealed the presence of a membrane potential in mature P. massiliensis virions and during its replication cycle, as demonstrated by the uptake of Mitotracker Deep Red 633 and TMRM dyes. This membrane potential was specifically associated with the virions and not with amoeba mitochondrial contamination, as confirmed by immunofluorescence staining and 3D analysis. Treatment with CCCP, a depolarizing agent, abolished the membrane potential, confirming the existence of an electrochemical gradient across the virion tegument. Bioinformatics analysis identified eight putative P. massiliensis proteins showing low sequence similarity to enzymes involved in the TCA cycle. These genes were all transcribed during viral replication, and the protein product of ORF132 was shown to function as a functional isocitrate dehydrogenase (IDH), a key enzyme in the TCA cycle. Low concentrations of acetyl-CoA enhanced the membrane potential, while high concentrations decreased it. This suggested a link between the membrane potential and the TCA cycle. Proteomic analysis identified two of the eight predicted TCA-related proteins in mature virions. While enzymatic activity assays showed functionality for ORF132 (IDH), other tested TCA-related proteins showed no enzymatic activity. Phylogenetic analysis of ORF132 revealed a close relationship with other orthologs in Pandoraviruses, but their evolutionary origin remained elusive. The low sequence similarity of the other seven ORFs with known proteins prevented a complete analysis of the putative TCA pathway.

The findings demonstrate, for the first time, the existence of a membrane potential in Pandoraviruses, possibly linked to a partial TCA cycle. The presence of a proton gradient, coupled with the identification of a functional isocitrate dehydrogenase and the transcriptional activity of other putative TCA cycle genes, suggests the possibility of an autonomous energy generation mechanism within the virus. While a complete TCA cycle was not demonstrated, the existence of a partial pathway is strongly indicated. The regulation of this potential TCA cycle by acetyl-CoA also supports its relevance in viral metabolism. The evolutionary origin of these genes remains unclear, but the data suggests they might be acquired via horizontal gene transfer. The implications of this energy production mechanism are significant. The ability to generate energy could enhance viral fitness by providing the reducing power (NADH or NADPH) necessary for early viral replication. This discovery challenges the traditional understanding of viruses and suggests a more complex and dynamic interaction with their hosts.

This study provides compelling evidence for the presence of a proton gradient and a partially functional TCA cycle within the Pandoravirus massiliensis. The identification of a functional isocitrate dehydrogenase and the transcriptional activity of other potential TCA cycle enzymes highlight a more complex metabolic capacity than previously recognized in viruses. These findings challenge traditional viral classification and raise questions about the fundamental definition of viruses. Future research should focus on characterizing the complete TCA pathway, determining the evolutionary origin of the involved genes, and investigating the role of this energy production mechanism in viral replication and host-virus interactions. The investigation of the crystal structures of the viral enzymes, especially IDH, will be key to the better understanding of this potential mechanism.

The study did not demonstrate a complete TCA cycle, only a partial one with evidence of a functional IDH. The low sequence similarity of the other putative TCA enzymes hindered phylogenetic analysis and definitive functional characterization. The functional analysis was mainly based on in vitro experiments, and the role of these genes in vivo still needs more research.