Mimivirus transcription and translation occur at well-defined locations within amoeba host cells.

Many giant viruses replicate in the cytoplasm in viral factories. How exactly these viral factories are established and where the different steps of the replication cycle occur remain largely obscure. We have developed a single-molecule messenger RNA fluorescence in situ hybridization (smFISH) protocol for giant viruses in an Acanthamoeba host. Combined with other labeling techniques (FUNCAT, DiD, rRNA FISH, and DAPI), we show the Mimivirus transcription and translation sites during an infection cycle in the amoeba host cell. Although viral mRNA localization changes depend on the infection stage, transcription occurs at well-defined spots within the viral factory. The original viral cores released within the cytoplasm most likely define these spots. When transported outside of the viral factory, the translation of viral mRNA takes place in a well-defined ring surrounding it. With this study, we obtained novel insights into giant virus replication, of which the methods are widely applicable to other viruses for the visualization and quantification of RNA molecules.IMPORTANCEGiant viruses have massive particle and genome sizes, which are known to infect unicellular eukaryotes. Although most viruses replicate in the host cell's nucleus, the giant Mimivirus replicates in viral factories established in the host cell's cytoplasm. Before this study, the location of the various steps in the Mimivirus replication cycle was largely unknown. By developing new protocols to label giant virus mRNA, protein synthesis, host cell membranes and rRNA, we demonstrate that Mimivirus transcription occurs at well-defined sites within the viral factory. In contrast, translation takes place directly outside of it. This is different from other viruses known to have a cytoplasmic life cycle. These results bring us a step closer to understanding how the genome complexity of viruses influences the virus-host interactions and viral replication strategies.