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.