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Genetic and ultrastructural characterization of Cafeteria roenbergensis virus and its virophage Mavirus Fischer, Matthias Gunther

Abstract

Giant viruses infecting unicellular eukaryotes have genomes that overlap in size and coding content with the smallest cellular life forms, thereby blurring the boundary between what is considered living and non-living. Due to their recent discovery, little is known about the biology and host range of giant viruses. In this dissertation, I characterize Cafeteria roenbergensis virus (CroV), the largest marine virus known to date. CroV infects the phagotrophic nanoflagellate C. roenbergensis, a widespread and ecologically important marine zooplankton species. CroV has a 730 kilobase pair DNA genome which is predicted to encode 544 proteins and 22 transfer RNAs. Four genes contained an intein insertion and several genes have not been found before in viruses, including an isoleucyl-tRNA synthetase and a histone acetyltransferase. A 38 kilobase pair region of putative bacterial origin encoded predicted enzymes for the biosynthesis of 3-deoxy-D-manno-octulosonate, a key component of the bacterial lipopolysaccharide layer. Microarray analysis revealed that at least 274 CroV genes were transcribed during infection and that different genes were expressed at early and late stages of viral replication. Promoter sequences specific for each stage were identified. Proteomic analyses showed that the virion is composed of at least 129 CroV-encoded proteins, including a large set of transcription enzymes and several DNA repair proteins. Phylogenetically, CroV was found to belong to the group of nucleocytoplasmic large DNA viruses and was most closely related to Acanthamoeba polyphaga mimivirus, although only a third of the CroV genes had homologues in Mimivirus. I also discovered a smaller virus, the Mavirus virophage, whose replication was dependent on co-infection by CroV and led to decreased CroV production and increased host-cell survival. Mavirus particles co-localized within the CroV virion factory, as shown by transmission electron microscopy of infected cells. Remarkably, the 19 kilobase pair DNA genome of Mavirus was most similar to the Maverick/Polinton eukaryotic DNA transposons, which led to the hypothesis that these transposons have originated from the endogenization of ancient virophages into eukaryotic genomes. This work describes the first giant virus infecting a zooplankton species and demonstrates a clear link between Mavirus and Maverick transposons.

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