Notaro, Anna (2018) Understanding the innovative viral glycosylation machinery using a combination of chemical and structural methodologies. [Tesi di dottorato]

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Abstract

The aim of this thesis is the study of the innovative glycosylation machinery of the Mimiviridae family, using Mimivirus, Moumouvirus australensis and Megavirus chilensis as prototypes of lineages A, B and C, respectively. In 2003 the discovery of Mimivirus, the first giant DNA virus infecting amoeba, challenged the traditional view of viruses. Mimiviruses are giant viruses due to the size of their virions, easily visible by light microscopy, with a diameter of 700 nm against 200 nm for “traditional virus”. Their genomes encode 1000 proteins and count up to 1.2 Mbp, so they are as complex as the smallest free-living bacteria. Mimiviruses exhibit heavily glycosylated fibrils surrounding their capsid that differ in length depending on the lineages. Surprisingly, it was evidenced that they encode the proteins involved in their fibrils glycosylation. The glycosylation of the fibrils was confirmed by the analysis of their sugar content, revealing that the major saccharide components were rhamnose, N-acetylglucosamine, and viosamine for Mimivirus and N-acetylglucosamine and N-acetylrhamnosamine for Megavirus chilensis. Until now, we lack information on the sugar composition of fibrils from members of the B lineage. In this thesis, the innovative glycosylation machinery of these giant DNA viruses was investigated combining three different strategies: carbohydrate chemistry, bioinformatic and biochemical methodologies. The carbohydrate chemistry methodologies allowed to elucidate the structures/composition of the glycans associated to the giant DNA viruses fibrils. Mimivirus fibrils are decorated with two distinct polysaccharides, called poly_1 and poly_2. Poly_1 is characterized by a linear disaccharide repeating unit made of 3)--L-Rha-(1→3)--D-GlcNAc-(1→, with a pyruvic acid branched at position 4,6 of GlcNAc. Poly_2 has a branched repeating unit with the sequence 2)--L-Rha-(1→3)--D-GlcNAc-(1→ in the linear backbone and rhamnose further branched at position 3 by viosamine methylated at position 2 and acetylated at position 4. Regarding the novelty of the identified structures, they have no equivalent in eukaryotes, while some components were reported in bacteria. Megavirus chilensis has a different sugar composition of its shorter fibrils, with N-acetylglucosamine, N-acetylrhamnosamine and N-acetylquinovosamine as major components. Purification results suggested that Megavirus fibrils were decorated by more than one polysaccharides/oligosaccharide species, one having this trisaccharide: -L-4OMe-RhaNAc-(1→3)--L-RhaNAc-(1→3)--L-RhaNAc-(1→. A preliminary analysis revealed that Moumouvirus australensis fibrils were decorated with glucosamine and quinovosamine in addition to the rare sugar, bacillosamine. Starting from this experimental data, it was possible to identify new genes involved in glycosylation. As a result, the published nine-gene cluster of Mimivirus was extended to thirteen genes. A different cluster of fourteen genes was identified in Moumouvirus australensis, representing the first glycosylation gene cluster identified for the B lineage. A comparison of the glycosylation genes in the Mimiviridae family reinforced our finding that fibrils glycosylation was lineage specific. However, Moumouvirus australensis is an exception as it exhibits a cluster of glycosylation genes that is missing in other member of the B lineage. Among the genes with the glycosylation cluster, the function of L142 was investigated in vitro, demonstrating that it is a N-acetyltransferase that acetylates the 4 amino group of viosamine. N-L142 represents the first virally encoded N-acetyltransferase. To conclude, the fibrils of Mimiviridae are heavily glycosylated and the type of sugars and their organization depends on their lineage. The majority of the genes responsible for sugar production, sugar modification and glycosyltransferases were identified, strongly suggesting that Mimiviridae are autonomous for their fibrils glycosylation.

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