Interações entre vírus gigantes e protozoários: caracterização do tupanvirus soda lake e do cedratvirus getuliensis

Abstract

Following the isolation of mimivirus, many other viruses as or more complex than APMV were identified, revealing notable differences related to structure, genome, replication cycle, and, consequently, host interactions. Recently, a extensive work of viral prospection using samples from diverse environments was performed by our research team, enabling the isolation of two new viruses, tupanvirus soda lake and Cedratvirus getuliensis. The initial characterization of tupanvirus showed a curious relationship determined by the ribosomal shutdown of host and non-host cells when inoculated with tupanvirus. Also remarkably, the discovery of cedratvirus getuliensis has represented the isolation of the third cedratvirus in the whole world, revealing the ascension of a new viral group and evidencing our limited knowledge about the biology of these viruses. Faced with this, the main goal of this thesis was to analyze the protozoan-viruses interactions based on the ribosomal RNA shutdown characterization and the presence of ribosomal sequences in tupanvirus soda lake, as well as the description of cedratvirus getuliensis replication cycle. Initially, to elucidate whether the canonical process of ribophagy/autophagy would be related with the observed ribosomal degradation promoted by tupanvirus, some experiments were conducted. The obtained results showed that the A. castellanii treatment with chloroquine and bafilomycin A, as well as the Atg8 gene silencing do not prevent the occurrence of tupanvirus induced shutdown in A. castellanii cells. Moreover, it was possible observe that the tupanvirus soda lake infection induces a remarkable acidification of amoebas cytoplasm and as a progressive degradation of the nucleus/nucleoli that was temporally associated with shutdown. Transmission electron microscopy also revealed the presence of small vesicles, in majority composed of just one membrane, containing ribosomes next to nuclear membrane. These vesicles, posteriorly, suffer aggregation and constitute growing structures that were depleted from the cytoplasm at the same time that shutdown could be detected. Taken together, these data suggest that the shutdown is not directly related to the ribophagy. Moreover, although the formation of vesicles containing ribosomes and the degradation of the nucleolus may be related to the occurrence of this phenomenon, the mechanism involved in the ribosomal degradation still need to be further investigated. In parallel to the experiments developed on the characterization of the ribosomal RNA shutdown, the presence of ribosomal sequences in tupanvirus were also investigated. Our analyzes revealed the presence of two copies that presented similarity with parts of 18S intronic regions, more specifically to group I self-splicing introns, in the genome of tupanvirus. Similar copies were also found in genomes of other mimiviruses, although never reported before. The phylogenetic analyzes of these two copies present evidenced that they had separated origins and, although they are located in intergenic regions, they are highly expressed during the replication cycle. However, the possible role of these copies in viral genomes still unclear. The other section of this thesis was devoted to the in-depth study of cedratvirus getuliensis replication cycle. Initially, the use of different endocytosis inhibitors evidenced that the entry of these viruses into A. castellanii occurs mainly by phagocytosis. Transmission electron microscopy images showed the formation of a perinuclear electronlucent viral factory. Inside and on the periphery of this structure occurs the morphogenesis that involved subsequent, complex and electron-dense structures. In addition, cellular alterations, such as mitochondria recruitment, lysosomes polarization, and intense membrane traffic were also observed along the replication of cedratvirus getuliensis. The end of the replication cycle was marked by the release of viral progeny mainly by cells lysis, but also by exocytosis. These data presented here revealed peculiar characteristics of both viral isolates and contribute to a better understanding about the host-virus interactions