Vírus gigantes de amebas: análise estrutural e funcional de fibrilas de superfície e investigação de mecanismos envolvidos no bloqueio da superinfecção

Abstract

The discovery of giant viruses of amoeba surprised and prompted the scientific community to search for new isolates after the description of mimivirus. Since then, several other mimivirus isolates have been discovered, described and classified, exhibiting clear genomic differences that currently subdivide them into three main genera: Mimivirus, Moumouvirus and Megavirus. During our efforts to isolate and characterize new giant viruses, a new virus was isolated from water samples collected from Pampulha Lagoon in Belo Horizonte. Through complete sequencing of its genome and phylogenetic analysis based on the gene encoding DNA polymerase subtype B, we identified the isolate as a megavirus and named it megavirus caiporensis. When analyzing the isolate by transmission electron microscopy, it was observed that the fibrils of these viruses are organized differently, forming clumps. It was also observed that during viral multiplication the cycle of the new isolate seemed to be slower when compared to other mimiviruses. From this point onwards, the analysis of the particle morphology, with emphasis on the fibrils, the study of the cycle and analysis of possible genes linked to these viruses’ fibrils, became the objective of this work. Additionally, we chose a representative of the genera Mimivirus and Moumouvirus to perform comparisons among our results. Through electron microscopy, we described for the first time that mimiviruses can present at least three distinct patterns of organization of their fibrils, linked to the genera/lineages to which they are closely related. It was showed that the different patterns are intrinsic to the viral particle, and not artifacts produced by preparations for microscopy. The study of the cycle of m. caiporensis was similar to that of other megaviruses and in late stages of the cycle, we observed newly formed virions inside vesicles. We studied the possibility of release by exocytosis, as is observed for other giant viruses, such as cedratviruses and pandoraviruses. Our analyses, combining viral infection and titration assays, electron microscopy, and immunofluorescence, showed that these viruses are not released by exocytosis, but rather that there is a continuous incorporation of particles by the amoeba infected by megavirus caiporensis, a phenomenon known as superinfection. Comparative analyses demonstrated that megavirus is not able to block superinfection in Acanthamoeba, unlike mimivirus and moumouvirus, and this process can be reversed by chemical inhibition of phagocytosis. Here, we describe for the first time superinfection and its inhibition by giant viruses that infect amoebas. This work contributes with information about the morphology, genomics, phylogeny, ecology, and evolution of mimiviruses. In addition, it contributes to the understanding of superinfection and its inhibition in mimiviruses, moumouviruses, and megaviruses, demonstrating that despite their evolutionary kinship, these viruses present profound differences in their interactions with their hosts.