Replicación y ensamblaje del Arbovirus Bunyamwera en células del insecto vector Aedes albopictus

Arboviruses, or arthropod-borne viruses, are important causes of epidemic and endemic disease in many countries all over the world. Most arboviruses are RNA viruses mainly of the families Bunyaviridae, Flaviviridae and Togaviridae. In nature, arboviruses are spread by arthropod vectors, predominantly mosquitoes, ticks, flies and midges to vertebrate hosts. Yellow fever, Dengue fever,West Nile fever, Rift Valley fever, La Crosse encephalitis, Chikungunya arthritis or Crimean-Congo haemorrhagic fever are some of the arbovirus-associated emerging and re-emerging diseases. With the only exception of Yellow fever virus there are no vaccines, nor efficient treatments, against pathogenic arboviruses. In their vertebrate hosts, arboviruses generally counteract innate defences and trigger a highly cytopathic infection that leads inevitably to cell death. However, in mosquito cells an early phase of efficient viral protein synthesis and virus production is followed by a persistent infection with low levels of viral protein expression and virus release. Little is known about how arthropod cells react to arbovirus infection, not even for Dengue, the most important human arbovirus with almost half of the world? s population at risk. In this work, we have studied how C6/36 cells, derived from Aedes albopictus, one of the relevant vectors for arboviruses, respond to Bunyamwera virus, a wellcharacterized arbovirus. Confocal, live cell microscopy and electron microscopy showed that Bunyamwera virus (BUNV) induces deep changes in mosquito cells. Early in infection these cells develop long projections and create new intercellular connections where cell organelles and viral proteins were detected. Live cell microscopy showed that these connections were developed before viral protein could be detected by immunofluorescence. Interestingly, their proliferation was accompanied by a progressive trapping of the nucleocapsid and RNA polymerase viral proteins into large cytoplasmic aggregates. A significant drop in the release of infectious virions then followed. Before that, numerous viruses assembled in peripheral Golgi stacks and they apparently exit the cells immediately since they did not accumulate intracellularly. This mechanism of assembly seems to cause little damage to the integrity of cell endomembranes. The characterization of the antiviral mechanisms operating in mosquito cells can be of great help in the fight against pathogenic arboviruses. The Orthobunyavirus non structural protein NSm is thought to be important in the assembly and morphogenesis of viruses and replication organelles. However, NSm proteins have not been well characterized and probably, as many other proteins from RNA viruses, they probably harbor multiple domains and functions. Confocal and electron microscopy studies with BUNV NSm deletion mutants showed that NSm protein is a key factor for the formation of the tubular domain of the replication organelle in mammalian cells. Our studies also suggested that these cylinders are not involved in viral genome replication that takes place in the globular domain of the organelle, but in connecting viral replication and assembly. NSm deletion mutants appeared to produce more aggressive infections in both mammalian and mosquito cells; viral particles were produced earlier and in higher amounts than with the wilt type virus. This caused a more severe cytotoxic effect in mammalian cells. However, mosquito cells were still able to survive and entered into the persistent phase of the infection. These results strongly suggest that NSm might delay infection in both cell lines and that this ? negative? regulation would no interphere with the development of persistence in mosquito cells. Despite of our extensive search by conventional EM methods, BUNV replication organelles have not yet been identified in mosquito cells. To this purpose, and for a better characterization of this organelles in infected mammalian cells, we have designed and generated a new tool that consist of a recombinant BUNV with the viral polymerase L protein fused to metallothionein (MT); this is a small metal-binding protein that works as a clonable tag for electron microscopy recently validated in our lab. We have also optimized the protocols for treating mammalian cells with gold salts and subsequent visualizacion of MT-tagged proteins in cells by metal-tagging transmission electron microscopy (METTEM). With the L-MT recombinant BUNV we will study the biogenesis and structure of replication complexes in replication organelles by METTEM, a method that allows visualization of proteins in their natural environment and at molecular scale resolution