CONICET | Buscador de Institutos y Recursos Humanos

To study the process of DENV encapsidation, the subcelullar localization of the C protein in infected cells was first investigated. Previous reports indicated that the C protein accumulates in the nucleus, however, using different techniques to preserve cellular membranes, the C protein was observed in ring-like patterns in the cytoplasm. A specific accumulation of the C protein surrounding organelles known as lipid droplets (LDs) was observed. LDs are dynamic structures derived from the ER that store neutral lipids and play crucial roles in lipid metabolism. Interestingly, viral infection resulted in an increase in the size and the number of LDs, suggesting a link between LD metabolism and DENV replication. In the C coding sequence of DENV there are overlapping elements that contain cis-acting RNA replication signals. Thus, to dissociate RNA synthesis from the encapsidation process, specific genetic tools were developed. Manipulation of infectious clones and generation of new reporter DENVs allowed us to define the molecular basis of C protein association to LDs. Specific amino acids on the á2 helix, located in the center of the C protein, were found to be crucial for both accumulation of C on LDs and DENV infectious particle formation. Although we found that the C protein decreased viral RNA synthesis. We propose that LDs play multiple roles during the viral life cycle; they could sequester the viral C protein early during infection to ensure efficient RNA synthesis and provide a scaffold for genome encapsidation. Our findings begin to unravel the complex mechanism by which dengue virus usurps cellular organelles to coordinate different steps of the viral life cycle.