RNA structure duplication in the DENV genome: functional redundancy or host specificity

DE BORBA, LUANA; FILOMATORI, CLAUDIA V.; MARSICO, FRANCO L.; CARBALLEDA, JUAN M.; BLAIR, CAROL D.; PALLARÉS, HORACIO M.; VILLORDO, SERGIO M.; SANCHEZ VARGAS, IRMA; GAMARNIK, ANDREA V.

Encuentro; 6th Pan-American Dengue Research Network Meeting; 2018

The dengue virus (DENV) genome contains a single open reading frame flanked by two highly structured untranslated regions (5' and 3'UTRs). The 3'UTR bears two pairs of duplicated RNA structures: two stem-loops (SLs) and two dumbbells (DBs), both involved in highly stable structures, including tertiary interactions like pseudoknots (PK). We have previously shown that the two SLs have different functions in vertebrates and invertebrate hosts. In this regard, a host adaptable SL structure has been identified, which was associated to the generation of different species of sfRNAs. Interestingly, DENV isolated from infected Aedes aegypti and Aedes albopictus, showed positive selection of mutations in one of the two DB structures. In order to evaluate the relevance of this selection, we introduced the identified mutations in the context of an infectious clone and assessed viral replication in mosquito and human cells. The phenotype of the viruses obtained indicated that mutations that destabilize the PK of DB2 increase the ability of the virus to replicate only in mosquito cells, explaining the natural selection of those mutations in mosquitoes, and providing evidence of different roles of this DB structure in the two hosts. Mechanistic studies designed to evaluate the function of the identified RNA elements supported the idea that the PK interaction of DB2 competes with genome cyclization, reducing RNA synthesis.Thus, disruption of the PK may be responsible for enhancement of viral replication in mosquito. Our data support an antagonistic role of the two DBs, while the DB1 has been involved in promoting genome cyclization (de Borba, et al. J. Virol., 2015), elements of DB2 compete with this RNA cyclization. In summary, our studies indicate that each structure of the two pairs, SLs and DBs, play different functions during viral replication. In addition, each of these RNA elements displays specific roles in the two hosts.