F1 motif of Dengue Virus polymerase is involved in initiation of viral RNA synthesis.

IGLESIAS, N.G.; FILOMATORI C.V.; GAMARNIK, A.V.

Puerto Madryn. Chubut. Argentina

Congreso; XLVI Reunión anual. Sociedad Argentina de Investgaciones en Bioquímica y Biología Molecular.; 2010

Sociedad Argentina de Investigación Bioquímica y de Biología Molecular

Dengue virus (DENV) is the most important mosquito-borne viral human pathogen worldwide. The viral genome is a single strand RNA molecule of positive polarity of about 11 kb long. The RNA encodes a single polyprotein that results in ten viral proteins. The coding sequence is flanked by highly structured 5’ and 3’UTRs. NS5 is the largest of the DENV proteins. It contains an N-terminal methyltransferase domain (MT) and a C-terminal RNA polymerase domain (RdRp). The RdRp activity depends on the presence of the RNA promoter (SLA), present at the 5’ end of the genome. We used recombinant DENV NS5 proteins and genetically modified viruses to investigate the relevance of positive-charged regions of the RdRp on RNA binding, polymerase activity, and viral replication in cell culture. Using in vitro activity assays, we analyzed 14 mutants of NS5. We found 4 mutants that were unable to synthesize RNA with any RNA template. In addition, we found one mutant located in the F motif, which showed a specific defect in RNA synthesis only when the viral RNA was used as template. These and other observation led us to propose that the F1 region of the F motif is involved in specific initiation of viral RNA synthesis. Furthermore, we investigated the replication of viruses carrying mutations using mammalian cells and found different phenotypes associated to specific NS5 changes.