Structural and functional studies of the promoter element for dengue virus RNA replication.

LODEIRO, MARÍA FERNANDA; FILOMATORI CLAUDIA; GAMARNIK ANDREA

JOURNAL OF VIROLOGY

AMER SOC MICROBIOLOGY

Año: 2009 vol. 83 p. 993 - 1008

0022-538X

The 5’ untranslated region of the dengue virus genome contains two defined elements essential for viral replication. At the 5’ end, a large stem-loop structure (SLA) functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as 5’UAR. Here, we analyzed the secondary structure of the SLA in solution and the structural requirements of this element for viral replication. Using infectious dengue virus clones, viral replicons, and in vitro polymerase assays, we defined two helical regions, a side stem-loop, a top loop, and a U bulge within SLA as crucial elements for viral replication. The determinants for SLA-polymerase recognition were found to be common in different DENV serotypes. In addition, structural elements within the SLA required for dengue virus RNA replication were also conserved among different mosquito and tick borne flavivirus genomes, suggesting possible common strategies for polymerase-promoter recognition in flaviviruses. Furthermore, a conserved oligo(U) track present downstream of the SLA was found to modulate RNA synthesis in transfected cells. In vitro polymerase assays indicated that a sequence of at least 10 residues following the SLA, upstream of 5’UAR, was necessary for efficient RNA synthesis using the viral 3’UTR template.