Flaviviruses constitute the most relevant group of arthropod-transmitted viruses, including important human pathogens such as the dengue, Zika, yellow fever, and West Nile viruses. The natural alternation of these viruses between vertebrate and invertebrate hosts shapes the viral genome population, which leads to selection of different viral variants with potential implications for epidemiological fitness and pathogenesis. However, the selective forces and mechanisms acting on the viral RNA during host adaptation are still largely unknown. Here, we found that two almost identical tandem RNA structures present at the viral 3′ untranslated region are under different selective pressures in the two hosts. Mechanistic studies indicated that the two RNA elements, known as dumbbells, contain sequences that overlap essential RNA cyclization elements involved in viral RNA synthesis. The data support a model in which the duplicated RNA structures differentially evolved to accommodate distinct functions for viral replication in the two hosts.

The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified new cis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3= untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells. Dengue virus (DENV) is a member of the Flaviviridae family that includes other important pathogens such as yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). The DENV genome is a plus-stranded RNA molecule that contains a single open reading frame flanked by highly structured 5= and 3= untranslated regions (UTRs) (1-3). RNA elements located within these regions are responsible for translation initiation and genome replication (4-7). The 5= UTR is about 100 nucleotides (nt) long and includes three different elements: (i) stem-loop A (SLA), which is the promoter for viral polymerase binding and activation (8-10); (ii) stem-loop B (SLB), which contains a sequence known as 5= upstream of the AUG region (5= UAR) that is complementary to a sequence present at the 3= UTR (3= UAR) and mediates longrange RNA-RNA interactions between the ends of the genome (11); and (iii) a spacer sequence between SLA and SLB rich in U's, which functions as an enhancer of viral replication (10). The viral 3= UTR is about 450 nucleotides long and comprises four defined domains: domain A1, which features a variable region (VR) (12); domains A2 and A3, which present two almost-identical dumbbell-like secondary structures (DB1 and DB2), which appear to work as enhancers for viral RNA replication (13-15); and domain A4, which contains a small hairpin (sHP) and the 3= stem-loop (3= SL), which are essential elements for viral replication (3,16). In addition to RNA structures defined in the UTRs that play different roles during infection, important RNA elements have been described in the protein coding region. In this regard, essential sequences that mediate long-range RNA-RNA interactions known as 5= cyclization sequence (5= CS) and 5= downstream of AUG region (5= DAR) are located within the capsid coding sequence (11,13,(17)(18)(19)(20)(21). Also, a hairpin known as cHP, located between 5= CS and 5= DAR, has been shown to be necessary for efficient RNA replication (22). The current model for viral RNA synthesis includes the interaction of the viral polymerase NS5 with the 5=-end SLA promoter and its transfer to the 3=-end initiation site by cyclization of the viral genome (9). Despite great advances in knowledge of cis-acting RNA elements in the flavivirus genomes, the molecular details and mechanisms by which many of them function during viral replication are still not well understood.Intrigued by dual roles of RNA sequences in v...

To help understand the mechanism of pathogenesis of dengue virus (DV), we set out to create an infectious cDNA of the Brazilian prototype strain of DV serotype 1 (DV1-BR/90). PCR-amplified fragments of DV1-BR/90 cDNA were readily assembled into a subgenomic cDNA that could be used to produce replicating RNAs (replicons), lacking the structural protein-encoding regions of the genome. However, assembly of a cDNA capable of producing infectious virus was only possible using a bacterial artificial chromosome plasmid, indicating that DV1 sequences were especially difficult to propagate in E. coli. While characterizing our cDNA we discovered a fortuitous temperature-sensitive mutation in the NS1 encoding region. Using our infectious cDNA and a renilla luciferase-expressing replicon we were able to demonstrate that this mutation produced a defect in RNA replication at 37 degrees C, demonstrating that the DV1 NS1 protein plays an essential role in RNA replication.