Interactions of Dengue Virus NS5 and NS3 with the 3' End of Its Negative-Strand RNA

Dengue virus is an important human pathogen that infects over 400 million people each year. Despite its global health significance, several essential aspects of the viral replication mechanism remain poorly understood. Flaviviruses carry out asymmetric viral RNA synthesis, wherein positive-strand RNA is synthesized in excess over negative-strand RNA. The template for positive-strand synthesis is the negative strand in a double-stranded RNA intermediate, yet little is known about how positive-strand RNA synthesis is initiated. Orthoflaviviruses, including dengue virus, require an RNA promoter, stem-loop A (SLA) at the 5' end of the viral genome for negative-strand RNA synthesis. Consequently, a complementary stem-loop structure is predicted at the 3' end of the negative strand (3'SLA), where positive-strand synthesis is initiated. To understand the functional role of 3'SLA, we investigated its structure and examined its interaction with the viral replication proteins NS5 polymerase and NS3 helicase. NS5 and NS3 differentially recognize the stem-loop structures of the positive and negative strands (5'SLA and 3'SLA, respectively), yet NS5 polymerase efficiently synthesizes RNA from both 5'SLA- and 3'SLA-containing templates. We further show that the stable 5' and 3'SLA elements readily form a duplex that mimics the replication intermediate under our in vitro conditions. Both NS5 and NS3 showed reduced binding to this dsRNA intermediate and NS3 was unable to unwind it, suggesting that additional factors may be required to regulate viral replication in infected cells.