Genome replication in flavivirus requires (—) strand RNA synthesis, (+) strand RNA synthesis, and 5’-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. Two major components of the replication complex are the viral non-structural (NS) proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (—) RNA synthesis, (+) RNA synthesis, and 5’-RNA capping and methylation.

Dengue viruses (DEN), mosquito-borne members of the familyFlaviviridae, are human pathogens of global significance. The effects of mycophenolic acid (MPA) and ribavirin (RBV) on DEN replication in monkey kidney (LLC-MK2) cells were examined. MPA (IC₅₀=0.4±0.3 μM) and RBV (IC₅₀=50.9±18 μM) inhibited DEN2 replication. Quantitative real-time RT-PCR of viral RNA and plaque assays of virions from DEN2-infected and MPA (10 μM)- and RBV (⩾200 μM)-treated cells showed a fivefold increase in defective viral RNA production by cells treated with each drug. Moreover, a dramatic reduction of intracellular viral replicase activity was seen byin vitroreplicase assays. Guanosine reversed the inhibition of these compounds, suggesting that one mode of antiviral action of MPA and RBV is by inhibition of inosine monophosphate dehydrogenase and thereby depletion of the intracellular GTP pool. In addition, RBV may act by competing with guanine-nucleotide precursors in viral RNA translation, replication and 5′ capping.

Using the massively parallel genetic algorithm for RNA folding, we show that the core region of the 3'-untranslated region of the dengue virus (DENV) RNA can form two dumbbell structures (5'- and 3'-DBs) of unequal frequencies of occurrence. These structures have the propensity to form two potential pseudoknots between identical five-nucleotide terminal loops 1 and 2 (TL1 and TL2) and their complementary pseudoknot motifs, PK2 and PK1. Mutagenesis using a DENV2 replicon RNA encoding the Renilla luciferase reporter indicated that all four motifs and the conserved sequence 2 (CS2) element within the 3'-DB are important for replication. However, for translation, mutation of TL1 alone does not have any effect; TL2 mutation has only a modest effect in translation, but translation is reduced by ∼60% in the TL1/TL2 double mutant, indicating that TL1 exhibits a cooperative synergy with TL2 in translation. Despite the variable contributions of individual TL and PK motifs in translation, WT levels are achieved when the complementarity between TL1/PK2 and TL2/PK1 is maintained even under conditions of inhibition of the translation initiation factor 4E function mediated by LY294002 via a noncanonical pathway. Taken together, our results indicate that the cis-acting RNA elements in the core region of DENV2 RNA that include two DB structures are required not only for RNA replication but also for optimal translation.