Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation.

The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5 element named SLA. We demonstrated that RNA-RNA interactions between 5 and 3 end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5 end of the genome and reaches the site of initiation at the 3 end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.[Keywords: Flavivirus; RNA-dependent RNA polymerase; RNA cyclization; viral RNA synthesis; AFM] Supplemental material is available at http://www.genesdev.org.

Little is known about the mechanism of flavivirus genome encapsidation. Here, functional elements of the dengue virus (DENV) capsid (C) protein were investigated. Study of the N-terminal region of DENV C has been limited by the presence of overlapping cis-acting RNA elements within the protein-coding region. To dissociate these two functions, we used a recombinant DENV RNA with a duplication of essential RNA structures outside the C coding sequence. By the use of this system, the highly conserved amino acids FNML, which are encoded in the RNA cyclization sequence 5=CS, were found to be dispensable for C function. In contrast, deletion of the N-terminal 18 amino acids of C impaired DENV particle formation. Two clusters of basic residues (R5-K6-K7-R9 and K17-R18-R20-R22) were identified as important. A systematic mutational analysis indicated that a high density of positive charges, rather than particular residues at specific positions, was necessary. Furthermore, a differential requirement of N-terminal sequences of C for viral particle assembly was observed in mosquito and human cells. While no viral particles were observed in human cells with a virus lacking the first 18 residues of C, DENV propagation was detected in mosquito cells, although to a level about 50-fold less than that observed for a wild-type (WT) virus. We conclude that basic residues at the N terminus of C are necessary for efficient particle formation in mosquito cells but that they are crucial for propagation in human cells. This is the first report demonstrating that the N terminus of C plays a role in DENV particle formation. In addition, our results suggest that this function of C is differentially modulated in different host cells. D engue virus (DENV) is a member of the genus Flavivirus in the familyFlaviviridae, together with other important pathogens such as yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). DENV is the most significant mosquito-borne human viral pathogen worldwide, infecting more than 50 million people each year (41). Despite the urgent medical need to control DENV infections, vaccines and antivirals are still unavailable.DENV has a plus-stranded RNA genome of about 11 kb. The single open reading frame encodes a polyprotein that is co-and posttranslationally processed by host and viral proteases. This processing yields three structural proteins (C, prM, and E) and at least seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (22). The coding sequence is flanked by highly structured 5= and 3= untranslated regions (UTRs) (11). In recent years, a number of cis-acting RNA elements that modulate viral RNA amplification have been identified in the viral genome (for a review, see reference 38). Viral RNA synthesis is catalyzed by the RNA-dependent RNA-polymerase activity of the viral protein NS5 and requires interaction with a 5=-end promoter element (9, 10, 13). During flavivirus morphogenesis, the newly synthesized viral RNA is r...