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.

Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5 and 3 ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5 and 3 CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5 and 3 untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5 and 3 UAR (upstream AUG region). In order to investigate the functional role of 5-3 UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5 or 3 UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.Outbreaks and epidemics caused by dengue virus continue to pose a public health problem in tropical and subtropical regions (60). It is estimated that more than 50 million human infections occur annually, and 2.5 billion people are at risk of dengue virus infection worldwide. Despite the wide morbidity and mortality associated with dengue virus infections, the molecular biology of this virus is not well understood, and at present, neither specific antiviral therapy nor licensed vaccine exists. Thus, defining the molecular determinants that regulate utilization of the viral RNA in the infected cell is of central importance for understanding the dengue virus life cycle.The genomes of positive-strand RNA viruses participate in at least three different processes in the cytoplasm of the infected host cell: they serve as mRNA to direct the synthesis of viral proteins, they act as a template for genome amplification, and they are packaged along with structural proteins during viral assembly. The molecular mechanisms controlling the utilization of the viral RNA in each step of the viral life cycle are still poorly understood. Several lines of evidence support the notion that viral RNA genomes could circularize to regulate initiation of translation and RNA synthesis at the 5Ј and 3Ј ends of the genome (4,15,18,22,23,30,31,33,38,44). However, the molecular nature of 5Ј-3Ј associations and the details of how different conformations of the RN...

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.

Dengue virus RNA-dependent RNA polymerase specifically binds to the viral genome by interacting with a promoter element known as stem-loop A (SLA). Although a great deal has been learned in recent years about the function of this promoter in dengue virus-infected cells, the molecular details that explain how the SLA interacts with the polymerase to promote viral RNA synthesis remain poorly understood. Using RNA binding and polymerase activity assays, we defined two elements of the SLA that are involved in polymerase interaction and RNA synthesis. Mutations at the top of the SLA resulted in RNAs that retained the ability to bind the polymerase but impaired promoter-dependent RNA synthesis. These results indicate that protein binding to the SLA is not sufficient to induce polymerase activity and that specific nucleotides of the SLA are necessary to render an active polymerase-promoter complex for RNA synthesis. We also report that protein binding to the viral RNA induces conformational changes downstream of the promoter element. Furthermore, we found that structured RNA elements at the 3 end of the template repress dengue virus polymerase activity in the context of a fully active SLA promoter. Using assays to evaluate initiation of RNA synthesis at the viral 3-UTR, we found that the RNA-RNA interaction mediated by 5-3-hybridization was able to release the silencing effect of the 3-stem-loop structure. We propose that the long range RNA-RNA interactions in the viral genome play multiple roles during RNA synthesis. Together, we provide new molecular details about the promoter-dependent dengue virus RNA polymerase activity. Dengue virus (DENV)3 is a member of the Flavivirus genus in the Flaviviridae family, together with other important human pathogens such as yellow fever virus, West Nile virus (WNV), Saint Luis encephalitis virus, and Japanese encephalitis virus (1). DENV is the most significant mosquito-borne human viral pathogen worldwide and is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. The lack of vaccines and antivirals against DENV leaves two billion people at risk, mainly in poor countries.DENV genome is a single-stranded RNA molecule of positive polarity of ϳ11 kb in length that encodes a long polyprotein that is co-and post-translationally processed by host and viral proteases to yield three structural proteins (C, prM, and E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The coding sequence is flanked by highly structured 5Ј-and 3Ј-UTRs. In recent years, a number of cis-acting RNA elements have been identified in the viral 5Ј-and 3Ј-UTRs that are essential for viral RNA amplification (for review see Ref.2). A model for DENV RNA synthesis has been proposed previously (3). This model involves binding of the viral RNA-dependent RNA polymerase (RdRp) to an RNA element present at the 5Ј end of the genome known as stemloop A (SLA). This element of 70 nucleotides folds into a Yshaped structure that is conserved among diff...