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...

The effects of polylysine (PLL) and PLL-asialoorosomucoid (AsOR) on DNA condensation have been analyzed by AFM. Different types of condensed DNA structures were observed, which show a sequence of conformational changes as circular plasmid DNA molecules condense progressively. The structures range from circular molecules with the length of the plasmid DNA to small toroids and short rods with approximately 1/6 to 1/8 the contour length of the uncondensed circular DNA. Single plasmid molecules of 6800 base pairs (bp) condense into single toroids of approximately 110 nm diameter, measured center-to-center. The results are consistent with a model for DNA condensation in which circular DNA molecules fold several times into progressively shorter rods. Structures intermediate between toroids and rods suggest that at least some toroids may form by the opening up of rods as proposed by Dunlap et al. [(1997) Nucleic Acids Res. 25, 3095]. Toroids and rods formed at lysine:nucleotide ratios of 5:1 and 6:1. This high lysine:nucleotide ratio is discussed in relation to entropic considerations and the overcharging of macroions. PLL-AsOR is much more effective than PLL alone for condensing DNA, because several PLL molecules are attached to a single AsOR molecule, resulting in an increased cation density.

The uptake of glucose oxidase (GOx) onto a polycationic redox polymer (PAA-Os)-modified surface, by adsorption from dilute aqueous GOx solutions, was followed by the quartz crystal microbalance (QCM) and shows double exponential kinetics. The electrochemistry of the layer-by-layer-deposited redox-active polymer was followed by cyclic voltammetry in glucose-free solutions, and the enzyme catalysis mediated by the redox polymer was studied in beta-D-glucose-containing solutions. AFM studies of the different layers showed the existence of large two dimension enzyme aggregates on the osmium polymer for 1 microM GOx and less aggregation for 50 nM GOx solutions. When the short alkanethiol, 2,2'-diaminoethyldisulfide was preadsorbed onto gold, a monoexponential adsorption law was observed, and single GOx enzyme molecules could be seen on the surface where the enzyme was adsorbed from 50 nM GOx in water.