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 5 untranslated region (5UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication. At the 5 end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as the 5 upstream AUG region (5UAR). Here, we analyzed the secondary structure of the SLA in solution and the structural requirements of this element for viral replication. Using infectious DENV clones, viral replicons, and in vitro polymerase assays, we defined two helical regions, a side stem-loop, a top loop, and a U bulge within SLA as crucial elements for viral replication. The determinants for SLA-polymerase recognition were found to be common in different DENV serotypes. In addition, structural elements within the SLA required for DENV RNA replication were also conserved among different mosquito-and tick-borne flavivirus genomes, suggesting possible common strategies for polymerase-promoter recognition in flaviviruses. Furthermore, a conserved oligo(U) track present downstream of the SLA was found to modulate RNA synthesis in transfected cells. In vitro polymerase assays indicated that a sequence of at least 10 residues following the SLA, upstream of the 5UAR, was necessary for efficient RNA synthesis using the viral 3UTR as template.Dengue fever is the most prevalent mosquito-borne viral disease in humans. Any of the four dengue virus (DENV) serotypes (DENV1 to DENV4) can produce clinical illness ranging from dengue fever, a nonspecific flu-like syndrome, to dengue hemorrhagic fever, a severe and sometimes fatal disease (14). The World Health Organization continues to report outbreaks of severe forms of the disease in the Americas and Asia. It is estimated that more than 50 million DENV infections occur annually. Despite the urgent need to control this virus, a licensed vaccine against DENV is not yet available. Incorporation of attenuating mutations into DENV clones has been shown to be a valuable tool for generating live vaccine candidates (32). In this regard, manipulation of the viral 5Ј untranslated region (5ЈUTR) and the 3ЈUTR has been shown to be a feasible strategy. For instance, Whitehead and collaborators have produced a recombinant DENV that harbors a 30-nucleotide deletion at the 3ЈUTR. DENV1 ⌬30 and DENV4 ⌬30 are promising candidates that are being tested in clinical trials (6, 33). In addition, mutations within the 5ЈUTR have also been explored to generate attenuated DENVs (8,27). Because a dengue vaccine must be able to protect against all four circulating virus serotypes, dissecting conserved cis-acting elements of the viral genome will aid to define mutations that can alter virulence in the four serotypes.DENVs are members of the genus Flavivirus in the Flaviviridae family, together with other important human pathogens, such as yellow fever virus, West Nile virus (WNV), and Japanese encephalitis virus (13). The viral genome is a singlestranded RNA molecule with positive ...

The core human mitochondrial transcription apparatus is currently regarded as an obligate three-component system comprising the bacteriophage T7-related mitochondrial RNA polymerase, the rRNA methyltransferase-related transcription factor, h-mtTFB2, and the high mobility group box transcription/DNA-packaging factor, h-mtTFA/TFAM. Using a faithful recombinant human mitochondrial transcription system from Escherichia coli, we demonstrate that specific initiation from the mtDNA promoters, LSP and HSP1, only requires mitochondrial RNA polymerase and h-mtTFB2 in vitro. When h-mtTFA is added to these basal components, LSP exhibits a much lower threshold for activation and a larger amplitude response than HSP1. In addition, when LSP and HSP1 are together on the same transcription template, h-mtTFA-independent transcription from HSP1 and h-mtTFA-dependent transcription from both promoters is enhanced and a higher concentration of h-mtTFA is required to stimulate HSP1. Promoter competition experiments revealed that, in addition to LSP competing transcription components away from HSP1, additional cis-acting signals are involved in these aspects of promoter regulation. Based on these results, we speculate that the human mitochondrial transcription system may have evolved to differentially regulate transcription initiation and transcription-primed mtDNA replication in response to the amount of h-mtTFA associated with nucleoids, which could begin to explain the heterogeneity of nucleoid structure and activity in vivo. Furthermore, this study sheds new light on the evolution of mitochondrial transcription components by showing that the human system is a regulated two-component system in vitro, and thus more akin to that of budding yeast than thought previously.h-mtTFA/TFAM | mtDNA | nucleoid | POLRMT | h-mtTFB2/TFB2M

The aim of the present study was to identify the signaling mechanisms to ghrelin-stimulated activation of the serine/threonine kinase Akt. In human embryonic kidney 293 (HEK293) cells transfected with GHS-R1a, ghrelin leads to the activation of Akt through the interplay of distinct signaling mechanisms: an early Gi/o protein-dependent pathway and a late pathway mediated by β-arrestins. The starting point is the Gi/o-protein dependent PI3K activation that leads to the membrane recruitment of Akt, which is phosphorylated at Y by c-Src with the subsequent phosphorylation at A-loop (T308) and HM (S473) by PDK1 and mTORC2, respectively. Once the receptor is activated, a second signaling pathway is mediated by β-arrestins 1 and 2, involving the recruitment of at least β-arrestins, c-Src and Akt. This β-arrestin-scaffolded complex leads to full activation of Akt through PDK1 and mTORC2, which are not associated to the complex. In agreement with these results, assays performed in 3T3-L1 preadipocyte cells indicate that β-arrestins and c-Src are implicated in the activation of Akt in response to ghrelin through the GHS-R1a. In summary this work reveals that c-Src is crucially involved in the ghrelin-mediated Akt activation. Furthermore, the results support the view that β-arrestins act as both scaffolding proteins and signal transducers on Akt activation.