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 ϳ60% 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. The dengue virus (DENV)3 is a mosquito-borne flavivirus (MBFV) in the Flaviviridae family that consists of over 70 members, many of which are significant human pathogens (1). The MBFV members are classified into three subgroups: DENV, yellow fever virus, and Japanese encephalitis virus (JEV) (2, 3). The four serotypes of DENV (DENV1 to -4) cause an estimated 50 million cases of infections, with ϳ10% of those leading to severe forms of the disease, dengue hemorrhagic fever and dengue shock syndrome (4 -6).The viral genome is a single-stranded RNA of positive (ϩ) polarity containing ϳ11 kilobases (10,723 nt for DENV2 New Guinea C strain, GenBank TM accession number M29095 (7)). The 3Ј-end is non-polyadenylated, and the 5Ј-end has a type I cap structure (for a review, see Ref. 8). Flanking the single long open reading frame are the 5Ј-and 3Ј-UTRs, which contain conserved cis-acting RNA secondary structure elements required for translation and replication (9 -18). The viral RNA is translated to form a polyprotein precursor, which is processed by host and viral proteases in the endoplasmic reticulum membrane. Protein processing gives rise to three structural (C, prM, and E) and seven nonstructural (NS) proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 in that order (for reviews, see Refs. 8,19,and 20) and references therein). According to the current model for replication, assembly of the viral replicase complex occurs in a cytoplasmic membrane organelle, followed by negative (Ϫ)-strand RNA synthesis starting at the 3Ј-end of the viral genome, resulting in a double-stranded replicative form. NS3 and NS5 are multifunctional pr...

The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The fulllength dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK 2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcriptderived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts.

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We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3 stem-loop structure (3-SL) that is conserved among all flavivirus species. The 3-SL constitutes the downstream terminal ϳ95 nucleotides of the 3 noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a humanvirulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.The four serotypes of dengue viruses (DEN1, DEN2, DEN3, and DEN4) cause dengue fever (DF) and dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS). The incidences of both DF and DHF/DSS are rising worldwide (10), and there is currently no vaccine available to prevent the spread of dengue or reduce the incidence of disease. A live virus vaccine is desirable for a variety of reasons, including reduced cost in comparison to that of subunit vaccines, the possibility for single-dose vaccination, and the need to elicit long-term immunity. Vaccine development has been hampered thus far by the lack of any animal model for DF or DHF/DSS and the perceived need to elicit a protective response to all four serotypes of dengue virus concurrently (10,16,27).The dengue viruses are members of the Flavivirus genus. Flaviviruses contain a positive-strand RNA genome that is ϳ11 kb long and includes a single open reading frame (ORF) encoding a polyprotein which is processed co-and posttranslationally to yield three structural and at least seven nonstructural (NS) proteins (6). The ORF is flanked at the 5Ј and 3Ј termini of the genome by noncoding region...

The flavivirus dengue 1 Western Pacific,74 (DEN1 WP) virus has a positive-stranded RNA genome of 10,735 nucleotides. DEN1 WP genomic RNA was amplified into three overlapping fragments by RT-PCR. These fragments were assembled into a full-length cDNA clone in the yeast-E. coli shuttle vector pRS424, using homologous recombination in yeast. RNA produced by in vitro transcription of this clone was infectious upon electroporation into LLCMK2 cells, as shown by cytopathic effects and detection of viral antigens by indirect immunofluorescence, and by propagation of the virus released into the culture media. Biological properties of the transcript-derived virus, such as the pattern of dengue-specific protein synthesis and growth rate in LLCMK2 or C6/36 cells, resembled those of the parent DEN1 WP virus.