Identification of Mutated Cyclization Sequences That Permit Efficient Replication of West Nile Virus Genomes: Use in Safer Propagation of a Novel Vaccine Candidate

Suzuki, Ritsuro; Fayzulin, Rafik; Frolov, Ilya; Mason, Peter W.

doi:10.1128/jvi.00662-08

Cited by 27 publications

(35 citation statements)

References 45 publications

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“…3, D and E). A similar conclusion was reported in which the 3Ј-CS1 involving the same nucleotides that constitute PK1 was mutated in the WNV replicon (43). Their results showed that specific bases in the 5Ј-and 3Ј-CS1 are important for replication, regardless of complementarity.…”

Section: Discussionsupporting

confidence: 65%

“…A long range RNA-RNA interaction between these complementary motifs resulting in circularization of the genome was postulated (40) and demonstrated by atomic force microscopy (13). The physical and functional RNA-RNA interaction between 5Ј-and 3Ј-CS1 was shown to be essential for RNA synthesis in vitro (10,41) and in replication of subgenomic replicons or infectious clones in cultured mammalian cells (11,12,14,42,43). In addition to the 5Ј-and 3Ј-CS1, two regions of complementarity in the 5Ј-and 3Ј-terminal sequences, termed the upstream of AUG region (UAR), were also identified as a requirement for cyclization and RNA replication (13,17,44).…”

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confidence: 99%

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Identification of Cis-Acting Elements in the 3′-Untranslated Region of the Dengue Virus Type 2 RNA That Modulate Translation and Replication

Manzano

Reichert

Polo

et al. 2011

Journal of Biological Chemistry

124

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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...

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Section: Discussionsupporting

confidence: 65%

mentioning

confidence: 99%

Identification of Cis-Acting Elements in the 3′-Untranslated Region of the Dengue Virus Type 2 RNA That Modulate Translation and Replication

Manzano

Reichert

Polo

et al. 2011

Journal of Biological Chemistry

124

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“…BHK(VEErep/C*-E/Pac) cells expressing the WNV C-prM/E proteins (8), BHK(VEErep/Pac-Ubi-C*) cells expressing the WNV C protein (52), and BHK(VEErep/Pac-Ubi-D2C) cells expressing the DENV2 C protein (see below) were propagated at 37°C in Dulbecco's MEM supplemented with 10% FBS and 10 g/ml puromycin as previously described (8). The WNV derived from an infectious cDNA engineered from a 2002 WNV isolate was previously described (49), and RepliVAX WN.2 has also been described (52). The DENV2 used for in vitro and animal studies consisted of a high-passage, mouse brainadapted derivative of the New Guinea C (NGC) strain.…”

Section: Methodsmentioning

confidence: 99%

Construction and Characterization of a Single-Cycle Chimeric Flavivirus Vaccine Candidate That Protects Mice against Lethal Challenge with Dengue Virus Type 2

Suzuki

Winkelmann²,

Mason

2009

J Virol

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We have previously described a novel flavivirus vaccine technology based on a single-cycle, capsid (C) gene-deleted flavivirus called RepliVAX. RepliVAX can be propagated in cells that express high levels of C but undergoes only a single cycle of infection in vaccinated hosts. Here we report that we have adapted our RepliVAX technology to produce a dengue vaccine by replacing the prM/E genes of RepliVAX WN (a West Nile virus [WNV] RepliVAX) with the same genes of dengue virus type 2 (DENV2). Our first RepliVAX construct for dengue virus (RepliVAX D2) replicated poorly in WNV C-expressing cells. However, addition of mutations in prM and E that were selected during blind passage of a RepliVAX D2 derivative was used to produce a second-generation RepliVAX D2 (designated D2.2) that displayed acceptable growth in WNV C-expressing cells. RepliVAX D2.2 grew better in DENV2 C-expressing cells than WNV C-expressing cells, but after several passages in DENV2 C-expressing cells it acquired further mutations that permitted efficient growth in WNV C-expressing cells. We tested the potency and efficacy of RepliVAX D2.2 in a well-described immunodeficient mouse model for dengue (strain AG129; lacking the receptors for both type I and type II interferons). These mice produced dose-dependent DENV2-neutralizing antibody responses when vaccinated with RepliVAX D2.2. When challenged with 240 50% lethal doses of DENV2, mice given a single inoculation of RepliVAX D2.2 survived significantly longer than sham-vaccinated animals, although some of these severely immunocompromised mice eventually died from the challenge. Taken together these studies indicate that the RepliVAX technology shows promise for use in the development of vaccines that can be used to prevent dengue.Dengue viruses (DENV) are the etiologic agents of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. The viruses are transmitted to humans by Aedes spp. mosquitoes. DENV infections are a serious cause of morbidity and mortality in most tropical and subtropical areas of the world (12). Dengue cases are estimated to occur in up to 100 million individuals annually, and there are over 2.5 billion people living in areas at risk for infection, making dengue the most important arbovirus disease in the world. DENV belongs to the Flavivirus genus in the family Flaviviridae and exists as four antigenically distinct serotypes (DENV1 to -4) (4). The four serotypes of DENV do not confer cross-protective immunity, and epidemiological evidence indicates that immunity to one serotype of DENV increases the chance of a more severe disease upon infection with a second serotype by about 10-fold (26).DENV are single-stranded, positive-sense RNA viruses and have an 11-kb genome characterized by a single open reading frame encoding three structural proteins (C, prM/M, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) and untranslated regions at its 5Ј and 3Ј termini (5Ј untranslated region [UTR] and 3ЈUTR). Viral RNA replication occurs in the ...

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“…To further demonstrate the functionality of the new replicon system, we tested a replicon with a mutation in the 5Ј CS that was previously shown to abrogate RNA replication (Fig. 1B [WNV-SPACER-EMCV-Rep CS m4/7-5Ј]) (15) and confirmed inhibition of replication.…”

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confidence: 99%

The 5′ and 3′ Downstream AUG Region Elements Are Required for Mosquito-Borne Flavivirus RNA Replication

Friebe

Shi

Harris

2011

J Virol

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Flaviviruses require complementarity between the 5 and 3 ends of the genome for RNA replication. For mosquito-borne flaviviruses, the cyclization sequences (CS) and upstream of AUG region (UAR) elements at the genomic termini are necessary for viral RNA replication, and a third motif, the downstream of AUG region (DAR), was recently designated for dengue virus. The 3 DAR sequence is also part of a hairpin (HP-3SL), and both complementarity between 5 and 3 DAR motifs and formation of the HP-3SL in the absence of the 5 end are conserved among mosquito-borne flaviviruses. Using West Nile virus as a model, we demonstrate that 5-3 DAR complementarity and HP-3SL formation are essential for viral RNA replication.Flaviviruses have an approximately 11-kb-long RNA genome of positive polarity (see reference 10). The viral RNA encodes one open reading frame (ORF), and the resulting polyprotein is cleaved co-and posttranslationally into 3 structural (N-terminal) and 7 nonstructural (NS) (C-terminal) proteins. The ORF is flanked on both sites by untranslated regions (5Ј and 3Ј UTR), which contain several cis-acting RNA elements that regulate RNA translation and replication. A prerequisite for viral RNA replication is genome circularization, and two essential sequence motifs complementary between the 5Ј and 3Ј ends have been reported for mosquito-borne flaviviruses: the 5Ј-3Ј cyclization sequences (CS) and 5Ј-3Ј upstream of AUG region (UAR) elements (1-3, 8, 9, 14, 16-19, 22, 23). Recently, a third motif in the dengue virus (DENV) 5Ј terminus, located downstream of the AUG region (DAR), was reported to be involved in RNA replication (7) and possibly genome circularization. The sequence at the 3Ј genomic end complementary to the 5Ј DAR motif (3Ј DAR) also appeared to have a role in RNA replication, but the data were less conclusive than that for the 5Ј DAR. Furthermore, the 3Ј DAR sequence is involved in the formation of a small hairpin at the bottom of the 3Ј stem-loop (HP-3ЈSL) in the absence of the 5Ј end, and nucleotides within the loop of the HP-3ЈSL are further predicted to form a pseudoknot with nucleotides in the 3ЈSL (13). Although it has been implicated in DENV translation (4) and replication (1,7,21), the role of the HP-3ЈSL in flavivirus replication has not been definitely delineated.To further define the role of the DAR sequences and the HP-3ЈSL in RNA replication of mosquito-borne flaviviruses, we used West Nile virus (WNV) as a model, since to date nothing has been definitively determined about the impact of either of these motifs in WNV RNA replication. To study the effect of the 5Ј DAR sequence on RNA replication, we modified a previously described WNV replicon (Fig. 1A [RlucRep]) in which the structural proteins were replaced by the gene encoding Renilla luciferase (11). This resulted in WNV-EMCV-Rep and WNV-SPACER-EMCV-Rep (Fig. 1A), which have an overall design analogous to that of DENV replicons 5ЈUTR-Cap-tr and 5ЈUTR-Cap-tr-SPACER (7). In the case of WNV, the 5Ј UTR and the first 93 capsid-coding nucleoti...

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Identification of Mutated Cyclization Sequences That Permit Efficient Replication of West Nile Virus Genomes: Use in Safer Propagation of a Novel Vaccine Candidate

Cited by 27 publications

References 45 publications

Identification of Cis-Acting Elements in the 3′-Untranslated Region of the Dengue Virus Type 2 RNA That Modulate Translation and Replication

Identification of Cis-Acting Elements in the 3′-Untranslated Region of the Dengue Virus Type 2 RNA That Modulate Translation and Replication

Construction and Characterization of a Single-Cycle Chimeric Flavivirus Vaccine Candidate That Protects Mice against Lethal Challenge with Dengue Virus Type 2

The 5′ and 3′ Downstream AUG Region Elements Are Required for Mosquito-Borne Flavivirus RNA Replication

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