A 5′ RNA element promotes dengue virus RNA synthesis on a circular genome

Filomatori, Claudia Verónica; Lodeiro, María F.; Álvarez, Diego E.; Samsa, Marcelo M.; Pietrasanta, Lı́a I.; Gamarnik, Andrea V.

doi:10.1101/gad.1444206

Cited by 334 publications

(448 citation statements)

References 41 publications

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“…It is located at the leading end of the first stem situated at the base of the predicted SLA structure at the 5′-end of the genome. The SLA is recognized by viral RNA polymerase (NS5) and participates in cyclization of the viral genome through the interaction with the 3′-end sequence, thereby promoting minus-strand viral RNA synthesis (Filomatori et al, 2006). Using the singleround FL-DENV/GFP RNA-launched system, we confirmed that the 5′-69M construct resulted in significantly less (P < 0.05) intracellular viral RNA levels than the FL-DENV/GFP containing wt 5′NCR construct (Fig.…”

Section: Discussionmentioning

confidence: 56%

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

et al. 2012

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Insertion of green fluorescent protein (GFP) encoding-gene into virus genes has provided a valuable tool for flavivirus research. This study aimed to develop dengue virus (DENV) replicons expressing GFP reporter that would provide a fast in vitro system to analyze functional roles of specific DENV sequences in viral replication. Two classes of recombinant replicon constructs were generated; one was a RNA-launched replicon with a GFP gene directly inserted into a fulllength DENV genome (FL-DENV/GFP), and the other consisted of 4 types of DNA-launched DENV subgenomic replicons with GFP replacement at various structural genes (Δ-DENV/GFP). The FL-DENV/GFP resulted in GFP expression in transfected cells with no viable DENV being recovered from the transfection. The Δ-DENV/GFP constructs with partial structural gene deletion (ΔC-, ΔCprM/M-, ΔprM/M-, or ΔE-) expressed bright and long lasting GFP. The GFP expression intensity in living cells correlated well with the level of RNA replication. Various mutations in the 5′noncoding region of DENV-2 previously shown to be important genetic determinants for virus replication and mouse virulence were incorporated into the 5 different replicon constructs. Characterizations of 29 mutants demonstrated that these replicons can provide a useful platform for a quick and powerful in vitro system to analyze genetic determinants of DENV replication. These constructs can also be useful for development of vectors expressing foreign genes for various researches.

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Section: Discussionmentioning

confidence: 56%

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

et al. 2012

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“…The 5Ј-UTRs of flavivirus RNAs are ϳ100 nt in length and fold to form stable secondary structures (16,26,27). The 5Ј-terminal region within ϳ160 nt forms two stem-loop (SL) structures, SLA and SLB (Fig.…”

Section: The Dengue Virus (Denv)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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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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“…NS5 has methyltransferase and RNAdependent RNA polymerase activities, whilst NS3 is a serine protease, NTPase/RNA helicase and 59 RNA triphosphatase (for a review, see Lindenbach & Rice, 2001;Luo et al, 2008). Consistent with their putative role in viral replication, both NS3 and NS5 exist as a complex in DENV-infected cells (Kapoor et al, 1995) and interact with the 39-and 59UTR of Japanese encephalitis virus and DENV RNA, respectively (Chen et al, 1997;Filomatori et al, 2006;Lodeiro et al, 2009;Luo et al, 2008). Additionally, some sequences present in the 39-and 59UTR are able to interact with cellular proteins such as La, polypyrimidine tract-binding protein (PTB), translation elongation factor 1a, Y box-binding protein 1, heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2/B2 and Q, protein disulfide isomerase, poly(A)-binding protein (PABP) and calreticulin (De Nova-Ocampo et al, 2002;García-Montalvo et al, 2004;Paranjape & Harris, 2007;Polacek et al, 2009;Yocupicio-Monroy et al, 2003.…”

Section: Introductionmentioning

confidence: 99%

Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells

Agis-Juárez

Galván²,

Medina

et al. 2009

Journal of General Virology

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The 39 untranslated region (39UTR) of the dengue virus (DENV) genome contain several sequences required for translation, replication and cyclization processes. This region also binds cellular proteins such as La, polypyrimidine tract-binding protein (PTB), Y box-binding protein 1, poly(A)-binding protein and the translation initiation factor eEF-1a. PTB is a cellular protein that interacts with the regulatory sequences of positive-strand RNA viruses such as several picornaviruses and hepatitis C virus. In the present report, it was demonstrated that PTB translocates from the nucleus to the cytoplasm during DENV infection. At 48 h post-infection, PTB, as well as the DENV proteins NS1 and NS3, were found to co-localize with the endoplasmic reticulum marker calnexin. Silencing of PTB expression inhibited virus translation and replication, whilst overexpression of PTB augmented these processes. Thus, these results provide evidence that, during infection, PTB moves from the nucleus to the cytoplasm and plays an important role in the DENV replicative cycle. INTRODUCTIONDengue virus (DENV), a member of the family Flaviviridae, is the causative agent of dengue fever, dengue haemorrhagic fever and dengue shock syndrome. The single-stranded, positive-polarity RNA genome of approximately 11 kb contains a type I cap at the 59 end and lacks a poly(A) tail at the 39 end. The single open reading frame (ORF) encodes a polyprotein that generates three structural proteins -envelope (E), membrane and capsid (C) -and seven non-structural proteins -NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Flanking the ORF, the viral RNA contains two untranslated regions (UTRs) involved in various functions such as initiation and regulation of virus translation, replication and assembly (Lindenbach & Rice, 2001;Proutski et al., 1999). The 39UTR of DENV and other mosquito-borne flaviviruses contains a conserved stemloop structure within the last~96 nt (Brinton & Dispoto, 1988;Brinton et al., 1986;Grange et al., 1985;Mohan & Padmanabhan, 1991). Additionally, there are two pairs of conserved sequences (59CS1, 39CS1, 59UAR and 39UAR) that together induce DENV cyclization (Alvarez et al., 2005a, b; Hahn et al., 1987). These motifs are essential for negativestrand RNA synthesis of DENV (Ackermann & Padmanabhan, 2001; Alvarez et al., 2005a, b;Villordo & Gamarnik, 2009;You & Padmanabhan, 1999) and other flaviviruses (Bredenbeek et al., 2003;Corver et al., 2003;Jones et al., 2005;Khromykh et al., 2001;Lo et al., 2003;Nomaguchi et al., 2004). On the other hand, sequences present within a large stem-loop structure located at the 59 end as well as a conserved oligo(U) track function as the promoter for viral polymerase activity (Lodeiro et al., 2009). Moreover, an RNA secondary structure present in the coding region of DENV type 2 (DENV-2) directs translation, start-codon selection and replication of the viral genome (Clyde & Harris, 2006;Clyde et al., 2008). Although it has been shown that the cyclization process does not require the presence of cellular or v...

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A 5′ RNA element promotes dengue virus RNA synthesis on a circular genome

Cited by 334 publications

References 41 publications

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

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

Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells

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