Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication

Abstract

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 ...

Duck egg drop syndrome virus: an emerging Tembusu-related flavivirus in China

“…Both in vivo and in vitro data support the idea that the SLA functions as the promoter element for the viral polymerase rather than other elements [45]. A study of DENV replication demonstrates that at least one U in the bottom bugle of the SLA is required for viral replication [49]. Oligo(U), located downstream of the SLA, functions as a spacer that enhances viral RNA synthesis and is shared by all of the flavivirus genomes [49,50].…”

“…A study of DENV replication demonstrates that at least one U in the bottom bugle of the SLA is required for viral replication [49]. Oligo(U), located downstream of the SLA, functions as a spacer that enhances viral RNA synthesis and is shared by all of the flavivirus genomes [49,50]. In most cases, a second short stem loop (SLB) that contains the 5′UAR sequence is formed and ends at the translational start codon [38,51].…”

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

“…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.…”

“…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).…”

Arginine methylation enhances the RNA chaperone activity of the West Nile virus host factor AUF1 p45

“…Second, the stem of the 3 ′ SL structure has to be opened such that the released 3 ′ -terminal nucleotides may serve as the initiating template of the replicase ( Fig. 1; Filomatori et al 2006Filomatori et al , 2011Dong et al 2008;Lodeiro et al 2009). Mainly from the mosquito-borne DV and WNV, solid experimental evidence exists showing that these prerequisites are achieved by long-range RNA-RNA interactions of conserved complementary sequence elements, which support a 5 ′ -3 ′ circularization of the viral genome.…”