RNA Sequences and Structures Required for the Recruitment and Activity of the Dengue Virus Polymerase

Abstract

Dengue virus RNA-dependent RNA polymerase specifically binds to the viral genome by interacting with a promoter element known as stem-loop A (SLA). Although a great deal has been learned in recent years about the function of this promoter in dengue virus-infected cells, the molecular details that explain how the SLA interacts with the polymerase to promote viral RNA synthesis remain poorly understood. Using RNA binding and polymerase activity assays, we defined two elements of the SLA that are involved in polymerase interaction and RNA synthesis. Mutations at the top of the SLA resulted in RNAs that retained the ability to bind the polymerase but impaired promoter-dependent RNA synthesis. These results indicate that protein binding to the SLA is not sufficient to induce polymerase activity and that specific nucleotides of the SLA are necessary to render an active polymerase-promoter complex for RNA synthesis. We also report that protein binding to the viral RNA induces conformational changes downstream of the promoter element. Furthermore, we found that structured RNA elements at the 3 end of the template repress dengue virus polymerase activity in the context of a fully active SLA promoter. Using assays to evaluate initiation of RNA synthesis at the viral 3-UTR, we found that the RNA-RNA interaction mediated by 5-3-hybridization was able to release the silencing effect of the 3-stem-loop structure. We propose that the long range RNA-RNA interactions in the viral genome play multiple roles during RNA synthesis. Together, we provide new molecular details about the promoter-dependent dengue virus RNA polymerase activity. Dengue virus (DENV)3 is a member of the Flavivirus genus in the Flaviviridae family, together with other important human pathogens such as yellow fever virus, West Nile virus (WNV), Saint Luis encephalitis virus, and Japanese encephalitis virus (1). DENV is the most significant mosquito-borne human viral pathogen worldwide and is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. The lack of vaccines and antivirals against DENV leaves two billion people at risk, mainly in poor countries.DENV genome is a single-stranded RNA molecule of positive polarity of ϳ11 kb in length that encodes a long polyprotein that is co-and post-translationally processed by host and viral proteases to yield three structural proteins (C, prM, and E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The coding sequence is flanked by highly structured 5Ј-and 3Ј-UTRs. In recent years, a number of cis-acting RNA elements have been identified in the viral 5Ј-and 3Ј-UTRs that are essential for viral RNA amplification (for review see Ref.2). A model for DENV RNA synthesis has been proposed previously (3). This model involves binding of the viral RNA-dependent RNA polymerase (RdRp) to an RNA element present at the 5Ј end of the genome known as stemloop A (SLA). This element of 70 nucleotides folds into a Yshaped structure that is conserved among diff...

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

“…This conclusion is also supported by a stem trace plot of the minigenome RNA maturation in an MPGAfold run (data not shown). However, there is strong experimental evidence that 5Ј-3Ј-UAR base pairing is essential for relieving the repression of RNA synthesis by RdRP in vitro by 3Ј-SL (74). The 3Ј-SL includes a part of 3Ј-UAR element, which upon a conformational change in the presence of the viral polymerase is likely to form the duplex 5Ј-3Ј-UAR (74).…”

“…However, there is strong experimental evidence that 5Ј-3Ј-UAR base pairing is essential for relieving the repression of RNA synthesis by RdRP in vitro by 3Ј-SL (74). The 3Ј-SL includes a part of 3Ј-UAR element, which upon a conformational change in the presence of the viral polymerase is likely to form the duplex 5Ј-3Ј-UAR (74). Conformational changes occurring within the 3Ј-SL due to 5Ј-3Ј-terminal RNA-RNA interaction as well as the NS5/RNA interactions by structure probing and footprinting methods have also been reported for WNV (75).…”

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

“…′ -3 ′ -hybridization of the UAR elements is supposed to be a main trigger for conformational changes that open the 3 ′ SL stem structure and thus release the genomic 3 ′ -end to be recognized by NS5 (Dong et al 2008;Filomatori et al 2011).…”

“…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.…”

Differential RNA Sequence Requirement for Dengue Virus Replication in Mosquito and Mammalian Cells

“…The promoter for RNA synthesis, known as stemloop A (SLA), is located at the 5= end of the genome (6)(7)(8). This promoter binds and activates the polymerase NS5 that then initiates RNA synthesis at the 3= end of a circularized viral genome.…”