NMR structure of Dengue West Nile Viruses stem-loop B: a key cis-acting element for Flavivirus Replication
Flaviviruses are major emerging human pathogenic viruses that pose a persistent and growing menace to global health. They are enveloped single-stranded RNA viruses with positive polarity transmitted by arthropod vectors like mosquitoes or ticks, responsible for a significant and growing number of human deaths and illnesses. The 5\xe2\x80\xb2- and 3\xe2\x80\xb2-untranslated regions (UTRs) are highly structured and contain conserved cis-acting RNA elements that participate in viral translation, replication and host adaptation. Despite their role in flaviviruses replication, few high-resolution structural studies have investigated the RNA elements required for viral replication. Here we report the NMR structures of stem-loop B from WNV and DENV4 viruses. Because this element is required for cyclization of the genome and the activity of the replicative viral enzymes, viral replication rates are sensitive to even small changes in these RNAs. Therefore, this work provides structural insight into a new drug target to reduce flavivirus replication rates.
Introduction<!>RNA synthesis, purification and NMR sample preparation<!>NMR Experiments<!>NMR structural constraints and structure calculations<!>Construct design and secondary structure of DENV4 SLB<!>Construct design and secondary structure of WNV SLB<!>Structure of WNV and Dengue 4 SLB RNAs<!>The structural stability of SLB affects viral replication<!>Discussion
<p>Flaviviruses are emerging pathogens responsible for a significant numbers of infections, disease and mortality [1], which include well-known human pathogens such as yellow fever virus (YFV), Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV) and about 70 other known viruses divided into tick-borne flaviviruses (TBFVs), mosquito-borne flaviviruses (MBFVs), no-known-vector flaviviruses (NKVs) and insect specific flaviviruses (ISFVs) [2]. The mosquito-borne flaviviruses are responsible for most human infections and diseases ranging in severity from mild fever to lethal hemorrhagic fever and encephalitis [3]. For example, the recent ZIKV outbreak, the only flavivirus transmitted sexually, was responsible form any birth defects in newborns and other clinical manifestations in children and adults in the Americas and Caribbean [4]. Effective vaccines or antiviral drugs are still lacking for most flaviviruses.</p><p>Flaviviruses have a positive-sense single-stranded RNA genome of approximately 11,000 nucleotides, which functions both as template for RNA synthesis and viral mRNA. The open reading frame (ORF) encodes a precursor poly protein of approximately 3,300 amino acids and is flanked by a relatively short 5′-untranslated region (5′-UTR) of about 100 nts and a much longer 3′-UTR stretching from 340 to 700 nts in length, with capped 5′-end but non polyadenylated 3′-end [5]. The single polypeptide is post-translationally cleaved by viral proteases to generate mature proteins which participate in replication, viral particle formation and modulation of the host immune system (Figure S1) [6].</p><p>Conserved RNA structures within both UTRs function as enhancers, promoters or repressors in viral translation, replication, host adaptation and encapsidation (Figure S1) [7]. Typically, cis-acting elements responsible for translational control are located in the 3ʹ-UTR, whereas elements required for initiation of RNA synthesis are found near the 5ʹ-end of the viral genome [8]. In MBFVs, the 3′-UTR contains a stem-loop (SL) domain, a dumbbell (DB) domain which facilitate host switching, and the 3ʹ-stem-loop domain (sHP-3ʹ-SL), a structure present in all flavivirues and required for viral replication [9]. During RNA replication, the NS5 viral RNA polymerase interacts with the 5ʹ terminal of the genomic RNA, while long range interactions between complementary sequences at both ends of the genome allow for genome cyclization [10]. Multiple studies by atomic force microscopy [11], structure probing and electrophoretic mobility shift assays [11] have confirmed the interaction between 3ʹ- and 5ʹ-UTRs. Thus, the flavivirus RNA genome has high structural plasticity to undergo multiple conformational rearrangements which are required for efficient viral replication [12].</p><p>The 5ʹ-UTRs of flaviviruses contain the highly conserved stem-loop A (SLA), which serves as a promoter element for viral RNA synthesis and is recognized by the RNA-dependent RNA polymerase (RdRP) [13]. At least three pairs of complementary viral genomic sequences, the 5′- and 3′-UAR (upstream AUG regions), 5′- and 3′-DAR (downstream AUG regions), and 5′- cyclization sequence (within the ORF) and 3′-CS (upstream of the 3′-SL), participate instead in genome cyclization. The 5′-UAR element in MBFVs forms a secondary structure, stem-loop B (SLB), in the linear conformation of the viral RNA [14].</p><p>The 5′-UAR-flanking stem (abbreviated hereafter as UFS) is critical for viral replication [14]. This U-rich stretch locks the 5′-UAR/SLB element in a double helix and facilitate binding of the RdRP to its promoter [14]. The UFS duplex regulates dynamic viral RNA polymerase recruitment, as a switch sensing the linear and circular forms of the genome [14, 15]. Thus, after the MBFVs genome RNA circularize, the UFS unfolds making the NS5 viral RNA polymerase less likely to bind to the 5′-end [15], suggesting that changes in the UFS duplex conformation support translocation of NS5 to the 3′ initiation site [14]. Consistent with this mechanism, stabilization of the secondary structure of the UFS duplex impairs circularization and viral replicative fitness [14, 15].</p><p>Structural studies of the RNA elements of flaviviruses are limited to the xrRNA1/2 crystal structures from Zika and Murray Valley encephalitis virus (MVEV) [16]. The secondary structures of the 3′-UTRs region of multiple flaviviruses (MBFVs) have been predicted from phylogenetic analyses, but only subgenomic flavivirus RNA (sfRNA) from DENV2 has been experimentally determined by SHAPE [17]. The 3D structures of individual subdomains and the overall structural architecture of the UTR's remain unknown.</p><p>We report herein the 3D structures of SLB from WNV and DENV4, determined using NMR spectroscopy, to investigate their role in flavivirus replication. We demonstrate that the U-rich region of MBFV forms a conserved RNA duplex which sequesters the 5′-UAR/SLB element. We further show that UFS mutants that structurally stabilize the UFS duplex reduce viral replication efficiency.</p><!><p>All RNA constructs were prepared by in vitro transcription using T7 RNA polymerase (in-house purified) and synthetic DNA templates (purchased from Integrated DNA Technologies). 2′-O-methyl groups were attached to the last two residues at the 5′-end of the template to reduce the addition of untemplated nucleotides at the 5′-end. The resulting RNAs were purified and NMR samples were prepared (as discussed in SI Methods). Final RNA concentrations used for NMR studies were 0.8–1 mM.</p><!><p>All experiments were recorded on Bruker Avance 800 or/and 600 MHz NMR spectrometers equipped with 1H/13C/15N triple resonance cryogenic probes. Before each experiment, the samples were freshly annealed using the protocols (as described in SI methods). The 1D 1H spectra in 95 % H2O/10 % D2O were recorded using the excitation sculpting pulse sequence. 2D 1H-1H nuclear Overhauser effect (NOESY) and phase sensitive NOESY spectra were recorded with mixing times of 100 and 300 ms, respectively, at both 15 °C and 25 °C. Exchangeable 2D 1H-1H NOESY and 2D [1H-15N] HSQC spectra were recorded in 95 % H2O/5 % D2O in 20 mM phosphate (pH 6.0) and 0.01 μM EDTA, at 25 °C and 15 °C. The non-exchangeable 2D 1H-1H NOESY, 1H-1H TOCSY (spin lock 60–80 ms), 2D [1H-13C] HSQC were recorded in 99.9 % D2O at 25°C. 1H-1H total correlation spectroscopy (TOCSY) were recorded for both RNA samples with mixing times of 40 ms and 80 ms. The NOESY spectra were recorded with various mixing times in the range of 100 to 300 ms to facilitate spectral assignments and quantification of cross peak intensities by comparison with peaks corresponding to fixed distances (e.g. H5-H6). 1H chemical shifts were referenced relative to external sodium 2,2-dimethyl-2-silapentane-5 sulfonate (DSS).</p><p>All NMR data were processed with either Topspin (www.bruker.com) or NMRPipe [18] and analyzed using Sparky [19]. In all 2D experiments, the time domain data consisted of 2048 complex points along the t2 dimension and 400–512 FIDs in t1 dimension with relaxation delay set at 1.0 s for all experiments.</p><!><p>Spectral assignments of both RNAs were facilitated by predicted RNA chemical shifts and using established NOE helical "walk" patterns [20, 21]. NMR structural constraints were used in the final structure calculation (as discussed in SI Methods). A summary of the NMR restraints used in our structure calculation is provided in Table S1.</p><p>Xplor-NIH [22] was used for all structure calculations, based on a simulated annealing protocol (as discussed in SI Methods). The 10-structure ensemble was validated based on < 0.1 ppm chemical shift overlap between the NOEs from deuterated D2O NOESY spectra and predicted values for A-type helical base-pairs. Convergence was established when we observed no NOE violation greater than 0.5 Å or dihedral angle violations greater than 5 degrees for the majority of structures within the ensemble. The 3D structures were visualized with PyMol [23] or Chimera [24] and structural quality was analyzed using Molprobity [25].</p><!><p>For the determination of the structure of the DENV4 SLB, we transcribed the RNA with one additional terminal GC base pair (Figure 1A) to improve in vitro transcription and obtained spectra of high quality, consistent with a well-folded RNA displaying a single conformation in solution. We observed all the imino proton peaks expected from the predicted secondary structure, except for fast exchanging imino protons for unpaired nucleotides and nucleotides that are paired but at the end of helical stretches. The wobble pairs G6-U36 and U5-G37 were confirmed by the characteristic strong cross peaks between GNH1 and UNH3 within the non-canonical base-pairing range (12–10 ppm) (Figure S2). Starting from these distinctive peaks, we assigned all imino protons for DENV4 and fully established its secondary structure for further NMR studies. We then recorded a series of D2O 2D NOESY experiments with increasing mixing times (between 100 and 300 ms) in order to assign non-exchangeable protons. Even within the purine rich internal loop, bases stack on top to each other leading to the characteristic sequential pattern of NOEs expected for regular helices, except for residue G30. Extensive overlap in the NMR spectra of ribose protons was resolved by deuteration of the H3′, H4′ and H5′/5″ positions in the sugar and the H5 positions in C/U, which simplified the spectra and sharpened the linewidth by reducing dipolar relaxation considerably (Figure 2). With the aid of deuteration and 3D C13-edited NOESY spectra, we assigned 92.6 % of resonances (except for H3′, H4′, H5′ and H5″ peaks within A11, A21 and G22).</p><!><p>We prepared a construct of SLB from WNV with two additional single stranded GA nucleotides at its 5′-end (Figure S3) to improve in vitro transcription; because of the single stranded nature, the GA addition is not expected to affect the structure or stability of the stem-loop. However, the 2D TOCSY spectra of the RNA displayed multiple conformations and were not consistent with a single, well folded structure (Figure S3). Despite exhaustive efforts to optimize temperature, sequence and buffer conditions, this RNA remained recalcitrant to NMR structural studies.</p><p>To investigate the source of the conformational flexibility, we replaced the apical loop AGACUC with a highly stable C(UUCG)G tetraloop (TL) and added a GC base pair (as illustrated in Figure S4). The spectral quality for this construct improved greatly and prompted us to do further analysis (as shown in Figure S5). We observed all the imino peaks in the 1D NMR spectrum (Figure 1E), except for some rapidly exchanging protons for unpaired residues, and recorded NOESY spectra to obtain unambiguous assignment (Figure S6). Imino chemical shifts were assigned for each base pair, with the exception of U26H3, predicted to base pair with A13, which neighbors the flexible A25 and was probably broadened by rapid exchange with solvent. The wobble pair G20-U17 was confirmed by strong cross peaks between G20H1 and U17H3 within the non-canonical base-pairing range (12–10 ppm) (Figure S6). Thus, the secondary structure of the TL variant of WNV was confirmed with NMR studies as shown in Figure 1D.</p><p>To investigate the structure of the apical loop, we prepared another construct of 14 nts, which corresponds to the WNV SLB apical loop (Figure S4). Analysis of the imino spectra show that this very short truncated RNA form a stem-loop that exists in a single conformation in solution (Figure S7). Overlaying the NOESY NMR spectra of the 14 nt WNV on the UUCG-containing WNV RNA spectra revealed that chemical shifts and NOESY cross peaks for both aromatic to ribose sugar protons (Figure S8A) and ribose to ribose sugar protons (Figure S8B) are highly similar between the two constructs, except of course for the apical loop residues (AGCUC) that were replaced by a tetraloop (CUUCGG).</p><p>For WNV SLB, most of the data required for structure determination were collected with the C(UUCG)G-containing tetraloop construct because of improved quality of the NMR spectra, as illustrated in Figure 1 and 2, essentially as was done for Dengue 4 SLB. Deuteration of the H3′, H4′ and H5′/5″ positions in the sugar and H5 position simplified the spectra and reduced the linewidth by reducing dipolar relaxation considerably (Figure 2).</p><p>After collecting and analysing the 2D NOESY data in both H2O and D2O at multiple mixing times for the short 14 nts apical loop RNA, we were able to obtain spectral assignments and added the distance restraints obtained from this segment to the UUCG-containing WNV RNA, to generate a hybrid constrain list by merging the two sets of NOEs obtained for each RNA. Thus, we generated a restrained table for WNV SLB (Table S1) by combining distance and torsion angle restraints from each segment to generate a structure for the complete WNV SLB RNA. These restraints were used in the final structure calculation of SLB WNV. This strategy of segmental RNA assignment and structure determination and annealing allowed us to generate a structure for WNV SLB despite the challenges presented by its conformational flexibility and the presence of multiple conformations in exchange.</p><!><p>We generated a restraint table (Table S1) for WNV SLB by combining distance and torsion angle restraints from each segment to generate a structure for the complete RNA, which was calculated using a standard simulated annealing protocol (see SI methods) from NIH-XPLOR [28]. An ensemble of the 10 lowest energy structures from a refined set of 150 structures was chosen for subsequent analysis with heavy atom RMSD of 1.69 Å. The resultant structural statistics are described in Table S1.</p><p>Alignment of individual sections of the RNA were all below 1.00 Å, with the exception of the highly flexible apical loop (AGAUC), which converged more poorly due to the paucity of NOEs between loop residues and shows a higher RMSD value of 2.81 Å (Table S1), consistent with an unstructured loop. The lower helix is better defined (Figure 3) whereas the upper helix retains some flexibility in the region A26-U28, and converges to a higher RMSD. This suggest that the apical helix is only partially stable because of the presence of irregularity in the structure whereas the lower helix is more rigid (Figure 3).</p><p>The structure of WNB SLB revealed a relatively unremarkable stem-loop with three local distortions: the apical loop (AGAUC), as well as the A24 and U27 bulges. All nucleotides in WNV SLB have 3′-endo sugar pucker conformation which is common in A-form helical RNA except for the U27 bulged nucleotide which exhibited strong intensity H2′-H1′ and H3′-H1′ TOCSY correlations (Figure S11A), suggesting 2′-endo sugar pucker conformation. The U27 base is inserted between the G26-C11 and G10-C28 base pairs with the only deviation from A-form helical pattern found in the C28 backbone angles (α= −80, β= −120 and γ = −178), required to accommodate U27. Similarly, the A24 bulge also showed strong intensity H2′-H1′ and H3′-H1′ TOCSY correlations (Figure S11A) suggestive of the 2′-endo sugar pucker conformation.</p><p>We also generated a full set of restraints for DENV4 SLB (Table S1). A structural ensemble of 10 structures was chosen based on lowest energy score from a refinement set of 150 structures, with a 1.90 Å heavy atom RMSD. Structural statistics are reported in Table S1. Superposition of individual sections of the RNA are all below 1.00 Å, with the exception of the highly flexible apical loop, residues 19–23 (GCAGA), which converged poorly with a higher RMSD value of 2.75 Å due to conformational flexibility (Table S1). In the purine rich internal loop, residues G10, A11 and A12 stack on top to each other; likewise, residues A31 and A32 stack on top of each other, except for G30 which is bulged out, but no evidence of stable cross-strand base pairing was found.</p><p>In DENV4 SLB, the lower helix stem is well-defined and converges better than the upper helix (Figure 3), which retains flexibility in the regions A18-U24 and converges with a higher RMSD. All nucleotides in DENV4 SLB show 3′-endo sugar pucker conformation except for G19 and G23 which are part of apical loop and exhibited strong H2′-H1′ and H3′-H1′ TOCSY correlations (Figure S11B), suggesting 2′-endo sugar pucker conformation for these nucleotides, as well as G30, which is a part of internal loop and exhibited mixed 2′-endo/3′-endo sugar pucker conformation, based on relatively strong H1′ to H2′, H3′, and H4′ cross-peaks (Figure S11B) and also exhibited deviations from A-form helical pattern in its backbone angles (α= 154, β= 176 and γ =173).</p><!><p>To investigate the relationship between the structural stability of SLB and its function in viral replication, we investigated two mutants, M2C and M4C, in which UA base pairs of the DENV4 UFS were increasingly replaced with GC base pairs. The M2C mutant comprised two UA-to-GC base pair replacements, whereas the M4C mutant comprised four replacements (Figure 4). Both changes were shown to reduce replicative fitness significantly, the second change more so (Figure 4).</p><p>We performed variable temperature (VT) NMR studies between 5 °C–50 °C for WT, M2C and M4C. For DENV4 SLB, we observed that, after 30 °C RNA, base pairing begins to be disrupted and, after 40 °C, base pairing frays enough that imino protons are not protected from solvent exchange (Figure 4). For the M2C mutant, base pairs are only partially disrupted upto 50 °C. For the M4C mutant, the NMR data shows no disruption to base pairing even at 50 °C, indicating that M4C mutant is highly stable compared to M2C and WT DENV4. Thus, the temperature-dependent NMR results demonstrate that both mutants have enhanced structural stability. These results are consistent with a model where increased stability of the UFS duplex limits vRNA replication (Figure 4), as discussed below.</p><!><p>Flaviviruses utilize highly conserved RNA elements within their UTRs to affect various essential replicative steps as well as evolution, diversification and host adaption [6]. In particular, long-range interactions and genome cyclization are general features of positive-strand RNA viruses necessary for viral RNA (vRNA) replication. The structure and dynamic properties of these cis-acting RNA elements are essential aspects of their function [11] by allowing for conversion of the genome between linear and circular form in an efficient manner in response to selection pressure during adaptation to new host organisms [14]. The UA-rich region in the UFS and the UFS-like element in MBFVs, two species of NKVs, and ISFVs is evolutionarily conserved in flaviviruses [14, 15], and might have emerged when ancient adapted into vertebrate hosts [15].</p><p>We have established the SLB structures found in the 5′-UTRs of WNV and DENV4 using solution NMR spectroscopy. Within the stem-loop, the AU-rich region forms a double helix which sequesters the 5′-UAR/SLB element within its two strands. Mutants containing 2 or more UA to GC base pair substitutions affect the stability of the UFS duplex and we show (Figure 4) that mutations that compromise vRNA replication reduce the structural stability of the stem-loop.</p><p>It follows from these results that small molecules that bind to these RNAs and further stabilize their structure would reduce viral replicative fitness. Currently there are no established drug therapies for any flavivirus and the lack of treatment options is significant given the continued threat of emerging outbreaks around the globe. The development of antiviral therapeutics will facilitate novel approaches for the management of flavivirus outbreaks and perhaps even serve in prophylaxis. In this context, our structural studies represent a first step toward the identification of small molecules that target SLB's to reduce flavivirus replication and this work provides insight into a potential new drug target to reduce flavivirus replication rates.</p>