The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain

International audienceViral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltrans-ferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conforma-tions during RNA synthesis

Molecular Basis for Nucleotide Conservation at the Ends of the Dengue Virus Genome

International audienceThe dengue virus (DV) is an important human pathogen from the Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5PolDV of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5PolDV contains a specific priming site for adenosine 59-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn 2+ is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5PolDV ensures the conservation of the 59-adenosine by strongly discriminating against viral templates containing an erroneous 39-end nucleotide in the presence of Mg 2+. In the presence of Mn2+, NS5Pol DV is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo

Insights into the RNA polymerase activity of the dengue virus NS5

Le virus de la dengue cause une maladie de type grippal qui peut dans certains cas évoluer vers des fièvreshémorragiques mortelles. Mon projet de thèse porte sur la réplication de ce virus. Je focalise sur la compréhension du mécanisme d'action de la protéine NS5 de ce virus. La protéine contient 2 domaines : 1) domaine méthyltransférase, essentiel pour la traduction des protéines virales, 2) domaine polymérase, synthétisant le génome ARN du virus. Premièrement, nous avons démontré que la polymérase joue un rôle principal dans la conservation de l'extrémité 3' et 5' du génome et de l'anti-génome. Puis, j'ai caractérisé l'influence du domaine méthyltransférase sur l'activité polymérase de la protéine NS5. J'ai développé un système d'études mécanistiques en utilisant des techniques biochimiques de cinétique pré-stationnaire pour la protéine NS5, et obtenu des paramètres cinétiques et thermodynamiques de cette protéine envers ses substrats. Avec ce même système, j'ai pu tester des activités de la polymérase NS5 avec des ARN coiffés et triphosphates de différente longueur, mimant les séquences à l'extrémité 5' du génome du virus de la dengue. L'activité polymérase de NS5 est influencée par la présence de la coiffe de l'ARN, ce qui m'a permis de proposer une distance physique correspondant à environ 13 nucléotides entre les sites actifs domaines méthyltransférase et polymérase. Mes travaux ouvrent la voie à la détermination de la structure 3D de NS5 avec ses ARN et des nucléotides 5'-triphosphate.Elucider son mécanisme d'action, c'est être capable d'inhiber son action et donc de pouvoir proposer des molécules capables d'arrêter la prolifération virale lors d'une infection.Dengue virus causes dengue fever, which may evolve towards life-threatening hemorrhagic fever. My research projectfocuses on dengue replication, and more precisely on the mechanism of NS5 at the molecular/atomic level. NS5 is a bifunctionalenzyme containing two domains: 1) a methyltransferase domain essential for translation of viral proteins, 2) apolymerase domain synthesizing the viral RNA genome. First, we demonstrated the main role of the polymerase in theconservation of 5' and 3' ends of dengue genome and anti-genome RNAs. Next, I showed the influence of themethyltransferase domain on the activity of the polymerase domain. I also developed a system allowing mechanistic studiesusing pre-steady state kinetics to characterize NS5 in depth. I have made use of this system to determine the catalyticparameters of NS5 towards its substrates. Using the same pre-steady state system, I was able to test the polymerase activityof NS5 with capped and uncapped 5'-triphosphate RNAs of different lengths corresponding to the 5'-end of the dengue RNAgenome. The polymerase activity of NS5 is significantly affected by the presence of the 5'-cap, which allowed me to designan experimental set-up pointing to a minimal physical distance of around 13 nucleotides between the methyltransferase andpolymerase active sites. My work will be useful to characterize the biophysics of NS5 in complex with its RNA and NTPsubstrates, and then to determine the crystal structure of such complex at play during viral RNA synthesis. Knowing thedetailed NS5 mechanism paves the way to inhibit its action and thus design drugs aiming at stopping a viral infection

Substrate selectivity of Dengue and Zika virus NS5 polymerase towards 2′-modified nucleotide analogues

International audienceIn targeting the essential viral RNA-dependent RNA-polymerase (RdRp), nucleotide analogues play a major role in antiviral therapies. In the Flaviviridae family, the hepatitis C virus (HCV) can be eradicated from chronically infected patients using a combination of drugs which generally include the 2'-modified uridine analogue Sofosbuvir, delivered as nucleotide prodrug. Dengue and Zika viruses are emerging flaviviruses whose RdRp is closely related to that of HCV, yet no nucleoside drug has been clinically approved for these acute infections. We have purified dengue and Zika virus full-length NS5, the viral RdRps, and used them to assemble a stable binary complex made of NS5 and virus-specific RNA primer/templates. The complex was used to assess the selectivity of NS5 towards nucleotide analogues bearing modifications at the 2'-position. We show that dengue and Zika virus RdRps exhibit the same discrimination pattern: 2'-O-Me > 2'-C-Me-2'-F > 2'-C-Me nucleoside analogues, unlike HCV RdRp for which the presence of the 2'-F is beneficial rendering the discrimination pattern 2'-O-Me > 2'-C-Me ≥ 2'-C-Me-2'-F. Both 2'-C-Me and 2'-C-Me-2'-F analogues act as non-obligate RNA chain terminators. The dengue and Zika NS5 nucleotide selectivity towards 2'-modified NTPs mirrors potency of the corresponding analogues in infected cell cultures

The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain (vol 42, pg 11642, 2014)

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Specificity for ATP as the initiating nucleotide.

<p>(<b>A</b>) Specific pppAG dinucleotide formation by NS5Pol_DV in the presence of Mg²⁺ on DV₁₀3′- templates (ACUAACAA-CU) with varying last nucleotides: lane 1 -CU, lanes 2 and 3 -CC, lane 4 -CA and lane 5 -CG) in presence of Mg²⁺. Corresponding initiating NTPs and GTP were used as substrates. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 5 mM MgCl₂, 500 nM NS5Pol_DV, 10 µM template, 500 µM of initiating NTPs, and 100 µM GTP (containing αGTP). For the reaction on the -CC template, 300 µM (lane 2) and 600 µM GTP (lane 3) was used. Reactions were started by the addition of MgCl₂ and incubated for 2 h. Samples were analyzed using PAGE and autoradiography. (<b>B</b>) pppAG dinucleotide formation by NS5Pol_DV in the presence of Mn²⁺. Reaction mixtures contained 2 mM MnCl₂, 500 nM NS5Pol_DV, 500 µM GTP, and 100 µM ATP (containing αATP) and either no template (lane 1), 1 µM DV₁₀3′+ (lane 2), or 1 µM DV₁₀3′- (lane 3). Reactions were started by the addition of MnCl₂ and incubated for 2 h. The identity of product bands is given on the right. (<b>C</b>) Specific non-templated pppAG dinucleotide formation and non-specific NG dinucleotide formation on DV₁₀3′- template variants (see under <b>A</b>) in the presence of Mn²⁺. Reaction mixtures contained 2 mM MnCl₂, 500 nM NS5Pol_DV, 1 µM template, 500 µM of NTPs, which were not labeled, and 100 µM GTP (containing either αGTP or γGTP as outlined below the gel) and either no template or DV₁₀3′- variants (given below the gel). Reactions were started by the addition MnCl₂ and samples were taken at given time points. The identity of product bands is given on the right side of the reaction kinetics.</p

Dengue virus RdRp conserves the correct 5′- and 3′-ends of the genome.

<p>DV RdRp conducts strict ATP-specific <i>de novo</i> initiation in the absence of a template and in the presence of the correct template using the indicated catalytic ions Mn²⁺ or Mg²⁺. The pppAG primer is then elongated. When DV RdRp encounters templates with incorrect 3′-end nucleotides it refuses <i>de novo</i> initiation (when Mg²⁺ is present) or corrects the error by preferentially generating and elongating pppAG (using Mn²⁺ as catalytic ion). The structure of the DV2 RdRp domain is shown in the background.</p

Role of the predicted priming loop T794-A799 in correct <i>de novo</i> initiation.

<p>(<b>A</b>) 3D-structural model of NS5Pol_DV used in this study (DV serotype 2 strain New Guinea C) derived from the structure of serotype 3 NS5Pol_DV (PDB code 2J7W <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#ppat.1002912-Yap1" target="_blank">[16]</a>). NS5Pol_DV adopts the typical closed right-hand structure of RdRps containing the palm (light green), fingers (light blue) and thumb (red) subdomains. Between fingers and thumb subdomains the template tunnel runs down to the active site harbored mainly by the palm subdomain. The side chains of the three conserved catalytic residues D533 (motif A), D663 and D664 (motif C) in the active site are shown in sticks (C-atoms light green, O-atoms red). The priming loop emerges from the thumb subdomain and closes the dsRNA exit tunnel and the active site. The close-up shows aromatic residues W795 and H798 (in sticks) within the putative priming loop T794 to A799. In the mutant TGGK the priming loop was replaced by two glycines situated between T793 and K800 (in sticks). (<b>B</b>) Activity of wt NS5Pol_DV and its deletion mutant TGGK NS5Pol_DV was determined on a specific minigenomic template. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a>. Initial velocities in cpm/min determined by filter-binding assays in the presence of [³H]-UTP and liquid scintillation counting, are compared in the presence of Mg²⁺ (left panel) and Mn²⁺ (right panel). The center panel shows agarose-formaldehyde gel analysis of reaction kinetics in the presence of [α-³²P]-UTP and Mg²⁺ ions. Product bands are labeled on the right sight of the gel. (<b>C</b>) <i>De novo</i> initiation of wt NS5Pol_DV and its deletion mutant TGGK was followed in the presence of Mn²⁺ using either 1 µM DV₁₀3′-, in the absence of a template, or 1 µM DV₁₀3′+ (from left to right as indicated). Reaction mixtures also contained 2 mM MnCl₂, 500 nM enzyme, 500 µM of NTPs, which were not labeled, and 100 µM labeled NTP (containing αGTP or γATP as indicated). Reactions were started by addition of MnCl₂ and samples were taken at given time points. Identities of labeled product bands are given on the right and left side of the reaction kinetics. pppGA and pppGAA internal <i>de novo</i> initiation products on DV₁₀3′+ are labeled by an asterisk.</p

pppAG-elongation on the correct antigenome 3′-end and on variants with an incorrect last nucleotide.

<p>pppAG-elongation by NS5Pol_DV on DV₁₀3′- templates (ACUAACAA-CN) varying the last nucleotide (correct -CU versus -CC, -CA and -CG). Control reactions were included without template. (<b>A</b>) pppAG elongation in the presence of Mn²⁺. pppAG (100 µM) and UTP (100 µM, containing αUTP) were used as substrates. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 2 mM MnCl₂, 500 nM NS5Pol_DV, and 1 µM template. Reactions were started by addition of MnCl₂ and UTP. Samples were taken at given time points and analyzed by PAGE and autoradiography. OligoG marker is shown on the left, the identity of product bands is given on the right. (<b>B</b>) pppAG-elongation in the presence of Mg²⁺. pppAG (100 µM) and UTP (100 µM, containing αUTP) were used as substrates. Reaction mixtures were prepared as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002912#s4" target="_blank">Materials and Methods</a> plus 5 mM MgCl₂, 5 µM NS5Pol_DV, and 1 µM template. Reactions were started by addition of MgCl₂ and UTP. Samples were taken at given time points and analyzed by PAGE and autoradiography. OligoG marker is shown on the right; the identity of product bands is given on the left.</p