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

In Vitro Reconstitution of SARS-Coronavirus mRNA Cap Methylation

SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5′ end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2′O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 7MeGpppA-RNAs. The latter are then selectively 2′O-methylated by the 2′O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 7MeGpppA2′OMe-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC50 values in the micromolar range, providing a validated basis for anti-coronavirus drug design

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

Structural and Functional Basis of the Fidelity of Nucleotide Selection by Flavivirus RNA-Dependent RNA Polymerases

International audienceViral RNA-dependent RNA polymerases (RdRps) play a central role not only in viral replication, but also in the genetic evolution of viral RNAs. After binding to an RNA template and selecting 5'-triphosphate ribonucleosides, viral RdRps synthesize an RNA copy according to Watson-Crick base-pairing rules. The copy process sometimes deviates from both the base-pairing rules specified by the template and the natural ribose selectivity and, thus, the process is error-prone due to the intrinsic (in)fidelity of viral RdRps. These enzymes share a number of conserved amino-acid sequence strings, called motifs A-G, which can be defined from a structural and functional point-of-view. A co-relation is gradually emerging between mutations in these motifs and viral genome evolution or observed mutation rates. Here, we review our current knowledge on these motifs and their role on the structural and mechanistic basis of the fidelity of nucleotide selection and RNA synthesis by Flavivirus RdRps

The RNA helicase, nucleotide 5′-triphosphatase, and RNA 5′-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

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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

An RNA cap (nucleoside-2′-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization

Viruses represent an attractive system with which to study the molecular basis of mRNA capping and its relation to the RNA transcription machinery. The RNA-dependent RNA polymerase NS5 of flaviviruses presents a characteristic motif of S-adenosyl-l-methionine-dependent methyltransferases at its N-terminus, and polymerase motifs at its C-terminus. The crystal structure of an N-terminal fragment of Dengue virus type 2 NS5 is reported at 2.4 Å resolution. We show that this NS5 domain includes a typical methyltransferase core and exhibits a (nucleoside-2′-O-)-methyltransferase activity on capped RNA. The structure of a ternary complex comprising S-adenosyl-l-homocysteine and a guanosine triphosphate (GTP) analogue shows that 54 amino acids N-terminal to the core provide a novel GTP-binding site that selects guanine using a previously unreported mechanism. Binding studies using GTP- and RNA cap-analogues, as well as the spatial arrangement of the methyltransferase active site relative to the GTP-binding site, suggest that the latter is a specific cap-binding site. As RNA capping is an essential viral function, these results provide a structural basis for the rational design of drugs against the emerging flaviviruses

Dengue Virus NS5 Transcribes Metabolite-Capped, RIG-I Sensitive vRNAs

International audienceEukaryotic nuclear and mitochondrial RNA polymerases (RNAPs) can cap RNAs by initiating with the non-canonical initiating nucleotide (NCIN) nicotinamide adenine dinucleotide (both NAD+ and NADH). Similarly, recently Dengue viral RNAs (vRNA) have been observed with NCIN caps, but how these vRNAs are capped is unknown. Here we show directly that Dengue virus's (DENV) RNAP NS5 can utilize both NAD+ and NADH, as well as flavin adenine dinucleotide and dephospho-coenzyme A, as initiating nucleotides during RNA transcription in place of DENV's well-conserved initiating adenine. In addition, we demonstrate that NCIN-capped dsRNAs can bind to the innate immune receptor RIG-I with KD's between 3 and 6 nM. RIG-I can also use these NCIN-capped dsRNAs to initiate robust interferon production. Taken with the previously published results that NAD+-capped mRNAs cannot be translated, we propose that NCIN-capped DENV vRNA represents a newly discovered mechanism of metabolite-mediated immunity that generates translation-deficient, highly immunogenic vRNAs

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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Arenaviridae NP-exonuclease inhibition by bisphosphonate

International audienceArenaviruses are emerging enveloped negative-sense RNA viruses which cause neurological and hemorrhagic diseases in humans. Currently there is no FDA approved vaccine or therapeutic agent except ribavirin which must be administered early during infection for optimum efficacy. A hallmark for arenavirus infection is a rapid and efficient immune-suppression mediated by the exonuclease domain encoded by the nucleoprotein. This exonuclease is therefore an attractive target for the design of novel antiviral drugs since exonuclease inhibitors might not only have a direct effect on the enzyme but could also boost viral clearance through stimulation of the innate immune system of the host cell. Here we have used an in silico screening and an enzymatic assay to identify a novel, specific but weak inhibitor of the arenavirus exonuclease with IC 50 values of 65.9 µM and 68.6 µM for Mopeia virus and Lymphocytic choriomeningitis virus, respectively. We further characterized this finding with crystallographic and docking approaches. This study serves as a proof of concept and may have assigned a new therapeutic purpose for the bisphosphonate family and therefore paves the way for the development of inhibitors against Arenaviridae