The structure of the binary methyltransferase-SAH complex from Zika virus reveals a novel conformation for the mechanism of mRNA capping

Zika virus, a flavivirus like Dengue and West Nile viruses, poses a significant risk as a pathogen in the category of emerging infectious diseases. Zika infections typically cause nonspecific, mild symptoms, but can also manifest as a neurological disorder like Guillain-Barré syndrome. Infection in pregnant women is linked to microcephaly in newborn infants. The methyltransferase domain of the non-structural protein 5 is responsible for two sequential methylations of the 5′-RNA cap. This is crucial for genome stability, efficient translation, and escape from the host immune response. Here we present the crystal structures of the Zika methyltransferase domain in complex with the methyl-donor SAM and its by-product SAH. The methyltransferase-SAH binary complex presents a new conformation of a “closed” or “obstructed” state that would restrict the binding of new RNA for capping. The combination and comparison of our new structures with recently published Zika methyltransferase structures provide a first glimpse into the structural mechanism of Zika virus mRNA capping

High-yield production of short GpppA- and (7Me)GpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2′O positions

Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, (7Me)G(5′)-ppp(5′)-A(2′OMe). The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and (7Me)GpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC(n) and (7Me)GpppAC(n) (1 ≤ n ≤ 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 ≤ n ≤ 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2′-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases

Divisible E-Cash from Constrained Pseudo-Random Functions

International audienceElectronic cash (e-cash) is the digital analogue of regular cash which aims at preservingusers’ privacy. Following Chaum’s seminal work, several new features were proposed for e-cash toaddress the practical issues of the original primitive. Among them,divisibilityhas proved very usefulto enable efficient storage and spendings. Unfortunately, it is also very difficult to achieve and, todate, quite a few constructions exist, all of them relying on complex mechanisms that can only beinstantiated in one specific setting. In addition security models are incomplete and proofs sometimeshand-wavy.In this work, we first provide a complete security model for divisible e-cash, and we study the linkswith constrained pseudo-random functions (PRFs), a primitive recently formalized by Boneh andWaters. We exhibit two frameworks of divisible e-cash systems from constrained PRFs achievingsome specific properties: either key homomorphism or delegability. We then formally prove theseframeworks, and address two main issues in previous constructions: two essential security notionswere either not considered at all or not fully proven. Indeed, we introduce the notion ofclearing,which should guarantee that only the recipient of a transaction should be able to do the deposit,and we show theexculpability, that should prevent an honest user to be falsely accused, was wrongin most proofs of the previous constructions. Some can easily be repaired, but this is not the casefor most complex settings such as constructions in the standard model. Consequently, we providethe first construction secure in the standard model, as a direct instantiation of our framework

Design, Synthesis and Discovery of N,N'-Carbazoyl-aryl-urea Inhibitors of Zika NS5 Methyltransferase and Virus Replication

The recent outbreaks of Zika virus (ZIKV) infection worldwide make the discovery of novel antivirals against flaviviruses a research priority. This work describes the identification of novel inhibitors of ZIKV through a structure‐based virtual screening approach using the ZIKV NS5‐MTase. A novel series of molecules with a carbazoyl‐aryl‐urea structure has been discovered and a library of analogues has been synthesized. The new compounds inhibit ZIKV MTase with IC50 between 23–48 μM. In addition, carbazoyl‐aryl‐ureas also proved to inhibit ZIKV replication activity at micromolar concentration

A Provably-Secure Unidirectional Proxy Re-Encryption Scheme Without Pairing in the Random Oracle Model

Proxy re-encryption (PRE) enables delegation of decryption rights by entrusting a proxy server with special information, that allows it to transform a ciphertext under one public key into a ciphertext of the same message under a different public key. It is important to note that, the proxy which performs the re-encryption learns nothing about the message encrypted under either public keys. Due to its transformation property, proxy re-encryption schemes have practical applications in distributed storage, encrypted email forwarding, Digital Rights Management (DRM) and cloud storage. From its introduction, several proxy re-encryption schemes have been proposed in the literature, and a majority of them have been realized using bilinear pairing. In Africacrypt 2010, the first PKI-based collusion resistant CCA secure PRE scheme without pairing was proposed in the random oracle model. In this paper, we point out an important weakness in the scheme. We also present the first collusion-resistant pairing-free unidirectional proxy re-encryption scheme which meets CCA security under a variant of the computational Diffie-Hellman hardness assumption in the random oracle model

Crystal structure of the RNA polymerase domain of the West Nile Virus non-structural protein 5

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue ( DENV) and West Nile ( WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase ( RdRp) activity of the non-structural protein 5 ( NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-angstrom resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNVRdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase ( MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain ( modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role

Structural biology in the fight against COVID-19

How can structural biology help us understand and combat SARS-CoV-2? Researchers in the field share their experiences and opinions and point to the challenges that lie ahead.Microscopic imaging and technolog