MDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA.

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.

A prenylated dsRNA sensor protects against severe COVID-19.

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.

An atomistic model of the coronavirus replication-transcription complex as a hexamer assembled around nsp15.

The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease.

Theoretical basis for stabilizing messenger RNA through secondary structure design.

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term 'superfolder' mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.

Structural Characterization of the Helicase nsp10 Encoded by Porcine Reproductive and Respiratory Syndrome Virus.

Currently, an effective therapeutic treatment for porcine reproductive and respiratory syndrome virus (PRRSV) remains elusive. PRRSV helicase nsp10 is an important component of the replication transcription complex that plays a crucial role in viral replication, making nsp10 an important target for drug development. Here, we report the first crystal structure of full-length nsp10 from the arterivirus PRRSV, which has multiple domains: an N-terminal zinc-binding domain (ZBD), a 1B domain, and helicase core domains 1A and 2A. Importantly, our structural analyses indicate that the conformation of the 1B domain from arterivirus nsp10 undergoes a dynamic transition. The polynucleotide substrate channel formed by domains 1A and 1B adopts an open state, which may create enough space to accommodate and bind double-stranded RNA (dsRNA) during unwinding. Moreover, we report a unique C-terminal domain structure that participates in stabilizing the overall helicase structure. Our biochemical experiments also showed that deletion of the 1B domain and C-terminal domain significantly reduced the helicase activity of nsp10, indicating that the four domains must cooperate to contribute to helicase function. In addition, our results indicate that nidoviruses contain a conserved helicase core domain and key amino acid sites affecting helicase function, which share a common mechanism of helicase translocation and unwinding activity. These findings will help to further our understanding of the mechanism of helicase function and provide new targets for the development of antiviral drugs.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory disease agent in pigs that causes enormous economic losses to the global swine industry. PRRSV helicase nsp10 is a multifunctional protein with translocation and unwinding activities and plays a vital role in viral RNA synthesis. Here, we report the first structure of full-length nsp10 from the arterivirus PRRSV at 3.0-Å resolution. Our results show that the 1B domain of PRRSV nsp10 adopts a novel open state and has a unique C-terminal domain structure, which plays a crucial role in nsp10 helicase activity. Furthermore, mutagenesis and structural analysis revealed conservation of the helicase catalytic domain across the order Nidovirales (families Arteriviridae and Coronaviridae). Importantly, our results will provide a structural basis for further understanding the function of helicases in the order Nidovirales.