Deletion of the s2m RNA Structure in the Avian Coronavirus Infectious Bronchitis Virus and Human Astrovirus Results in Sequence Insertions.

Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.

Revealing druggable cryptic pockets in the Nsp1 of SARS-CoV-2 and other ß-coronaviruses by simulations and crystallography.

Non-structural protein 1 (Nsp1) is a main pathogenicity factor of α- and ß-coronaviruses. Nsp1 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suppresses the host gene expression by sterically blocking 40S host ribosomal subunits and promoting host mRNA degradation. This mechanism leads to the downregulation of the translation-mediated innate immune response in host cells, ultimately mediating the observed immune evasion capabilities of SARS-CoV-2. Here, by combining extensive molecular dynamics simulations, fragment screening and crystallography, we reveal druggable pockets in Nsp1. Structural and computational solvent mapping analyses indicate the partial crypticity of these newly discovered and druggable binding sites. The results of fragment-based screening via X-ray crystallography confirm the druggability of the major pocket of Nsp1. Finally, we show how the targeting of this pocket could disrupt the Nsp1-mRNA complex and open a novel avenue to design new inhibitors for other Nsp1s present in homologous ß-coronaviruses.

RNAdegformer: accurate prediction of mRNA degradation at nucleotide resolution with deep learning.

Messenger RNA-based therapeutics have shown tremendous potential, as demonstrated by the rapid development of messenger RNA based vaccines for COVID-19. Nevertheless, distribution of mRNA vaccines worldwide has been hampered by mRNA's inherent thermal instability due to in-line hydrolysis, a chemical degradation reaction. Therefore, predicting and understanding RNA degradation is a crucial and urgent task. Here we present RNAdegformer, an effective and interpretable model architecture that excels in predicting RNA degradation. RNAdegformer processes RNA sequences with self-attention and convolutions, two deep learning techniques that have proved dominant in the fields of computer vision and natural language processing, while utilizing biophysical features of RNA. We demonstrate that RNAdegformer outperforms previous best methods at predicting degradation properties at nucleotide resolution for COVID-19 mRNA vaccines. RNAdegformer predictions also exhibit improved correlation with RNA in vitro half-life compared with previous best methods. Additionally, we showcase how direct visualization of self-attention maps assists informed decision-making. Further, our model reveals important features in determining mRNA degradation rates via leave-one-feature-out analysis.

Parsing the role of NSP1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response.

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics.

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured "superfolder" mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

Degradation of SARS-CoV-2 specific ribonucleic acid in samples for nucleic acid amplification detection.

The degradation of SARS-CoV-2 specific ribonucleic acid (RNA) was investigated by a numerical modeling approach based on nucleic acid amplification test (NAAT) results utilizing the SmartAmp technique. The precision of the measurement was verified by the relative standard deviation (RSD) of repeated measurements at each calibration point. The precision and detection limits were found to be 6% RSD (seven repeated measurements) and 94 copies/tube, respectively, at the lowest calibration point. RNA degradation curves obtained from NAAT data on four different temperatures were in good agreement with the first-order reaction model. By referring to rate constants derived from the results, the Arrhenius model was applied to predict RNA degradation behavior. If the initial RNA concentration was high enough, such as in samples taken from infected bodies, the NAAT results were expected to be positive during testing. On the other hand, if initial RNA concentrations were relatively low, such as RNA in residual viruses on environmental surfaces, special attention should be paid to avoid false-negative results. The results obtained in this study provide a practical guide for RNA sample management in the NAAT of non-human samples.

Chemical modifications to mRNA nucleobases impact translation elongation and termination.

Messenger RNAs (mRNAs) serve as blueprints for protein synthesis by the molecular machine the ribosome. The ribosome relies on hydrogen bonding interactions between adaptor aminoacyl-transfer RNA molecules and mRNAs to ensure the rapid and faithful translation of the genetic code into protein. There is a growing body of evidence suggesting that chemical modifications to mRNA nucleosides impact the speed and accuracy of protein synthesis by the ribosome. Modulations in translation rates have downstream effects beyond protein production, influencing protein folding and mRNA stability. Given the prevalence of such modifications in mRNA coding regions, it is imperative to understand the consequences of individual modifications on translation. In this review we present the current state of our knowledge regarding how individual mRNA modifications influence ribosome function. Our comprehensive comparison of the impacts of 16 different mRNA modifications on translation reveals that most modifications can alter the elongation step in the protein synthesis pathway. Additionally, we discuss the context dependence of these effects, highlighting the necessity of further study to uncover the rules that govern how any given chemical modification in an mRNA codon is read by the ribosome.

Systematic identification of NF90 target RNAs by iCLIP analysis.

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNAs directing numerous essential roles in cellular physiology. Nuclear Factor 90 (NF90) is an RBP encoded by the interleukin enhancer-binding factor 3 (ILF3) gene that has been found to influence RNA metabolism at several levels, including pre-RNA splicing, mRNA turnover, and translation. To systematically identify the RNAs that interact with NF90, we carried out iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) analysis in the human embryonic fibroblast cell line HEK-293. Interestingly, many of the identified RNAs encoded proteins involved in the response to viral infection and RNA metabolism. We validated a subset of targets and investigated the impact of NF90 on their expression levels. Two of the top targets, IRF3 and IRF9 mRNAs, encode the proteins IRF3 and IRF9, crucial regulators of the interferon pathway involved in the SARS-CoV-2 immune response. Our results support a role for NF90 in modulating key genes implicated in the immune response and offer insight into the immunological response to the SARS-CoV-2 infection.

Chemically modified mRNA beyond COVID-19: Potential preventive and therapeutic applications for targeting chronic diseases.

Chemically modified mRNA represents a unique, efficient, and straightforward approach to produce a class of biopharmaceutical agents. It has been already approved as a vaccination-based method for targeting SARS-CoV-2 virus. The COVID-19 pandemic has highlighted the prospect of synthetic modified mRNA to efficiently and safely combat various diseases. Recently, various optimization advances have been adopted to overcome the limitations associated with conventional gene therapeutics leading to wide-ranging applications in different disease conditions. This review sheds light on emerging directions of chemically modified mRNAs to prevent and treat widespread chronic diseases, including metabolic disorders, cancer vaccination and immunotherapy, musculoskeletal disorders, respiratory conditions, cardiovascular diseases, and liver diseases.

Discovery and initial characterization of YloC, a novel endoribonuclease in Bacillus subtilis.

The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3' exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3' exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved "YicC family" of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.

A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems.

Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.

SARS-CoV-2 RNA stability in dry swabs for longer storage and transport at different temperatures.

During the current COVID-19 pandemic, different methods have been used to evaluate patients with suspected SARS-CoV-2 infection. In this study, we experimentally evaluate the ability of spiked saliva-moist swabs and spiked swabs without any transport medium to retain SARS-CoV-2 for storage and transport at different environmental settings during different incubation time periods. Our results show that at ambient temperature of 20°C, SARS-CoV-2 RNA remains stable for up to 9 days allowing a long-time span for transport and storage without compromising clinical results. Additionally, this study demonstrates that saliva-moist swabs can also be stored at -20°C and +4°C for up to 26 days without affecting RT-qPCR results. Our data are relevant for low-and middle-income countries, which have limited access to rapid refrigerated transport and storage of samples representing an economical alternative. Finally, our study demonstrates the practical and economic advantage of using swabs without transport medium.

The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression.

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.

Duplex formation between the template and the nascent strand in the transcription-regulating sequences is associated with the site of template switching in SARS - CoV-2.

Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis, where the polymerase switches template from a 3' proximal genome body sequence to a 5' untranslated leader sequence. This leads to a fusion between the common 5' leader sequence and a 3' proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesizing the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs. We speculate on the effect of reported variant nucleotide substitutions on our models.

Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2'-O-methyl transfer by SARS-CoV-2 nsp16.

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.

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.

SARS-CoV-2 infection triggers widespread host mRNA decay leading to an mRNA export block.

The transcriptional induction of interferon (IFN) genes is a key feature of the mammalian antiviral response that limits viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Here, we performed single-molecule RNA visualization to examine the expression and localization of host mRNAs during SARS-CoV-2 infection. Our data show that the biogenesis of type I and type III IFN mRNAs is inhibited at multiple steps during SARS-CoV-2 infection. First, translocation of the interferon regulatory factor 3 (IRF3) transcription factor to the nucleus is limited in response to SARS-CoV-2, indicating that SARS-CoV-2 inhibits RLR-MAVS signaling and thus weakens transcriptional induction of IFN genes. Second, we observed that IFN mRNAs primarily localize to the site of transcription in most SARS-CoV-2 infected cells, suggesting that SARS-CoV-2 either inhibits the release of IFN mRNAs from their sites of transcription and/or triggers decay of IFN mRNAs in the nucleus upon exiting the site of transcription. Lastly, nuclear-cytoplasmic transport of IFN mRNAs is inhibited during SARS-CoV-2 infection, which we propose is a consequence of widespread degradation of host cytoplasmic basal mRNAs in the early stages of SARS-CoV-2 replication by the SARS-CoV-2 Nsp1 protein, as well as the host antiviral endoribonuclease, RNase L. Importantly, IFN mRNAs can escape SARS-CoV-2-mediated degradation if they reach the cytoplasm, making rescue of mRNA export a viable means for promoting the immune response to SARS-CoV-2.