Multi-omics characterization of RNA binding proteins reveals disease comorbidities and potential drugs in COVID-19.

The COVID-19 has led to a devastating global health crisis, which emphasizes the urgent need to deepen our understanding of the molecular mechanism and identifying potential antiviral drugs. Here, we comprehensively analyzed the transcriptomic and proteomic profiles of 178 COVID-19 patients, ranging from asymptomatic to critically ill. Our analyses found that the RNA binding proteins (RBPs) were likely to be perturbed in infection. Interactome analysis revealed that RBPs interact with virus proteins and the viral interacting RBPs were likely to locate in central regions of human protein-protein interaction network. Functional enrichment analysis revealed that the viral interacting RBPs were likely to be enriched in RNA transport, apoptosis and viral genome replication-related pathways. Based on network proximity analyses of 299 human complex-disease genes and COVID-19-related RBPs in the human interactome, we revealed the significant associations between complex diseases and COVID-19. Network analysis also implicated potential antiviral drugs for treatment of COVID-19. In summary, our integrative characterization of COVID-19 patients may thus help providing evidence regarding pathophysiology and potential therapeutic strategies for COVID-19.

SARS-CoV-2 Z-RNA activates the ZBP1-RIPK3 pathway to promote virus-induced inflammatory responses.

SARS-CoV-2 infection can trigger strong inflammatory responses and cause severe lung damage in COVID-19 patients with critical illness. However, the molecular mechanisms by which the infection induces excessive inflammatory responses are not fully understood. Here, we report that SARS-CoV-2 infection results in the formation of viral Z-RNA in the cytoplasm of infected cells and thereby activates the ZBP1-RIPK3 pathway. Pharmacological inhibition of RIPK3 by GSK872 or genetic deletion of MLKL reduced SARS-CoV-2-induced IL-1ß release. ZBP1 or RIPK3 deficiency leads to reduced production of both inflammatory cytokines and chemokines during SARS-CoV-2 infection both in vitro and in vivo. Furthermore, deletion of ZBP1 or RIPK3 alleviated SARS-CoV-2 infection-induced immune cell infiltration and lung damage in infected mouse models. These results suggest that the ZBP1-RIPK3 pathway plays a critical role in SARS-CoV-2-induced inflammatory responses and lung damage. Our study provides novel insights into how SARS-CoV-2 infection triggers inflammatory responses and lung pathology, and implicates the therapeutic potential of targeting ZBP1-RIPK3 axis in treating COVID-19.

ZBP1: A Powerful Innate Immune Sensor and Double-Edged Sword in Host Immunity.

Z-conformation nucleic acid binding protein 1 (ZBP1), a powerful innate immune sensor, has been identified as the important signaling initiation factor in innate immune response and the multiple inflammatory cell death known as PANoptosis. The initiation of ZBP1 signaling requires recognition of left-handed double-helix Z-nucleic acid (includes Z-DNA and Z-RNA) and subsequent signaling transduction depends on the interaction between ZBP1 and its adapter proteins, such as TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), receptor-interacting serine/threonine-protein kinase 1 (RIPK1), and RIPK3. ZBP1 activated innate immunity, including type-I interferon (IFN-I) response and NF-κB signaling, constitutes an important line of defense against pathogenic infection. In addition, ZBP1-mediated PANoptosis is a double-edged sword in anti-infection, auto-inflammatory diseases, and tumor immunity. ZBP1-mediated PANoptosis is beneficial for eliminating infected cells and tumor cells, but abnormal or excessive PANoptosis can lead to a strong inflammatory response that is harmful to the host. Thus, pathogens and host have each developed multiplex tactics targeting ZBP1 signaling to maintain strong virulence or immune homeostasis. In this paper, we reviewed the mechanisms of ZBP1 signaling, the effects of ZBP1 signaling on host immunity and pathogen infection, and various antagonistic strategies of host and pathogen against ZBP1. We also discuss existent gaps regarding ZBP1 signaling and forecast potential directions for future research.

Discovery of Genes that Modulate Flavivirus Replication in an Interferon-Dependent Manner.

Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions induced by IFN. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent manner, opening new perspectives to target weakness points in the life cycle of these viruses.

IFITM3 restricts virus-induced inflammatory cytokine production by limiting Nogo-B mediated TLR responses.

Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that limits viral pathogenesis and exerts poorly understood immunoregulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 promotes MyD88-dependent, TLR-mediated IL-6 production following exposure to cytomegalovirus (CMV). IFITM3 also restricts IL-6 production in response to influenza and SARS-CoV-2. In dendritic cells, IFITM3 binds to the reticulon 4 isoform Nogo-B and promotes its proteasomal degradation. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesis in vivo, and in the case of TLR2 responses, this process involves alteration of TLR2 cellular localization. Nogo-B deletion abrogates inflammatory cytokine responses and associated disease in virus-infected IFITM3-deficient mice. Thus, we uncover Nogo-B as a driver of viral pathogenesis and highlight an immunoregulatory pathway in which IFITM3 fine-tunes the responsiveness of myeloid cells to viral stimulation.

LRTCLS: low-rank tensor completion with Laplacian smoothing regularization for unveiling the post-transcriptional machinery of N6-methylation (m6A)-mediated diseases.

Recently, N6-methylation (m6A) has recently become a hot topic due to its key role in disease pathogenesis. Identifying disease-related m6A sites aids in the understanding of the molecular mechanisms and biosynthetic pathways underlying m6A-mediated diseases. Existing methods treat it primarily as a binary classification issue, focusing solely on whether an m6A-disease association exists or not. Although they achieved good results, they all shared one common flaw: they ignored the post-transcriptional regulation events during disease pathogenesis, which makes biological interpretation unsatisfactory. Thus, accurate and explainable computational models are required to unveil the post-transcriptional regulation mechanisms of disease pathogenesis mediated by m6A modification, rather than simply inferring whether the m6A sites cause disease or not. Emerging laboratory experiments have revealed the interactions between m6A and other post-transcriptional regulation events, such as circular RNA (circRNA) targeting, microRNA (miRNA) targeting, RNA-binding protein binding and alternative splicing events, etc., present a diverse landscape during tumorigenesis. Based on these findings, we proposed a low-rank tensor completion-based method to infer disease-related m6A sites from a biological standpoint, which can further aid in specifying the post-transcriptional machinery of disease pathogenesis. It is so exciting that our biological analysis results show that Coronavirus disease 2019 may play a role in an m6A- and miRNA-dependent manner in inducing non-small cell lung cancer.

Differential Leukocyte Expression of IFITM1 and IFITM3 in Patients with Severe Pandemic Influenza A(H1N1) and COVID-19.

Interferon-induced transmembrane (IFITM) proteins mediate protection against enveloped viruses by blocking membrane fusion at endosomes. IFITM1 and IFITM3 are crucial for protection against influenza, and various single nucleotide polymorphisms altering their function have been linked to disease susceptibility. However, bulk IFITM1 and IFITM3 mRNA expression dynamics and their correlation with clinical outcomes have not been extensively addressed in patients with respiratory infections. In this study, we evaluated the expression of IFITM1 and IFITM3 in peripheral leukocytes from healthy controls and individuals with severe pandemic influenza A(H1N1) or coronavirus disease 2019 (COVID-19). Comparisons between participants grouped according to their clinical characteristics, underlying disease, and outcomes showed that the downregulation of IFITM1 was a distinctive characteristic of severe pandemic influenza A(H1N1) that correlated with outcomes, including mortality. Conversely, increased IFITM3 expression was a common feature of severe pandemic influenza A(H1N1) and COVID-19. Using a high-dose murine model of infection, we confirmed not only the downregulation of IFITM1 but also of IFITM3 in the lungs of mice with severe influenza, as opposed to humans. Analyses in the comparative cohort also indicate the possible participation of IFITM3 in COVID-19. Our results add to the evidence supporting a protective function of IFITM proteins against viral respiratory infections in humans.

Investigating the Human Host-ssRNA Virus Interaction Landscape Using the SMEAGOL Toolbox.

Viruses have evolved numerous mechanisms to exploit the molecular machinery of their host cells, including the broad spectrum of host RNA-binding proteins (RBPs). However, the RBP interactomes of most viruses are largely unknown. To shed light on the interaction landscape of RNA viruses with human host cell RBPs, we have analysed 197 single-stranded RNA (ssRNA) viral genome sequences and found that the majority of ssRNA virus genomes are significantly enriched or depleted in motifs for specific human RBPs, suggesting selection pressure on these interactions. To facilitate tailored investigations and the analysis of genomes sequenced in future, we have released our methodology as a fast and user-friendly computational toolbox named SMEAGOL. Our resources will contribute to future studies of specific ssRNA virus-host cell interactions and support the identification of antiviral drug targets.

The SARS-CoV-2 targeted human RNA binding proteins network biology to investigate COVID-19 associated manifestations.

The global coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus has had unprecedented social and economic ramifications. Identifying targets for drug repurposing could be an effective means to present new and fast treatments. Furthermore, the risk of morbidity and mortality from COVID-19 goes up when there are coexisting medical conditions, however, the underlying mechanisms remain unclear. In the current study, we have adopted a network-based systems biology approach to investigate the RNA binding proteins (RBPs)-based molecular interplay between COVID-19, various human cancers, and neurological disorders. The network based on RBPs commonly involved in the three disease conditions consisted of nine RBPs connecting 10 different cancer types, 22 brain disorders, and COVID-19 infection, ultimately hinting at the comorbidities and complexity of COVID-19. Further, we underscored five miRNAs with reported antiviral properties that target all of the nine shared RBPs and are thus therapeutically valuable. As a strategy to improve the clinical conditions in comorbidities associated with COVID-19, we propose perturbing the shared RBPs by drug repurposing. The network-based analysis presented hereby contributes to a better knowledge of the molecular underpinnings of the comorbidities associated with COVID-19.

Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures.

Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.

ZAP isoforms regulate unfolded protein response and epithelial- mesenchymal transition.

Human ZAP inhibits many viruses, including HIV and coronaviruses, by binding to viral RNAs to promote their degradation and/or translation suppression. However, the regulatory role of ZAP in host mRNAs is largely unknown. Two major alternatively spliced ZAP isoforms, the constitutively expressed ZAPL and the infection-inducible ZAPS, play overlapping yet different antiviral and other roles that need further characterization. We found that the splicing factors hnRNPA1/A2, PTBP1/2, and U1-snRNP inhibit ZAPS production and demonstrated the feasibility to modulate the ZAPL/S balance by splice-switching antisense oligonucleotides in human cells. Transcriptomic analysis of ZAP-isoform-specific knockout cells revealed uncharacterized host mRNAs targeted by ZAPL/S with broad cellular functions such as unfolded protein response (UPR), epithelial-mesenchymal transition (EMT), and innate immunity. We established that endogenous ZAPL and ZAPS localize to membrane compartments and cytosol, respectively, and that the differential localization correlates with their target-RNA specificity. We showed that the ZAP isoforms regulated different UPR branches under resting and stress conditions and affected cell viability during ER stress. We also provided evidence for a different function of the ZAP isoforms in EMT-related cell migration, with effects that are cell-type dependent. Overall, this study demonstrates that the competition between splicing and IPA is a potential target for the modulation of the ZAPL/S balance, and reports new cellular transcripts and processes regulated by the ZAP isoforms.

Positive Regulation of the Antiviral Activity of Interferon-Induced Transmembrane Protein 3 by S-Palmitoylation.

The interferon-induced transmembrane protein 3 (IFITM3), a small molecule transmembrane protein induced by interferon, is generally conserved in vertebrates, which can inhibit infection by a diverse range of pathogenic viruses such as influenza virus. However, the precise antiviral mechanisms of IFITM3 remain unclear. At least four post-translational modifications (PTMs) were found to modulate the antiviral effect of IFITM3. These include positive regulation provided by S-palmitoylation of cysteine and negative regulation provided by lysine ubiquitination, lysine methylation, and tyrosine phosphorylation. IFITM3 S-palmitoylation is an enzymatic addition of a 16-carbon fatty acid on the three cysteine residues within or adjacent to its two hydrophobic domains at positions 71, 72, and 105, that is essential for its proper targeting, stability, and function. As S-palmitoylation is the only PTM known to enhance the antiviral activity of IFITM3, enzymes that add this modification may play important roles in IFN-induced immune responses. This study mainly reviews the research progresses on the antiviral mechanism of IFITM3, the regulation mechanism of S-palmitoylation modification on its subcellular localization, stability, and function, and the enzymes that mediate the S-palmitoylation modification of IFITM3, which may help elucidate the mechanism by which this IFN effector restrict virus replication and thus aid in the design of therapeutics targeted at pathogenic viruses.

The interplay between lncRNAs, RNA-binding proteins and viral genome during SARS-CoV-2 infection reveals strong connections with regulatory events involved in RNA metabolism and immune response.

Rationale: Viral infections are complex processes based on an intricate network of molecular interactions. The infectious agent hijacks components of the cellular machinery for its profit, circumventing the natural defense mechanisms triggered by the infected cell. The successful completion of the replicative viral cycle within a cell depends on the function of viral components versus the cellular defenses. Non-coding RNAs (ncRNAs) are important cellular modulators, either promoting or preventing the progression of viral infections. Among these ncRNAs, the long non-coding RNA (lncRNA) family is especially relevant due to their intrinsic functional properties and ubiquitous biological roles. Specific lncRNAs have been recently characterized as modulators of the cellular response during infection of human host cells by single stranded RNA viruses. However, the role of host lncRNAs in the infection by human RNA coronaviruses such as SARS-CoV-2 remains uncharacterized. Methods: In the present work, we have performed a transcriptomic study of a cohort of patients with different SARS-CoV-2 viral load and analyzed the involvement of lncRNAs in supporting regulatory networks based on their interaction with RNA-binding proteins (RBPs). Results: Our results revealed the existence of a SARS-CoV-2 infection-dependent pattern of transcriptional up-regulation in which specific lncRNAs are an integral component. To determine the role of these lncRNAs, we performed a functional correlation analysis complemented with the study of the validated interactions between lncRNAs and RBPs. This combination of in silico functional association studies and experimental evidence allowed us to identify a lncRNA signature composed of six elements - NRIR, BISPR, MIR155HG, FMR1-IT1, USP30-AS1, and U62317.2 - associated with the regulation of SARS-CoV-2 infection. Conclusions: We propose a competition mechanism between the viral RNA genome and the regulatory lncRNAs in the sequestering of specific RBPs that modulates the interferon response and the regulation of RNA surveillance by nonsense-mediated decay (NMD).

Bovine lactoferrin increases the poly(I:C)-induced antiviral response in vitro.

Bovine lactoferrin (bLF) is a naturally occurring glycoprotein with antibacterial and antiviral activities. We evaluated whether bLF can prevent viral infections in the human intestinal epithelial cell line Caco-2. To assess antiviral responses, we measured the levels of interferon (IFN) expression, IFN-stimulated gene expression, and infection with a pseudotyped virus bearing either severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or vesicular stomatitis virus (VSV)-G protein after treatment of cells with both bLF and polyinosinic-polycytidylic acid, an analog of double-stranded RNA that mimics viral infection. Combination treatment of cells with both bLF and polyinosinic-polycytidylic acid increased mRNA and protein expression of several IFN genes (IFNB, IFNL1, and IFNL2) and IFN-stimulated genes (ISG15, MX1, IFITM1, and IFITM3) in Caco-2 cells. However, treatment with bLF alone did not induce an antiviral response. Furthermore, combination treatment suppressed infection of the SARS-CoV-2 pseudotyped virus more efficiently than did bLF treatment alone, even though combination treatment increased the expression of mRNA encoding ACE2. These results indicate that bLF increases the antiviral response associated with the double-stranded RNA-stimulated signaling pathway. Our results also suggest that bLF and double-stranded RNA analogs can be used to treat viral infections, including those caused by SARS-CoV-2.

SARS-CoV-2 couples evasion of inflammatory response to activated nucleotide synthesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-κB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized small-molecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes.

SARS-CoV-2 N Protein Antagonizes Stress Granule Assembly and IFN Production by Interacting with G3BPs to Facilitate Viral Replication.

SARS-CoV-2 is the causative agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19) and poses a significant threat to global health. N protein (NP), which is a major pathogenic protein among betacoronaviruses, binds to the viral RNA genome to allow viral genome packaging and viral particle release. Recent studies showed that NP antagonizes interferon (IFN) induction and mediates phase separation. Using live SARS-CoV-2 viruses, this study provides solid evidence showing that SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming G3BP1-mediated antiviral innate immunity. G3BP1 conditional knockout mice (g3bp1fl/fL, Sftpc-Cre) exhibit significantly higher lung viral loads after SARS-CoV-2 infection than wild-type mice. Our findings contribute to the growing body of knowledge regarding the pathogenicity of NPSARS-CoV-2 and provide insight into new therapeutics targeting NPSARS-CoV-2. IMPORTANCE In this study, by in vitro assay and live SARS-CoV-2 virus infection, we provide solid evidence that the SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming antiviral innate immunity mediated by G3BP1 in A549 cell lines and G3BP1 conditional knockout mice (g3bp1-cKO) mice, which provide in-depth evidence showing the mechanism underlying NP-related SARS-CoV-2 pathogenesis through G3BPs.

Dysregulation of RNA interference components in COVID-19 patients.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus causing severe respiratory illness (COVID-19). This virus was initially identified in Wuhan city, a populated area of the Hubei province in China, and still remains one of the major global health challenges. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing that plays a crucial role in innate viral defense mechanisms by inhibiting the virus replication as well as expression of various viral proteins. Dicer, Drosha, Ago2, and DGCR8 are essential components of the RNAi system, which is supposed to be dysregulated in COVID-19 patients. This study aimed to assess the expression level of the mentioned mRNAs in COVID-19patients compared to healthy individuals. RESULTS: Our findings demonstrated that the expression of Dicer, Drosha, and Ago2 was statistically altered in COVID-19 patients compared to healthy subjects. Ultimately, the RNA interference mechanism as a crucial antiviral defense system was suggested to be dysregulated in COVID-19 patients.

SARS-CoV-2 Variants of Concern Hijack IFITM2 for Efficient Replication in Human Lung Cells.

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human lung cells, cardiomyocytes, and gut organoids. To date, several "variants of concern" (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the five current SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, and Omicron) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had modest effects, while knockdown of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than 4 orders of magnitude. In addition, an antibody directed against the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells, thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominant Omicron variant. IMPORTANCE Recent data indicate that SARS-CoV-2 requires endogenously expressed IFITM proteins for efficient infection. However, the results were obtained with an early SARS-CoV-2 isolate. Thus, it remained to be determined whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that outcompeted the original strains in the meantime. This includes the Omicron VOC, which currently dominates the pandemic. Here, we show that depletion of endogenous IFITM2 expression almost entirely prevents productive infection of Alpha, Beta, Gamma, Delta, and Omicron SARS-CoV-2 VOCs in human lung cells. In addition, an antibody targeting the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs, including the currently dominant Omicron variant, are strongly dependent on IFITM2 for efficient replication, suggesting a key proviral role of IFITMs in viral transmission and pathogenicity.

A natural product compound inhibits coronaviral replication in vitro by binding to the conserved Nsp9 SARS-CoV-2 protein.

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.

Virus-specific editing identification approach reveals the landscape of A-to-I editing and its impacts on SARS-CoV-2 characteristics and evolution.

Upon SARS-CoV-2 infection, viral intermediates specifically activate the IFN response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to viral A-to-I RNA editing. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, varied between tissue and cell types, and was correlated with the intensity of innate immune response. On average, 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans during the epidemic. Our study highlights the ability of SARS-CoV-2 to hijack components of the host antiviral machinery to edit its genome and fuel its evolution, and also provides a framework and resource for studying viral RNA editing.