When viral RNA met the cell: a story of protein-RNA interactions

RNA is a central molecule for the RNA virus life cycle as it functions not only as messenger for the synthesis of proteins, but also as storage of genetic information as genome. Given the central role of viral RNA in infection, it is expected that it must function as a hub for critical host-virus interactions. To test this, my laboratory has developed new approaches that have been applied to several viruses such as Sindbis virus, SARS-CoV-2 and human immunodeficiency virus (HIV).We have discovered a new universe of host-virus interactions with central regulatory roles in infection. Interestingly, these viruses, despite having different sequences and infection cycles, engage with a largely shared pool of cellular RNA-binding proteins. My laboratory is currently focused on understanding the regulatory mechanisms underpinning these master regulators with molecular detail. We envision that these central host-virus interactions are promising targets for broad-spectrum antiviral strategies. I am funded by an ERC Consolidator grant (vRNP-capture 101001634) and an MRC research grant (MR/R021562/1).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

New Short RNA Motifs Potentially Relevant in the SARS-CoV-2 Genome

Background: The coronavirus disease has led to an exhaustive exploration of the SARS-CoV-2 genome. Despite the amount of information accumulated, the prediction of short RNA motifs encoding peptides mediating protein-protein or protein-drug interactions has received limited attention. Objective(s): The study aims to predict short RNA motifs that are interspersed in the SARS-CoV-2 genome. Method(s): A method in which 14 trinucleotide families, each characterized by being composed of triplets with identical nucleotides in all possible configurations, was used to find short peptides with biological relevance. The novelty of the approach lies in using these families to search how they are distributed across genomes of different CoV genera and then to compare the distributions of these families with each other. Result(s): We identified distributions of trinucleotide families in different CoV genera and also how they are related, using a selection criterion that identified short RNA motifs. The motifs were reported to be conserved in SARS-CoVs;in the remaining CoV genomes analysed, motifs contained, exclusively, different configurations of the trinucleotides A, T, G and A, C, G. Eighty-eight short RNA motifs, ranging in length from 12 to 49 nucleotides, were found: 50 motifs in the 1a polyprotein-encoding orf, 27 in the 1b polyprotein-encoding orf, 5 in the spike-encoding orf, and 6 in the nucleocapsid-encoding orf. Although some motifs (~27%) were found to be intercalated or attached to functional peptides, most of them have not yet been associated with any known functions. Conclusion(s): Some of the trinucleotide family distributions in different CoV genera are not random;they are present in short peptides that, in many cases, are intercalated or attached to functional sites of the proteome.Copyright © 2022 Bentham Science Publishers.

Chemi-Informatic Approach to Investigate Putative Pharmacoactive Agents of Plant Origin to Eradicate COVID-19

Background: The scientific community has supported the medicinal flora of ancient as well as modern times in extracting chemicals, which holds therapeutic potential. In many previous studies, Amentoflavone discovered as an anti-viral agent, and it is present as a bioactive constituent in many plants of different families like Selaginellaceae, Euphorbiaceae, and Calophyllaceae. Withania somnifera (Ashwagandha) is already considered a significant anti-viral agent in traditional medicine, and it is the main source of Somniferine-A and Withanolide-B. Objective(s): In this study, phytochemicals such as withanolide-b, somniferine-a, stigmasterol, amentoflavone, and chavicine were analyzed to screen protein inhibitors, out of them;such proteins are involved in the internalization and interaction of SARS-CoV-2 with human cytological domains. This will help in developing a checkpoint for SARS-CoV-2 internalization. Method(s): Chemi-informatic tools like basic local alignment search tool (BLAST), AutoDock-vina, SwissADME, MDWeb, Molsoft, ProTox-II, and LigPlot were used to examine the action of pharmacoactive agents against SARS-CoV-2. The tools used in the study were based on the finest algorithms like artificial neural networking, machine learning, and artificial intelligence. Result(s): On the basis of binding energies less than equal to-8.5 kcal/mol, amentoflavone, stigmasterol, and somniferine-A were found to be the most effective against COVID-19 disease as these chemical agents exhibit hydrogen bond interactions and competitively inhibit major proteins (SARS-CoV-2 Spike, Human ACE-2 receptor, Human Furin protease, SARS-CoV-2 RNA binding protein) that are involved in its infection and pathogenesis. Simulation analysis provides more validity to the selection of the drug candidate Amentoflavone. ADMET properties were found to be in the feasible range for putative drug candidates. Conclusion(s): Computational analysis was successfully used for searching pharmacoactive phytochemicals like Amentoflavone, Somniferine-A, and Stigmasterol that can bring control over COVID-19 expansion. This new methodology was found to be efficient, as it reduces monetary expenditures and time consumption. Molecular wet-lab validations will provide approval for finalizing our selected drug model for controlling the COVID-19 pandemic.Copyright © 2022 Bentham Science Publishers.

ZFP36 ring finger protein like 1 significantly suppresses human coronavirus OC43 replication.

CCCH-type zinc figure proteins (ZFP) are small cellular proteins that are structurally maintained by zinc ions. Zinc ions coordinate the protein structure in a tetrahedral geometry by binding to cystine-cystine or cysteines-histidine amino acids. ZFP's unique structure enables it to interact with a wide variety of molecules including RNA; thus, ZFP modulates several cellular processes including the host immune response and virus replication. CCCH-type ZFPs have shown their antiviral efficacy against several DNA and RNA viruses. However, their role in the human coronavirus is little explored. We hypothesized that ZFP36L1 also suppresses the human coronavirus. To test our hypothesis, we used OC43 human coronavirus (HCoV) strain in our study. We overexpressed and knockdown ZFP36L1 in HCT-8 cells using lentivirus transduction. Wild type, ZFP36L1 overexpressed, and ZFP36L1 knockdown cells were each infected with HCoV-OC43, and the virus titer in each cell line was measured over 96 hours post-infection (p.i.). Our results show that HCoV-OC43 replication was significantly reduced with ZFP36L1 overexpression while ZFP36L1 knockdown significantly enhanced virus replication. ZFP36L1 knockdown HCT-8 cells started producing infectious virus at 48 hours p.i. which was an earlier timepoint as compared to wild -type and ZFP36L1 overexpressed cells. Wild-type and ZFP36L1 overexpressed HCT-8 cells started producing infectious virus at 72 hours p.i. Overall, the current study showed that overexpression of ZFP36L1 suppressed human coronavirus (OC43) production.

Pro-Viral and Anti-Viral Roles of the RNA-Binding Protein G3BP1.

Viruses depend on host cellular resources to replicate. Interaction between viral and host proteins is essential for the pathogens to ward off immune responses as well as for virus propagation within the infected cells. While different viruses employ unique strategies to interact with diverse sets of host proteins, the multifunctional RNA-binding protein G3BP1 is one of the common targets for many viruses. G3BP1 controls several key cellular processes, including mRNA stability, translation, and immune responses. G3BP1 also serves as the central hub for the protein-protein and protein-RNA interactions within a class of biomolecular condensates called stress granules (SGs) during stress conditions, including viral infection. Increasing evidence suggests that viruses utilize distinct strategies to modulate G3BP1 function-either by degradation, sequestration, or redistribution-and control the viral life cycle positively and negatively. In this review, we summarize the pro-viral and anti-viral roles of G3BP1 during infection among different viral families.

Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the ∼30-kb RNA genome in the ∼90-nm virion. Previous studies suggest that each virion contains 35 to 40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined in vitro with short fragments of the viral genome, forms 15-nm particles similar to the vRNP structures observed within virions. These vRNPs depend on regions of N protein that promote protein-RNA and protein-protein interactions. Phosphorylation of N protein in its disordered serine/arginine region weakens these interactions to generate less compact vRNPs. We propose that unmodified N protein binds structurally diverse regions in genomic RNA to form compact vRNPs within the nucleocapsid, while phosphorylation alters vRNP structure to support other N protein functions in viral transcription.

VIRTUAL SCREENING AND MOLECULAR DOCKING ANALYSIS ON THREE SARS-COV-2 DRUG TARGETS BY MULTIPLE COMPUTATIONAL APPROACH

Objective: SARS-CoV-2 is a pandemic virus characterized by upper respiratory tract infection and can range from mild symptoms to severe complications. In this case, drug repurposing and computer-aided studies have become very important to find emergency solutions. In this study, drug-target interactions on three nonstructural protein structures of SARS-CoV-2 of 8820 drug candidates or drug molecules obtained from the DrugBank database were analyzed. Material and Method: Comprehensive virtual screening and molecular docking studies from 8820 drug molecules or candidates obtained from the DrugBank database were performed on the RNA binding protein, 2'-O-methyltransferase, and endoribonuclease of SARS-CoV-2;and potential drug candidates were determined for each target. Virtual screening studies have been done with High-Throughput Virtual Screening (HTVS), Standard Precision (SP), Extra Precision (XP), and Molecular Mechanics Generalized Born Surface Area (MM-GBSA). Also, information about the clinical findings, transmission, pathogenesis, and treatment of SARS-CoV-2 has been given. Result and Discussion: Drug-target interactions on three nonstructural protein structures of SARS-CoV-2 of 8820 drug candidates or drug molecules obtained from the DrugBank database were analyzed. Potential compound recommendations for each drug target were presented. Information was given about key amino acids where active sites of drug target proteins interact with ligands. This study is expected to be useful in target-based drug development studies on the proteins of SARS-CoV-2.

Subacute cutaneous lupus erythematosus after COVID-19 vaccination

Numerous cutaneous reactions have been reported secondary to COVID-19 vaccinations. The most commonly reported include local site reactions, delayed large local reactions, urticaria and morbilliform eruptions. Here we report a case of de novo subacute cutaneous lupus erythematosus (SCLE) after COVID-19 immunization. A 56-year-old woman presented with a 3-month history of a rash. The onset was 1 week following the first dose of the AstraZeneca COVID-19 vaccine. She reported lesions characterized by erythema, pruritus and a burning sensation. She also described mouth dryness. Examination revealed scaly annular erythematous plaques on the chest, arms, legs and scalp. Blood results were positive for anti-Ro antibodies and strongly positive for anti-nuclear antibodies (1: 2560 titre). Anti-Smith, anti-centromere and double- stranded DNA antibodies were negative. Skin biopsy revealed the histological appearance of an interface of dermatitis. Direct immunofluorescence was negative. These clinical and histopathological findings are consistent with a diagnosis of SCLE. The patient was treated with hydroxychloroquine, a weaning course of prednisolone, topical steroids and topical tacrolimus. Her hydroxychloroquine dose was 200 mg twice daily for the first 3 months and then increased to 400 mg twice daily. This resulted in an improvement of her presentation although she has yet to achieve complete remission. It has been suggested that enhanced interferon responses with COVID-19 vaccination and interactions of the SARS-CoV-2 spike protein with cytoplasmic RNA-binding proteins could contribute to disease flares in lupus. There are two other recent reports of SCLE developing or being exacerbated by COVID-19 vaccination. More research is required to determine how COVID-19 vaccinations affect patients with autoimmune skin diseases.

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).

Role of Stress Granules in Suppressing Viral Replication by the Infectious Bronchitis Virus Endoribonuclease.

Infectious bronchitis virus (IBV), a γ-coronavirus, causes the economically important poultry disease infectious bronchitis. Cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation. Previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. Here, we aimed to delineate the molecular mechanisms regulating the SG response to pathogenic IBV strain infection. We found that most chicken embryo kidney (CEK) cells formed no SGs during IBV infection and IBV replication inhibited arsenite-induced SG formation. This inhibition was not caused by changes in the integrity or abundance of SG proteins during infection. IBV nonstructural protein 15 (Nsp15) endoribonuclease activity suppressed SG formation. Regardless of whether Nsp15 was expressed alone, with recombinant viral infection with Newcastle disease virus as a vector, or with EndoU-deficient IBV, the Nsp15 endoribonuclease activity was the main factor inhibiting SG formation. Importantly, uridine-specific endoribonuclease (EndoU)-deficient IBV infection induced colocalization of IBV N protein/dsRNA and SG-associated protein TIA1 in infected cells. Additionally, overexpressing TIA1 in CEK cells suppressed IBV replication and may be a potential antiviral factor for impairing viral replication. These data provide a novel foundation for future investigations of the mechanisms by which coronavirus endoribonuclease activity affects viral replication. IMPORTANCE Endoribonuclease is conserved in coronaviruses and affects viral replication and pathogenicity. Infectious bronchitis virus (IBV), a γ-coronavirus, infects respiratory, renal, and reproductive systems, causing millions of dollars in lost revenue to the poultry industry worldwide annually. Mutating the viral endoribonuclease poly(U) resulted in SG formation, and TIA1 protein colocalized with the viral N protein and dsRNA, thus damaging IBV replication. These results suggest a new antiviral target design strategy for coronaviruses.

Photocatalytic Chemical Crosslinking for Profiling RNA–Protein Interactions in Living Cells

The dynamic interactions between RNAs and proteins play crucial roles in regulating diverse cellular processes. Proteome‐wide characterization of these interactions in their native cellular context remains desirable but challenging. Herein, we developed a photocatalytic crosslinking (PhotoCAX) strategy coupled with mass spectrometry (PhotoCAX‐MS) and RNA sequencing (PhotoCAX‐seq) for the study of the composition and dynamics of protein‐RNA interactions. By integrating the blue light‐triggered photocatalyst with a dual‐functional RNA–protein crosslinker (RP‐linker) and the phase separation‐based enrichment strategy, PhotoCAX‐MS revealed a total of 2044 RBPs in human HEK293 cells. We further employed PhotoCAX to investigate the dynamic change of RBPome in macrophage cells upon LPS‐stimulation, as well as the identification of RBPs interacting directly with the 5′ untranslated regions of SARS‐CoV‐2 RNA. [ FROM AUTHOR] Copyright of Angewandte Chemie International Edition is the property of John Wiley & Sons, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Proteomic approaches define rocaglates as translation remodelers with multiple protein targets

Intro: Deregulated protein synthesis is a common trait across solid and hematologic malignancies and an attractive target for cancer therapy. Rocaglates compounds that inhibit eukaryotic initiation factor 4A1 (eIF4A1), the essential DEAD-box RNA helicase that resolves mRNA 5'UTR secondary structures during cap-dependent translation initiation. Rocaglates' unique mechanism of action causes sequence-selective mRNA binding by eIF4A1, clamping the inactive helicase onto the transcript. This suppresses translation globally and affects many oncogenic and pro-survival transcripts in particular. Zotatifin, the first-in class synthetic rocaglate, is currently in Phase I clinical trials for the treatment of solid tumors and as an antiviral against SARS-CoV2. Currently, eIF4A1 and DDX3 are the only reported targets of rocaglate-mediated RNA clamping. Employing unbiased proteomic approaches, we have discovered that rocaglates, thought to act as pure eIF4A/translation inhibitors, extensively remodel the translation machinery and translatome. Additionally, mass-spec interrogation for proteins interacting with specific RNA sequences reveals novel targets of rocaglate-mediated, sequence-specific RNA clamping. Methods: We conducted original mass-spectrometry analyses of translational reprogramming by rocaglates. TMT-pSILAC assessed acute changes in protein production, while MATRIX, which captures high-resolution profiles of the translation machinery, revealed translation factors that drive reprogramming in response to rocaglate exposure. We validated results biochemically, in cellulo, and in vivo using patient-derived xenograft (PDX) mouse models. To probe existing and novel rocaglate RNA-clamping targets, we developed unbiased “clampome” assays - in cellulo protein-RNA-pull downs followed by mass-spec analysis of proteins with increased binding to RNA in the presence of rocaglates. Results: We find rocaglates, including zotatifin, have effects far more complex than simple “translational inhibition” as currently defined. Indeed, translatome analysis by TMT-pSILAC revealed myriad up-regulated proteins that drive hitherto unrecognized cytotoxic mechanisms. The GEF-H1 guanine exchange factor, for example, drives anti-survival RHOA/JNK activation, suggesting novel candidate biomarkers of rocaglate clinical outcomes. Translation-machinery analysis by MATRIX identifed rocaglate-induced dependence on specific translation factors including eEF1ϵ1 that drive remodeling. Novel rocaglate RNA-binding targets revealed by clampome studies remain under detailed evaluation as mediators of drug activities. Discussion: Our original proteome-level interrogation revealed that the complete cellular response to these historical “translation inhibitors” is mediated by comprehensive translational landscape remodeling. Effects on a broader suite of RNA binding proteins than eIF4A1 alone we suggest mediate the potent antitumor activities of these unique compounds, elucidation of which permits development of novel precision approaches to targeted translational deregulation in cancer.

hnRNP C modulates MERS-CoV and SARS-CoV-2 replication by governing the expression of a subset of circRNAs and cognitive mRNAs.

ABSTRACTHost circular RNAs (circRNAs) play critical roles in the pathogenesis of viral infections. However, how viruses modulate the biogenesis of host proviral circRNAs to facilitate their replication remains unclear. We have recently shown that Middle East respiratory syndrome coronavirus (MERS-CoV) infection increases co-expression of circRNAs and their cognate messenger RNAs (mRNAs), possibly by hijacking specific host RNA binding proteins (RBPs). In this study, we systemically analysed the interactions between the representative circRNA-mRNA pairs upregulated upon MERS-CoV infection and host RBPs. Our analysis identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a key host factor that governed the expression of numerous MERS-CoV-perturbed circRNAs, including hsa_circ_0002846, hsa_circ_0002061, and hsa_circ_0004445. RNA immunoprecipitation assay showed that hnRNP C could bind physically to these circRNAs. Specific knockdown of hnRNP C by small interfering RNA significantly (P < 0.05 to P < 0.0001) suppressed MERS-CoV replication in human lung adenocarcinoma (Calu-3) and human small airway epithelial (HSAEC) cells. Both MERS-CoV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection increased the total and phosphorylated forms of hnRNP C to activate the downstream CRK-mTOR pathway. Treatment of MERS-CoV- (IC50: 0.618 µM) or SARS-CoV-2-infected (IC50: 1.233 µM) Calu-3 cells with the mTOR inhibitor OSI-027 resulted in significantly reduced viral loads. Collectively, our study identified hnRNP C as a key regulator of MERS-CoV-perturbed circRNAs and their cognate mRNAs, and the potential of targeting hnRNP C-related signalling pathways as an anticoronaviral strategy.

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids.

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.

Recombinant SARS-CoV-2 Nucleocapsid Protein: Expression, Purification, and Its Biochemical Characterization and Utility in Serological Assay Development to Assess Immunological Responses to SARS-CoV-2 Infection.

The SARS-CoV-2 nucleocapsid protein (N) binds a single-stranded viral RNA genome to form a helical ribonucleoprotein complex that is packaged into virion particles. N is relatively conserved among coronaviruses and consists of the N-terminal domain (NTD) and C-terminal domain (CTD), which are flanked by three disorganized regions. N is highly immunogenic and has been widely used to develop a serological assay as a diagnostic tool for COVID-19 infection, although there is a concern that the natural propensity of N to associate with RNA might compromise the assay's specificity. We expressed and purified from bacterial cells two recombinant forms of SARS-CoV-2 N, one from the soluble fraction of bacterial cell lysates that is strongly associated with bacterial RNAs and the other that is completely devoid of RNAs. We showed that both forms of N can be used to develop enzyme-linked immunosorbent assays (ELISAs) for the specific detection of human and mouse anti-N monoclonal antibodies (mAb) as well as feline SARS-CoV-2 seropositive serum samples, but that the RNA-free form of N exhibits a slightly higher level of sensitivity than the RNA-bound form to react to anti-N mouse mAb. Using the electrophoretic mobility shift assay (EMSA), we also showed that N preferentially binds ssRNA in a sequence-independent manner and that both NTD and CTD of N contribute to RNA-binding activity. Collectively, our study describes methods to express, purify, and biochemically characterize the SARS-CoV-2 N protein and to use it for the development of serological assays to detect SARS-CoV-2 infection.

Global analysis of protein-RNA interactions in SARS-CoV-2-infected cells reveals key regulators of infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs, and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Among them are several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

Mouse Ifit1b is a cap1-RNA-binding protein that inhibits mouse coronavirus translation and is regulated by complexing with Ifit1c.

Knockout mouse models have been extensively used to study the antiviral activity of IFIT (interferon-induced protein with tetratricopeptide repeats). Human IFIT1 binds to cap0 (m7GpppN) RNA, which lacks methylation on the first and second cap-proximal nucleotides (cap1, m7GpppNm, and cap2, m7GpppNmNm, respectively). These modifications are signatures of "self" in higher eukaryotes, whereas unmodified cap0-RNA is recognized as foreign and, therefore, potentially harmful to the host cell. IFIT1 inhibits translation at the initiation stage by competing with the cap-binding initiation factor complex, eIF4F, restricting infection by certain viruses that possess "nonself" cap0-mRNAs. However, in mice and other rodents, the IFIT1 orthologue has been lost, and the closely related Ifit1b has been duplicated twice, yielding three paralogues: Ifit1, Ifit1b, and Ifit1c. Although murine Ifit1 is similar to human IFIT1 in its cap0-RNA-binding selectivity, the roles of Ifit1b and Ifit1c are unknown. Here, we found that Ifit1b preferentially binds to cap1-RNA, whereas binding is much weaker to cap0- and cap2-RNA. In murine cells, we show that Ifit1b can modulate host translation and restrict WT mouse coronavirus infection. We found that Ifit1c acts as a stimulatory cofactor for both Ifit1 and Ifit1b, promoting their translation inhibition. In this way, Ifit1c acts in an analogous fashion to human IFIT3, which is a cofactor to human IFIT1. This work clarifies similarities and differences between the human and murine IFIT families to facilitate better design and interpretation of mouse models of human infection and sheds light on the evolutionary plasticity of the IFIT family.

B cell-specific XIST complex enforces X-inactivation and restrains atypical B cells.

The long non-coding RNA (lncRNA) XIST establishes X chromosome inactivation (XCI) in female cells in early development and thereafter is thought to be largely dispensable. Here, we show XIST is continually required in adult human B cells to silence a subset of X-linked immune genes such as TLR7. XIST-dependent genes lack promoter DNA methylation and require continual XIST-dependent histone deacetylation. XIST RNA-directed proteomics and CRISPRi screen reveal distinctive somatic cell-type-specific XIST complexes and identify TRIM28 that mediates Pol II pausing at promoters of X-linked genes in B cells. Single-cell transcriptome data of female patients with either systemic lupus erythematosus or COVID-19 infection revealed XIST dysregulation, reflected by escape of XIST-dependent genes, in CD11c+ atypical memory B cells (ABCs). XIST inactivation with TLR7 agonism suffices to promote isotype-switched ABCs. These results indicate cell-type-specific diversification and function for lncRNA-protein complexes and suggest expanded roles for XIST in sex-differences in biology and medicine.