The Role of SARS-CoV Non-structural protein 1 in Regulating RNA stability

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected more than 600 million people across 219 countries during the past three years. SARS-CoV-2 consists of a positive-strand RNA genome that encodes structural and nonstructural proteins and shares a 79% sequence homology with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1). Nonstructural proteins are necessary for viral replication and suppression of the host cell immune response. Nonstructural protein 1 (nsp1), a small protein conserved among most beta-coronaviruses, inhibits host messenger RNA (mRNA) translation by binding to ribosomal mRNA channels. Nsp1 also triggers degradation of host mRNA while viral RNA remains intact. We have previously shown that nsp1 localizes within stress granules (SGs), non-membranous vesicles of stalled mRNA that form in response to viral infection. We also found that upon induction of stress, SGs disperses within 60- 120 minutes in the presence of nsp1. Since SGs are known to store and protect translationally stalled mRNAs that are target of nsp1, we sought to analyze the level of mRNAs accumulation in SGs in the presence of nsp1. The goal of this project is to identify the impact of nsp1 on stress granule formation during SARS-CoV infection. We used human embryonic kidney cells (HEK293) and transfected them with DNA expressing SARSCoV- 1 nsp1 or a control plasmid. Cells were then incubated at 37degreeC under 5% CO2 concentration for 16 hours. Following incubation, cells were subjected to 30 min of oxidative stress using sodium arenite. Cells were collected and lysed using lysis buffer, then centrifuge at 18 000xg to collect SG pellets used for RNA isolation. Isolated mRNAs were quantified using quantitative RT-PCR. We specifically targeted mRNAs that tend to show a preferential accumulation in SGs without any viral infection. When nsp1 was expressed, we found majority of mRNAs have shown a 2-fold decrease in accumulation in SGs. These results suggest there is a direct effect of nsp1 in dispersing of RNA from SGs. We are currently investigating the effect of viral leader sequence in their accumulation in SGs in the presence of nsp1. This project was supported by the DRP award from SC INBRE (NIGMS, P20GM103499).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Multiple functions of stress granules in viral infection at a glance.

Stress granules (SGs) are distinct RNA granules induced by various stresses, which are evolutionarily conserved across species. In general, SGs act as a conservative and essential self-protection mechanism during stress responses. Viruses have a long evolutionary history and viral infections can trigger a series of cellular stress responses, which may interact with SG formation. Targeting SGs is believed as one of the critical and conservative measures for viruses to tackle the inhibition of host cells. In this systematic review, we have summarized the role of SGs in viral infection and categorized their relationships into three tables, with a particular focus on Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Moreover, we have outlined several kinds of drugs targeting SGs according to different pathways, most of which are potentially effective against SARS-CoV-2. We believe this review would offer a new view for the researchers and clinicians to attempt to develop more efficacious treatments for virus infection, particularly for the treatment of SARS-CoV-2 infection.

Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells.

Stress granule formation is induced by numerous environmental stressors, including sodium arsenite treatment and viral infection. Accordingly, stress granules can inhibit viral propagation and function as part of the antiviral host response to numerous viral infections. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antagonizes stress granule formation, in part, via interaction between SARS-CoV-2 nucleocapsid (N) protein and Ras-GTPase-activating SH3-domain-binding protein 1 (G3BP1). However, it is unclear whether there are differential effects in different cell types. In this study, we assessed interaction between the N protein of SARS-CoV-2 S clade and G3BP1/2 in Vero and Calu-3 cells and investigated the effect of various SARS-CoV-2 strains on sodium arsenite-induced stress granule formation. Our data show that SARS-CoV-2 S clade N protein interacts with both G3BP1 and G3BP2 more strongly in Calu-3 vs. Vero cells. Consistent with this observation, infection with SARS-CoV-2 S clade induces stress granule formation in Vero but not in Calu-3 cells. However, infection with SARS-CoV-2 S clade, as well as other SARS-CoV-2 variants, inhibits sodium arsenite-induced stress granule formation in both cell lines. Taken together, our results show differential effects of SARS-CoV-2 infection on stress granule formation that is dependent on host cell type, rather than virus strain type.

Melatonin: Regulation of Viral Phase Separation and Epitranscriptomics in Post-Acute Sequelae of COVID-19.

The relentless, protracted evolution of the SARS-CoV-2 virus imposes tremendous pressure on herd immunity and demands versatile adaptations by the human host genome to counter transcriptomic and epitranscriptomic alterations associated with a wide range of short- and long-term manifestations during acute infection and post-acute recovery, respectively. To promote viral replication during active infection and viral persistence, the SARS-CoV-2 envelope protein regulates host cell microenvironment including pH and ion concentrations to maintain a high oxidative environment that supports template switching, causing extensive mitochondrial damage and activation of pro-inflammatory cytokine signaling cascades. Oxidative stress and mitochondrial distress induce dynamic changes to both the host and viral RNA m6A methylome, and can trigger the derepression of long interspersed nuclear element 1 (LINE1), resulting in global hypomethylation, epigenetic changes, and genomic instability. The timely application of melatonin during early infection enhances host innate antiviral immune responses by preventing the formation of "viral factories" by nucleocapsid liquid-liquid phase separation that effectively blockades viral genome transcription and packaging, the disassembly of stress granules, and the sequestration of DEAD-box RNA helicases, including DDX3X, vital to immune signaling. Melatonin prevents membrane depolarization and protects cristae morphology to suppress glycolysis via antioxidant-dependent and -independent mechanisms. By restraining the derepression of LINE1 via multifaceted strategies, and maintaining the balance in m6A RNA modifications, melatonin could be the quintessential ancient molecule that significantly influences the outcome of the constant struggle between virus and host to gain transcriptomic and epitranscriptomic dominance over the host genome during acute infection and PASC.

Phillygenin activates PKR/eIF2α pathway and induces stress granule to exert anti-avian infectious bronchitis virus.

The prevalence of avian infectious bronchitis virus (IBV) is still one of causes inducing severe losses of production in the poultry industry worldwide. Vaccination does not completely prevent IBV infection and spread due to immune failure and viral mutations. ForsythiaeFructus and its compounds have been widely used in a lot of prescriptions of the traditional Chinese medicine for a long history, and it is well-known as safety and efficiency in heat-clearing and detoxifying. This study aims to investigate the anti-IBV activity and mechanism of phillygenin. The results showed that phillygenin inhibited IBV replication by disturbing multiple stages of the virus life cycle, including viral adsorption, invasion, internalization, and release in Vero cells. After being treated with 100, 125 and 150 µg/mL phillygenin, the expression of G3BP1 was significantly increased and the phosphorylation of PKR/eIF2α was activated, which increased stress granule, thereby triggering the antiviral response in Vero cells. The anti-virus activity of PHI was decreased when G3BP1 was interfered by si-RNA, and G3BP1 was down-regulated when PKR/eIF2α was interfered by si-RNA. In conclusion, our findings indicate that phillygenin activates PKR/eIF2α pathway and induces stress granule formation to exert anti-IBV, which holds promise to develop into a novel anti-IBV drug. Further study in vivo is needed to explore phillygenin as a potential and effective drug to prevent IB in poultry.

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.

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.

The PERK/PKR-eIF2α Pathway Negatively Regulates Porcine Hemagglutinating Encephalomyelitis Virus Replication by Attenuating Global Protein Translation and Facilitating Stress Granule Formation.

The replication of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is closely associated with the endoplasmic reticulum (ER) of infected cells. The unfolded protein response (UPR), which is mediated by ER stress (ERS), is a typical outcome in coronavirus-infected cells and is closely associated with the characteristics of coronaviruses. However, the interaction between virus-induced ERS and coronavirus replication is poorly understood. Here, we demonstrate that infection with the betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) induced ERS and triggered all three branches of the UPR signaling pathway both in vitro and in vivo. In addition, ERS suppressed PHEV replication in mouse neuro-2a (N2a) cells primarily by activating the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) axis of the UPR. Moreover, another eIF2α phosphorylation kinase, interferon (IFN)-induced double-stranded RNA-dependent protein kinase (PKR), was also activated and acted cooperatively with PERK to decrease PHEV replication. Furthermore, we demonstrate that the PERK/PKR-eIF2α pathways negatively regulated PHEV replication by attenuating global protein translation. Phosphorylated eIF2α also promoted the formation of stress granules (SGs), which in turn repressed PHEV replication. In summary, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets (e.g., PERK, PKR, and eIF2α) for antiviral drugs. IMPORTANCE Coronavirus diseases are caused by different coronaviruses of importance in humans and animals, and specific treatments are extremely limited. ERS, which can activate the UPR to modulate viral replication and the host innate response, is a frequent occurrence in coronavirus-infected cells. PHEV, a neurotropic betacoronavirus, causes nerve cell damage, which accounts for the high mortality rates in suckling piglets. However, it remains incompletely understood whether the highly developed ER in nerve cells plays an antiviral role in ERS and how ERS regulates viral proliferation. In this study, we found that PHEV infection induced ERS and activated the UPR both in vitro and in vivo and that the activated PERK/PKR-eIF2α axis inhibited PHEV replication through attenuating global protein translation and promoting SG formation. A better understanding of coronavirus-induced ERS and UPR activation may reveal the pathogenic mechanism of coronavirus and facilitate the development of new treatment strategies for these diseases.

SARS-CoV-2 impairs the disassembly of stress granules and promotes ALS-associated amyloid aggregation.

The nucleocapsid (N) protein of SARS-CoV-2 has been reported to have a high ability of liquid-liquid phase separation, which enables its incorporation into stress granules (SGs) of host cells. However, whether SG invasion by N protein occurs in the scenario of SARS-CoV-2 infection is unknow, neither do we know its consequence. Here, we used SARS-CoV-2 to infect mammalian cells and observed the incorporation of N protein into SGs, which resulted in markedly impaired self-disassembly but stimulated cell cellular clearance of SGs. NMR experiments further showed that N protein binds to the SG-related amyloid proteins via non-specific transient interactions, which not only expedites the phase transition of these proteins to aberrant amyloid aggregation in vitro, but also promotes the aggregation of FUS with ALS-associated P525L mutation in cells. In addition, we found that ACE2 is not necessary for the infection of SARS-CoV-2 to mammalian cells. Our work indicates that SARS-CoV-2 infection can impair the disassembly of host SGs and promote the aggregation of SG-related amyloid proteins, which may lead to an increased risk of neurodegeneration.

SARS-CoV-2 Nucleocapsid Protein Targets a Conserved Surface Groove of the NTF2-like Domain of G3BP1.

Stress granule (SG) formation mediated by Ras GTPase-activating protein-binding protein 1 (G3BP1) constitutes a key obstacle for viral replication, which makes G3BP1 a frequent target for viruses. For instance, the SARS-CoV-2 nucleocapsid (N) protein interacts with G3BP1 directly to suppress SG assembly and promote viral production. However, the molecular basis for the SARS-CoV-2 N - G3BP1 interaction remains elusive. Here we report biochemical and structural analyses of the SARS-CoV-2 N - G3BP1 interaction, revealing differential contributions of various regions of SARS-CoV-2 N to G3BP1 binding. The crystal structure of the NTF2-like domain of G3BP1 (G3BP1NTF2) in complex with a peptide derived from SARS-CoV-2 N (residues 1-25, N1-25) reveals that SARS-CoV-2 N1-25 occupies a conserved surface groove of G3BP1NTF2 via surface complementarity. We show that a φ-x-F (φ, hydrophobic residue) motif constitutes the primary determinant for G3BP1NTF2-targeting proteins, while the flanking sequence underpins diverse secondary interactions. We demonstrate that mutation of key interaction residues of the SARS-CoV-2 N1-25 - G3BP1NTF2 complex leads to disruption of the SARS-CoV-2 N - G3BP1 interaction in vitro. Together, these results provide a molecular basis of the strain-specific interaction between SARS-CoV-2 N and G3BP1, which has important implications for the development of novel therapeutic strategies against SARS-CoV-2 infection.

Host Cellular RNA Helicases Regulate SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the largest RNA genome, approximately 30 kb, among RNA viruses. The DDX DEAD box RNA helicase is a multifunctional protein involved in all aspects of RNA metabolism. Therefore, host RNA helicases may regulate and maintain such a large viral RNA genome. In this study, I investigated the potential role of several host cellular RNA helicases in SARS-CoV-2 infection. Notably, DDX21 knockdown markedly accumulated intracellular viral RNA and viral production, as well as viral infectivity of SARS-CoV-2, indicating that DDX21 strongly restricts the SARS-CoV-2 infection. In addition, MOV10 RNA helicase also suppressed the SARS-CoV-2 infection. In contrast, DDX1, DDX5, and DDX6 RNA helicases were required for SARS-CoV-2 replication. Indeed, SARS-CoV-2 infection dispersed the P-body formation of DDX6 and MOV10 RNA helicases as well as XRN1 exonuclease, while the viral infection did not induce stress granule formation. Accordingly, the SARS-CoV-2 nucleocapsid (N) protein interacted with DDX1, DDX3, DDX5, DDX6, DDX21, and MOV10 and disrupted the P-body formation, suggesting that SARS-CoV-2 N hijacks DDX6 to carry out viral replication. Conversely, DDX21 and MOV10 restricted SARS-CoV-2 infection through an interaction of SARS-CoV-2 N with host cellular RNA helicases. Altogether, host cellular RNA helicases seem to regulate the SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has a large RNA genome, of approximately 30 kb. To regulate and maintain such a large viral RNA genome, host RNA helicases may be involved in SARS-CoV-2 replication. In this study, I have demonstrated that DDX21 and MOV10 RNA helicases limit viral infection and replication. In contrast, DDX1, DDX5, and DDX6 are required for SARS-CoV-2 infection. Interestingly, SARS-CoV-2 infection disrupted P-body formation and attenuated or suppressed stress granule formation. Thus, SARS-CoV-2 seems to hijack host cellular RNA helicases to play a proviral role by facilitating viral infection and replication and by suppressing the host innate immune system.

Phase separation by the SARS-CoV-2 nucleocapsid protein: Consensus and open questions.

In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review, we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein-an essential viral protein required for viral replication-and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs.

The PERK/PKR-eIF2α Pathway Negatively Regulates Porcine Hemagglutinating Encephalomyelitis Virus Replication by Attenuating Global Protein Translation and Facilitating Stress Granule Formation

The replication of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERSCoV), and the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is closely associated with the endoplasmic reticulum (ER) of infected cells. The unfolded protein response (UPR), which is mediated by ER stress (ERS), is a typical outcome in coronavirus-infected cells and is closely associated with the characteristics of coronaviruses. However, the interaction between virus-induced ERS and coronavirus replication is poorly understood. Here, we demonstrate that infection with the betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) induced ERS and triggered all three branches of the UPR signaling pathway both in vitro and in vivo. In addition, ERS suppressed PHEV replication in mouse neuro-2a (N2a) cells primarily by activating the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) axis of the UPR. Moreover, another eIF2a phosphorylation kinase, interferon (IFN)-induced double-stranded RNA-dependent protein kinase (PKR), was also activated and acted cooperatively with PERK to decrease PHEV replication. Furthermore, we demonstrate that the PERK/PKR-eIF2a pathways negatively regulated PHEV replication by attenuating global protein translation. Phosphorylated eIF2a also promoted the formation of stress granules (SGs), which in turn repressed PHEV replication. In summary, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets (e.g., PERK, PKR, and eIF2a) for antiviral drugs. [ FROM AUTHOR] Copyright of Journal of Virology is the property of American Society for Microbiology 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.)

Proximity-dependent biotinylation detects associations between SARS coronavirus nonstructural protein 1 and stress granule-associated proteins.

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 is a critical viral protein that suppresses host gene expression by blocking the assembly of the ribosome on host mRNAs. To understand the mechanism of inhibition of host gene expression, we sought to identify cellular proteins that interact with nsp1. Using proximity-dependent biotinylation followed by proteomic analyses of biotinylated proteins, here we captured multiple dynamic interactions of nsp1 with host cell proteins. In addition to ribosomal proteins, we identified several pre-mRNA processing proteins that interact with nsp1, including splicing factors and transcription termination proteins, as well as exosome, and stress granule (SG)-associated proteins. We found that the interactions with transcription termination factors are primarily governed by the C-terminal region of nsp1 and are disrupted by the mutation of amino acids K164 and H165 that are essential for its host shutoff function. We further show that nsp1 interacts with Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and colocalizes with G3BP1 in SGs under sodium arsenite-induced stress. Finally, we observe that the presence of nsp1 disrupts the maturation of SGs over a long period. Isolation of SG core at different times shows a gradual loss of G3BP1 in the presence of nsp1.

Insights into the SARS-CoV-2-Mediated Alteration in the Stress Granule Protein Regulatory Networks in Humans.

The rapidly and constantly evolving coronavirus, SARS-CoV-2, imposes a great threat to human health causing severe lung disease and significant mortality. Cytoplasmic stress granules (SGs) exert anti-viral activities due to their involvement in translation inhibition and innate immune signaling. SARS-CoV-2 sequesters important SG nucleator proteins and impairs SG formation, thus evading the host response for efficient viral replication. However, the significance of SGs in COVID-19 infection remains elusive. In this study, we utilize a protein-protein interaction network approach to systematically dissect the crosstalk of human post-translational regulatory networks governed by SG proteins due to SARS-CoV-2 infection. We uncovered that 116 human SG proteins directly interact with SARS-CoV-2 proteins and are involved in 430 different brain disorders including COVID-19. Further, we performed gene set enrichment analysis to identify the drugs against three important key SG proteins (DYNC1H1, DCTN1, and LMNA) and also looked for potential microRNAs (miRNAs) targeting these proteins. We identified bexarotene as a potential drug molecule and miRNAs, hsa-miR-615-3p, hsa-miR-221-3p, and hsa-miR-124-3p as potential candidates for the treatment of COVID-19 and associated manifestations.

Genomics-guided targeting of stress granule proteins G3BP1/2 to inhibit SARS-CoV-2 propagation.

SARS-CoV-2 nucleocapsid (N) protein undergoes RNA-induced phase separation (LLPS) and sequesters the host key stress granule (SG) proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and 2 (G3BP1 and G3BP2) to inhibit SG formation. This will allow viral packaging and propagation in host cells. Based on a genomic-guided meta-analysis, here we identify upstream regulatory elements modulating the expression of G3BP1 and G3BP2 (collectively called G3BP1/2). Using this strategy, we have identified FOXA1, YY1, SYK, E2F-1, and TGFBR2 as activators and SIN3A, SRF, and AKT-1 as repressors of G3BP1/2 genes. Panels of the activators and repressors were then used to identify drugs that change their gene expression signatures. Two drugs, imatinib, and decitabine have been identified as putative modulators of G3BP1/2 genes and their regulators, suggesting their role as COVID-19 mitigation agents. Molecular docking analysis suggests that both drugs bind to G3BP1/2 with a much higher affinity than the SARS-CoV-2 N protein. This study reports imatinib and decitabine as candidate drugs against N protein and G3BP1/2 protein.

A proposed role for the SARS-CoV-2 nucleocapsid protein in the formation and regulation of biomolecular condensates.

To date, the recently discovered SARS-CoV-2 virus has afflicted >6.9 million people worldwide and disrupted the global economy. Development of effective vaccines or treatments for SARS-CoV-2 infection will be aided by a molecular-level understanding of SARS-CoV-2 proteins and their interactions with host cell proteins. The SARS-CoV-2 nucleocapsid (N) protein is highly homologous to the N protein of SARS-CoV, which is essential for viral RNA replication and packaging into new virions. Emerging models indicate that nucleocapsid proteins of other viruses can form biomolecular condensates to spatiotemporally regulate N protein localization and function. Our bioinformatic analyses, in combination with pre-existing experimental evidence, suggest that the SARS-CoV-2 N protein is capable of forming or regulating biomolecular condensates in vivo by interaction with RNA and key host cell proteins. We discuss multiple models, whereby the N protein of SARS-CoV-2 may harness this activity to regulate viral life cycle and host cell response to viral infection.

In silico identification of MicroRNAs targeting the key nucleator of stress granules, G3BP: Promising therapeutics for SARS-CoV-2 infection.

Stress granules (SGs) are non-membrane ribonucleoprotein condensates formed in response to environmental stress conditions via liquid-liquid phase separation (LLPS). SGs are involved in the pathogenesis of aging and aging-associated diseases, cancers, viral infection, and several other diseases. GTPase-activating protein (SH3 domain)-binding protein 1 and 2 (G3BP1/2) is a key component and commonly used marker of SGs. Recent studies have shown that SARS-CoV-2 nucleocapsid protein via sequestration of G3BPs inhibits SGs formation in the host cells. In this study, we have identified putative miRNAs targeting G3BP in search of modulators of the G3BP expression. These miRNAs could be considered as new therapeutic targets against COVID-19 infection via the regulation of SG assembly and dynamics.