Transcription factor Dmrt1 triggers the SPRY1-NF-κB pathway to maintain testicular immune homeostasis and male fertility.

Bacterial or viral infections, such as Brucella, mumps virus, herpes simplex virus, and Zika virus, destroy immune homeostasis of the testes, leading to spermatogenesis disorder and infertility. Of note, recent research shows that SARS-CoV-2 can infect male gonads and destroy Sertoli and Leydig cells, leading to male reproductive dysfunction. Due to the many side effects associated with antibiotic therapy, finding alternative treatments for inflammatory injury remains critical. Here, we found that Dmrt1 plays an important role in regulating testicular immune homeostasis. Knockdown of Dmrt1 in male mice inhibited spermatogenesis with a broad inflammatory response in seminiferous tubules and led to the loss of spermatogenic epithelial cells. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) revealed that Dmrt1 positively regulated the expression of Spry1, an inhibitory protein of the receptor tyrosine kinase (RTK) signaling pathway. Furthermore, immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP) analysis indicated that SPRY1 binds to nuclear factor kappa B1 (NF-κB1) to prevent nuclear translocation of p65, inhibit activation of NF-κB signaling, prevent excessive inflammatory reaction in the testis, and protect the integrity of the blood-testis barrier. In view of this newly identified Dmrt1- Spry1-NF-κB axis mechanism in the regulation of testicular immune homeostasis, our study opens new avenues for the prevention and treatment of male reproductive diseases in humans and livestock.

Plant miRNA osa-miR172d-5p suppressed lung fibrosis by targeting Tab1.

Lung fibrosis, including idiopathic pulmonary fibrosis, is an intractable disease accompanied by an irreversible dysfunction in the respiratory system. Its pathogenesis involves the transforming growth factorß (TGFß)-induced overproduction of the extracellular matrix from fibroblasts; however, limited countermeasures have been established. In this study, we identified osa-miR172d-5p, a plant-derived microRNA (miR), as a potent anti-fibrotic miR. In silico analysis followed by an in vitro assay based on human lung fibroblasts demonstrated that osa-miR172d-5p suppressed the gene expression of TGF-ß activated kinase 1 (MAP3K7) binding protein 1 (Tab1). It also suppressed the TGFß-induced fibrotic gene expression in human lung fibroblasts. To assess the anti-fibrotic effect of osa-miR172d-5p, we established bleomycin-induced lung fibrosis models to demonstrate that osa-miR172d-5p ameliorated lung fibrosis. Moreover, it suppressed Tab1 expression in the lung tissues of bleomycin-treated mice. In conclusion, osa-miR172d-5p could be a potent candidate for the treatment of lung fibrosis, including idiopathic pulmonary fibrosis.

Comprehensive classification of proteins based on structures that engage lipids by COMPOSEL.

Structures can now be predicted for any protein using programs like AlphaFold and Rosetta, which rely on a foundation of experimentally determined structures of architecturally diverse proteins. The accuracy of such artificial intelligence and machine learning (AI/ML) approaches benefits from the specification of restraints which assist in navigating the universe of folds to converge on models most representative of a given protein's physiological structure. This is especially pertinent for membrane proteins, with structures and functions that depend on their presence in lipid bilayers. Structures of proteins in their membrane environments could conceivably be predicted from AI/ML approaches with user-specificized parameters that describe each element of the architecture of a membrane protein accompanied by its lipid environment. We propose the Classification Of Membrane Proteins based On Structures Engaging Lipids (COMPOSEL), which builds on existing nomenclature types for monotopic, bitopic, polytopic and peripheral membrane proteins as well as lipids. Functional and regulatory elements are also defined in the scripts, as shown with membrane fusing synaptotagmins, multidomain PDZD8 and Protrudin proteins that recognize phosphoinositide (PI) lipids, the intrinsically disordered MARCKS protein, caveolins, the ß barrel assembly machine (BAM), an adhesion G-protein coupled receptor (aGPCR) and two lipid modifying enzymes - diacylglycerol kinase DGKε and fatty aldehyde dehydrogenase FALDH. This demonstrates how COMPOSEL communicates lipid interactivity as well as signaling mechanisms and binding of metabolites, drug molecules, polypeptides or nucleic acids to describe the operations of any protein. Moreover COMPOSEL can be scaled to express how genomes encode membrane structures and how our organs are infiltrated by pathogens such as SARS-CoV-2.

SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Adaptor protein MyD88 confers the susceptibility to stress via amplifying immune danger signals.

Increasing evidence supports the pathogenic role of neuroinflammation in psychiatric diseases, including major depressive disorder (MDD) and neuropsychiatric symptoms of Coronavirus disease 2019 (COVID-19); however, the precise mechanism and therapeutic strategy are poorly understood. Here, we report that myeloid differentiation factor 88 (MyD88), a pivotal adaptor that bridges toll-like receptors to their downstream signaling by recruiting the signaling complex called 'myddosome', was up-regulated in the medial prefrontal cortex (mPFC) after exposure to chronic social defeat stress (CSDS) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. The inducible expression of MyD88 in the mPFC primed neuroinflammation and conferred stress susceptibility via amplifying immune danger signals, such as high-mobility group box 1 and SARS-CoV-2 spike protein. Overexpression of MyD88 aggravated, whereas knockout or pharmacological inhibition of MyD88 ameliorated CSDS-induced depressive-like behavior. Notably, TJ-M2010-5, a novel synthesized targeting inhibitor of MyD88 dimerization, alleviated both CSDS- and SARS-CoV-2 spike protein-induced depressive-like behavior. Taken together, our findings indicate that inhibiting MyD88 signaling represents a promising therapeutic strategy for stress-related mental disorders, such as MDD and COVID-19-related neuropsychiatric symptoms.

Dok3 restrains neutrophil production of calprotectin during TLR4 sensing of SARS-CoV-2 spike protein.

Increased neutrophils and elevated level of circulating calprotectin are hallmarks of severe COVID-19 and they contribute to the dysregulated immune responses and cytokine storm in susceptible patients. However, the precise mechanism controlling calprotectin production during SARS-CoV-2 infection remains elusive. In this study, we showed that Dok3 adaptor restrains calprotectin production by neutrophils in response to SARS-CoV-2 spike (S) protein engagement of TLR4. Dok3 recruits SHP-2 to mediate the de-phosphorylation of MyD88 at Y257, thereby attenuating downstream JAK2-STAT3 signaling and calprotectin production. Blocking of TLR4, JAK2 and STAT3 signaling could prevent excessive production of calprotectin by Dok3-/- neutrophils, revealing new targets for potential COVID-19 therapy. As S protein from SARS-CoV-2 Delta and Omicron variants can activate TLR4-driven calprotectin production in Dok3-/- neutrophils, our study suggests that targeting calprotectin production may be an effective strategy to combat severe COVID-19 manifestations associated with these emerging variants.

Dipeptidylpeptidase (DPP)-4 inhibitor therapy increases circulating levels of anti-inflammatory soluble frizzle receptor protein (sFRP)-5 which is decreased in severe COVID-19 disease.

Obesity and type 2 diabetes (T2D) show an increased risk for a severe COVID-19 disease. Treatment with DPP4 inhibitor (DPP4i) results in reduced mortality and better clinical outcome. Here, we aimed to identify potential mechanisms for the observed DPP4i effect in COVID-19. Comparing T2D subjects with and without DPP4i treatment, we identified a significant increase of the anti-inflammatory adipokine sFRP5 in relation to DPP4 inhibition. sFRP5 is a specific antagonist to Wnt5a, a glycopeptide secreted by adipose tissue macrophages acting pro-inflammatory in various diseases. We therefore examined sFRP5 levels in patients hospitalised for severe COVID-19 and found significant lower levels compared to healthy controls. Since sFRP5 might consequently be a molecular link for the beneficial effects of DPP4i in COVID-19, we further aimed to identify the exact source of sFRP5 in adipose tissue on cellular level. We therefore isolated pre-adipocytes, mature adipocytes and macrophages from adipose tissue biopsies and performed western-blotting. Results showed a sFRP5 expression specifically in mature adipocytes of subcutaneous and omental adipose tissue. In summary, our data suggest that DPP4i increase serum levels of anti-inflammatory sFRP5 which might be beneficial in COVID-19, reflecting a state of sFRP5 deficiency.

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.

SARS-CoV-2 Nsp5 Activates NF-κB Pathway by Upregulating SUMOylation of MAVS.

The COVID-19 is an infectious disease caused by SARS-CoV-2 infection. A large number of clinical studies found high-level expression of pro-inflammatory cytokines in patients infected with SARS-CoV-2, which fuels the rapid development of the disease. However, the specific molecular mechanism is still unclear. In this study, we found that SARS-CoV-2 Nsp5 can induce the expression of cytokines IL-1ß, IL-6, TNF-α, and IL-2 in Calu-3 and THP1 cells. Further research found that Nsp5 enhances cytokine expression through activating the NF-κB signaling pathway. Subsequently, we investigated the upstream effectors of the NF-κB signal pathway on Nsp5 overexpression and discovered that Nsp5 increases the protein level of MAVS. Moreover, Nsp5 can promote the SUMOylation of MAVS to increase its stability and lead to increasing levels of MAVS protein, finally triggering activation of NF-κB signaling. The knockdown of MAVS and the inhibitor of SUMOylation treatment can attenuate Nsp5-mediated NF-κB activation and cytokine induction. We identified a novel role of SARS-CoV-2 Nsp5 to enhance cytokine production by activating the NF-κB signaling pathway.

YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion.

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.

SARS-CoV-2 ORF10 suppresses the antiviral innate immune response by degrading MAVS through mitophagy.

The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused severe morbidity and mortality in humans. It is urgent to understand the function of viral genes. However, the function of open reading frame 10 (ORF10), which is uniquely expressed by SARS-CoV-2, remains unclear. In this study, we showed that overexpression of ORF10 markedly suppressed the expression of type I interferon (IFN-I) genes and IFN-stimulated genes. Then, mitochondrial antiviral signaling protein (MAVS) was identified as the target via which ORF10 suppresses the IFN-I signaling pathway, and MAVS was found to be degraded through the ORF10-induced autophagy pathway. Furthermore, overexpression of ORF10 promoted the accumulation of LC3 in mitochondria and induced mitophagy. Mechanistically, ORF10 was translocated to mitochondria by interacting with the mitophagy receptor Nip3-like protein X (NIX) and induced mitophagy through its interaction with both NIX and LC3B. Moreover, knockdown of NIX expression blocked mitophagy activation, MAVS degradation, and IFN-I signaling pathway inhibition by ORF10. Consistent with our observations, in the context of SARS-CoV-2 infection, ORF10 inhibited MAVS expression and facilitated viral replication. In brief, our results reveal a novel mechanism by which SARS-CoV-2 inhibits the innate immune response; that is, ORF10 induces mitophagy-mediated MAVS degradation by binding to NIX.

Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities.

Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, most current large-scale methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a viral peptide discovery approach covering 23 coronavirus strains that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an ΦxFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its ΦxFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction dampened SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.

SARS-CoV-2 nucleocapsid protein interacts with immunoregulators and stress granules and phase separates to form liquid droplets.

The current work investigated SARS-CoV-2 Nucleocapsid (NCAP or N protein) interactors in A549 human lung cancer cells using a SILAC-based mass spectrometry approach. NCAP interactors included proteins of the stress granule (SG) machinery and immunoregulators. NCAP showed specific interaction with the SG proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following oxidative stress and heat shock. Treatment of recombinant NCAP with RNA isolated from A549 cells exposed to oxidative stress-stimulated NCAP to undergo liquid-liquid phase separation (LLPS). RNA degradation using RNase A treatment completely blocked the LLPS property of NCAP as well as its SG association. The RNA intercalator mitoxantrone also disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS-CoV-2 NCAP.

Ultraviolet-A light increases mitochondrial anti-viral signaling protein in confluent human tracheal cells via cell-cell signaling.

Mitochondrial antiviral signaling (MAVS) protein mediates innate antiviral responses, including responses to certain coronaviruses such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We have previously shown that ultraviolet-A (UVA) therapy can prevent virus-induced cell death in human ciliated tracheal epithelial cells (HTEpC) infected with coronavirus-229E (CoV-229E), and results in increased intracellular levels of MAVS. In this study, we explored the mechanisms by which UVA light can activate MAVS, and whether local UVA light application can activate MAVS at locations distant from the light source (e.g. via cell-to-cell communication). MAVS levels were compared in HTEpC exposed to 2 mW/cm2 narrow band (NB)-UVA for 20 min and in unexposed controls at 30-40% and at 100% confluency, and in unexposed HTEpC treated with supernatants or lysates from UVA-exposed cells or from unexposed controls. MAVS was also assessed in different sections of confluent monolayer plates where only one section was exposed to NB-UVA. Our results showed that UVA increases the expression of MAVS protein. Further, cells in a confluent monolayer exposed to UVA conferred an elevation in MAVS in cells adjacent to the exposed section, and also in cells in the most distant sections which were not exposed to UVA. In this study, human ciliated tracheal epithelial cells exposed to UVA demonstrate increased MAVS protein, and also appear to transmit this influence to confluent cells not exposed to UVA, likely via cell-cell signaling.

Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

Identification of ZDHHC17 as a Potential Drug Target for Swine Acute Diarrhea Syndrome Coronavirus Infection.

The recent emergence and spread of zoonotic viruses highlights that animal-sourced viruses are the biggest threat to global public health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an HKU2-related bat coronavirus that was spilled over from Rhinolophus bats to swine, causing large-scale outbreaks of severe diarrhea disease in piglets in China. Unlike other porcine coronaviruses, SADS-CoV possesses broad species tissue tropism, including primary human cells, implying a significant risk of cross-species spillover. To explore host dependency factors for SADS-CoV as therapeutic targets, we employed genome-wide CRISPR knockout library screening in HeLa cells. Consistent with two independent screens, we identified the zinc finger DHHC-type palmitoyltransferase 17 (ZDHHC17 or ZD17) as an important host factor for SADS-CoV infection. Through truncation mutagenesis, we demonstrated that the DHHC domain of ZD17 that is involved in palmitoylation is important for SADS-CoV infection. Mechanistic studies revealed that ZD17 is required for SADS-CoV genomic RNA replication. Treatment of infected cells with the palmitoylation inhibitor 2-bromopalmitate (2-BP) significantly suppressed SADS-CoV infection. Our findings provide insight on SADS-CoV-host interactions and a potential therapeutic application. IMPORTANCE The recent emergence of deadly zoonotic viral diseases, including Ebola virus and SARS-CoV-2, emphasizes the importance of pandemic preparedness for the animal-sourced viruses with potential risk of animal-to-human spillover. Over the last 2 decades, three significant coronaviruses of bat origin, SARS-CoV, MERS-CoV, and SARS-CoV-2, have caused millions of deaths with significant economy and public health impacts. Lack of effective therapeutics against these coronaviruses was one of the contributing factors to such losses. Although SADS-CoV, another coronavirus of bat origin, was only known to cause fatal diarrhea disease in piglets, the ability to infect cells derived from multiple species, including human, highlights the potential risk of animal-to-human spillover. As part of our effort in pandemic preparedness, we explore SADS-CoV host dependency factors as targets for host-directed therapeutic development and found zinc finger DHHC-type palmitoyltransferase 17 is a promising drug target against SADS-CoV replication. We also demonstrated that a palmitoylation inhibitor, 2-bromopalmitate (2-BP), can be used as an inhibitor for SADS-CoV treatment.

SARS-CoV-2 suppresses IFNß production mediated by NSP1, 5, 6, 15, ORF6 and ORF7b but does not suppress the effects of added interferon.

Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.

SARS-CoV-2 Nsp5 Demonstrates Two Distinct Mechanisms Targeting RIG-I and MAVS To Evade the Innate Immune Response.

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with astonishing mortality and morbidity. The high replication and transmission of SARS-CoV-2 are remarkably distinct from those of previous closely related coronaviruses, and the underlying molecular mechanisms remain unclear. The innate immune defense is a physical barrier that restricts viral replication. We report here that the SARS-CoV-2 Nsp5 main protease targets RIG-I and mitochondrial antiviral signaling (MAVS) protein via two distinct mechanisms for inhibition. Specifically, Nsp5 cleaves off the 10 most-N-terminal amino acids from RIG-I and deprives it of the ability to activate MAVS, whereas Nsp5 promotes the ubiquitination and proteosome-mediated degradation of MAVS. As such, Nsp5 potently inhibits interferon (IFN) induction by double-stranded RNA (dsRNA) in an enzyme-dependent manner. A synthetic small-molecule inhibitor blunts the Nsp5-mediated destruction of cellular RIG-I and MAVS and processing of SARS-CoV-2 nonstructural proteins, thus restoring the innate immune response and impeding SARS-CoV-2 replication. This work offers new insight into the immune evasion strategy of SARS-CoV-2 and provides a potential antiviral agent to treat CoV disease 2019 (COVID-19) patients. IMPORTANCE The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.

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.

Differential roles of RIG-I like receptors in SARS-CoV-2 infection.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) sense viral RNA and activate antiviral immune responses. Herein we investigate their functions in human epithelial cells, the primary and initial target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A deficiency in MDA5, RIG-I or mitochondrial antiviral signaling protein (MAVS) enhanced viral replication. The expression of the type I/III interferon (IFN) during infection was impaired in MDA5-/- and MAVS-/-, but not in RIG-I-/-, when compared to wild type (WT) cells. The mRNA level of full-length angiotensin-converting enzyme 2 (ACE2), the cellular entry receptor for SARS-CoV-2, was ~ 2.5-fold higher in RIG-I-/- than WT cells. These data demonstrate MDA5 as the predominant SARS-CoV-2 sensor, IFN-independent induction of ACE2 and anti-SARS-CoV-2 role of RIG-I in epithelial cells.