CD19+ B cell values predict the increase of anti-SARS CoV2 antibodies in fingolimod-treated and COVID-19-vaccinated patients with multiple sclerosis.

BACKGROUND: Treatment with fingolimod for multiple sclerosis (MS) reduces the efficacy of COVID-19 vaccination. The aim of this exploratory study was to evaluate whether main lymphocyte subsets and demographic features correlated to the subsequent increase in anti-SARS-CoV2 antibodies following the third dose of COVID-19 vaccination in fingolimod-treated MS patients. METHODS: This was a prospective single-center observational exploratory study including a subgroup of adult patients with MS (pwMS) in treatment with fingolimod who underwent COVID-19 vaccination. The association of anti-SARS-CoV2 antibody levels (reported as the Log10 of the difference between the post and pre third dose levels) with the total number and percentage of CD3+ T and CD19+ B was assessed by a linear regression model adjusted for age and sex. RESULTS: We found that peripheral blood CD19+ B lymphocytes before the third dose of vaccination in pwMS treated with fingolimod predict the subsequent increase of anti-SARS-CoV2 antibodies. CONCLUSION: This work suggests that evaluating the percentage of CD19+ B cells may be important to identify patients at risk of not producing SARS-CoV-2 antibodies, with possible reduced protection from COVID-19.

PCIF1-mediated deposition of 5'-cap N6,2'-O-dimethyladenosine in ACE2 and TMPRSS2 mRNA regulates susceptibility to SARS-CoV-2 infection.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a major health problem worldwide. Due to the fast emergence of SARS-CoV-2 variants, understanding the molecular mechanisms of viral pathogenesis and developing novel inhibitors are essential and urgent. Here, we investigated the potential roles of N6,2'-O-dimethyladenosine (m6Am), one of the most abundant modifications of eukaryotic messenger ribonucleic acid (mRNAs), in SARS-CoV-2 infection of human cells. Using genome-wide m6Am-exo-seq, RNA sequencing analysis, and Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, we demonstrate that phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1), a cap-specific adenine N6-methyltransferase, plays a major role in facilitating infection of primary human lung epithelial cells and cell lines by SARS-CoV-2, variants of concern, and other coronaviruses. We show that PCIF1 promotes infection by sustaining expression of the coronavirus receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) via m6Am-dependent mRNA stabilization. In PCIF1-depleted cells, both ACE2/TMPRSS2 expression and viral infection are rescued by re-expression of wild-type, but not catalytically inactive, PCIF1. These findings suggest a role for PCIF1 and cap m6Am in regulating SARS-CoV-2 susceptibility and identify a potential therapeutic target for prevention of infection.

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.

Genetic variants in the NF-κB signaling pathway (NFKB1, NFKBIA, NFKBIZ) and risk of critical outcome among COVID-19 patients.

The NF-κB signaling pathway is a key regulator of inflammation in the response to SARS-CoV-2 infection. This pathway has been implicated in the hyperinflammatory state that characterizes the severe forms of COVID-19. The genetic variation of the NF-κB components might thus explain the predisposition to critical outcomes of this viral disease. We aimed to study the role of the common NFKB1 rs28362491, NFKBIA rs696 and NFKBIZ rs3217713 variants in the risk of developing severe COVID-19 with ICU admission. A total of 470 Spanish patients requiring respiratory support in the ICU were studied (99 deceased and 371 survivors). Compared to healthy population controls (N = 300), the NFKBIA rs696 GG genotype was increased in the patients (p = 0.045; OR = 1.37). The NFKBIZ rs3217713 insertion homozygosis was associated with a significant risk of death (p = 0.02; OR = 1.76) and was also related to increased D-dimer values (p = 0.0078, OR = 1.96). This gene has been implicated in sepsis in mice and rats. Moreover, we found a trend toward lower expression of the NFKBIZ transcript in total blood from II patients. In conclusion, variants in the NF-κB genes might be associated with the risk of developing severe COVID-19, with a significant effect of the NFKBIZ gene on mortality. Our results were based on a limited number of patients and require validation in larger cohorts from other populations.

Therapeutic role of mTOR inhibitors in control of SARS-CoV-2 viral replication.

By the end of 2019, COVID-19 was reported in Wuhan city of China, and through human-human transmission, this virus spread worldwide and became a pandemic. Initial symptoms of the disease include fever, cough, loss of smell, taste, and shortness of breath, but a decrease in the oxygen levels in the body leads, and pneumonia may ultimately lead to the patient's death. However, the symptoms vary from patient to patient. To understand COVID-19 disease pathogenesis, researchers have tried to understand the cellular pathways that could be targeted to suppress viral replication. Thus, this article reviews the markers that could be targeted to inhibit viral replication by inhibiting the translational initiation complex/regulatory kinases and upregulating host autophagic flux that may lead to a reduction in the viral load. The article also highlights that mTOR inhibitors may act as potential inhibitors of viral replication. mTOR inhibitors such as metformin may inhibit the interaction of SARS-CoV-2 Nsp's and ORFs with mTORC1, LARP1, and 4E-BP. They may also increase autophagic flux by decreasing protein degradation via inhibition of Skp2, further promoting viral cell death. These events result in cell cycle arrest at G1 by p27, ultimately causing cell death.

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.

Specific Antibody and the T-Cell Response Elicited by BNT162b2 Boosting After Two ChAdOx1 nCoV-19 in Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID) patients have markedly decreased immune response to vaccinations. In this study we evaluated humoral and T cell-mediated responses against severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2) with additional flow cytometric changes in CVID patients receiving booster vaccination with BNT162b2 after two ChAdOx1 nCoV-19. The BNT162b2 vaccine raised the anti-spike protein S immunoglobulin G over the cut-off value from 70% to 83% in CVID, anti-neutralizing antibody had been raised over a cut-off value from 70% to 80% but levels after boosting were significantly less in both tests than in healthy controls (*p=0.02; **p=0.009 respectively). Anti-SARS-CoV-2 immunoglobulin A became less positive in CVID after boosting, but the difference was not significant. The cumulative interferon-γ positive T cell response by ELISpot was over the cut-off value in 53% of the tested individuals and raised to 83% after boosting. This and flow cytometric control of cumulative CD4+ and CD8+ virus-specific T cell absolute counts in CVID were also statistically not different from healthy individuals after boosting. Additional flow cytometric measures for CD45+ lymphocytes, CD3+, and CD19+ cells have not shown significant differences from controls except for lower CD4+T cell counts at both time points (**p=0.003; **p=0.002), in parallel CD4+ virus-specific T-cell ratio was significantly lower in CVID patients at the first time point (*p: 0.03). After boosting, in more than 33% of both CVID patients and also in their healthy controls we detected a decrease in absolute CD45+, CD3+, CD3+CD4+, and CD3+CD8+, CD19+, and CD16+56+ cell counts. CD16+CD56+ cell counts were significantly lower compared to controls before and after boosting (*p=0.02, *p=0.02). CVID patients receiving immunosuppressive therapy throughout the previous year or autologous stem cell transplantation two years before vaccination had worse responses in anti-spike, anti-neutralizing antibody, CD3+CD4+T, CD19+ B, and natural killer cell counts than the whole CVID group. Vaccinations had few side effects. Based on these data, CVID patients receiving booster vaccination with BNT162b2 after two ChadOx1 can effectively elevate the levels of protection against COVID-19 infection, but the duration of the immune response together with COVID-19 morbidity data needs further investigation among these patients.

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.

XAF1 Protects Host against Emerging RNA Viruses by Stabilizing IRF1-Dependent Antiviral Immunity.

XIAP-associated factor 1 (XAF1) is an interferon (IFN)-stimulated gene (ISG) that enhances IFN-induced apoptosis. However, it is unexplored whether XAF1 is essential for the host fighting against invaded viruses. Here, we find that XAF1 is significantly upregulated in the host cells infected with emerging RNA viruses, including influenza, Zika virus (ZIKV), and SARS-CoV-2. IFN regulatory factor 1 (IRF1), a key transcription factor in immune cells, determines the induction of XAF1 during antiviral immunity. Ectopic expression of XAF1 protects host cells against various RNA viruses independent of apoptosis. Knockout of XAF1 attenuates host antiviral innate immunity in vitro and in vivo, which leads to more severe lung injuries and higher mortality in the influenza infection mouse model. XAF1 stabilizes IRF1 protein by antagonizing the CHIP-mediated degradation of IRF1, thus inducing more antiviral IRF1 target genes, including DDX58, DDX60, MX1, and OAS2. Our study has described a protective role of XAF1 in the host antiviral innate immunity against RNA viruses. We have also elucidated the molecular mechanism that IRF1 and XAF1 form a positive feedback loop to induce rapid and robust antiviral immunity. IMPORTANCE Rapid and robust induction of antiviral genes is essential for the host to clear the invaded viruses. In addition to the IRF3/7-IFN-I-STAT1 signaling axis, the XAF1-IRF1 positive feedback loop synergistically or independently drives the transcription of antiviral genes. Moreover, XAF1 is a sensitive and reliable gene that positively correlates with the viral infection, suggesting that XAF1 is a potential diagnostic marker for viral infectious diseases. In addition to the antitumor role, our study has shown that XAF1 is essential for antiviral immunity. XAF1 is not only a proapoptotic ISG, but it also stabilizes the master transcription factor IRF1 to induce antiviral genes. IRF1 directly binds to the IRF-Es of its target gene promoters and drives their transcriptions, which suggests a unique role of the XAF1-IRF1 loop in antiviral innate immunity, particularly in the host defect of IFN-I signaling such as invertebrates.

The MAVS Immune Recognition Pathway in Viral Infection and Sepsis.

Significance: It is estimated that close to 50 million cases of sepsis result in over 11 million annual fatalities worldwide. The pathognomonic feature of sepsis is a dysregulated inflammatory response arising from viral, bacterial, or fungal infections. Immune recognition of pathogen-associated molecular patterns is a hallmark of the host immune defense to combat microbes and to prevent the progression to sepsis. Mitochondrial antiviral signaling protein (MAVS) is a ubiquitous adaptor protein located at the outer mitochondrial membrane, which is activated by the cytosolic pattern recognition receptors, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation associated gene 5 (MDA5), following binding of viral RNA agonists. Recent Advances: Substantial progress has been made in deciphering the activation of the MAVS pathway with its interacting proteins, downstream signaling events (interferon [IFN] regulatory factors, nuclear factor kappa B), and context-dependent type I/III IFN response. Critical Issues: In the evolutionary race between pathogens and the host, viruses have developed immune evasion strategies for cleavage, degradation, or blockade of proteins in the MAVS pathway. For example, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M protein and ORF9b protein antagonize MAVS signaling and a protective type I IFN response. Future Directions: The role of MAVS as a sensor for nonviral pathogens, host cell injury, and metabolic perturbations awaits better characterization in the future. New technical advances in multidimensional single-cell analysis and single-molecule methods will accelerate the rate of new discoveries. The ultimate goal is to manipulate MAVS activities in the form of immune-modulatory therapies to combat infections and sepsis. Antioxid. Redox Signal. 35, 1376-1392.

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.

Lipopolysaccharide Responsive Beige-like Anchor Protein Deficiency in a Patient with Autoimmune Lymphoproliferative Syndrome-like Disease Phenotype: A Case Report and Literature Review.

LPS-responsive beige-like anchor protein (LRBA) deficiency is a primary immunodeficiency caused by a mutation in the LRBA gene. Affected individuals present with a variety of clinical symptoms including hypogammaglobulinemia, recurrent infections, splenomegaly, hepatomegaly, and autoimmune cytopenias. Except for hypogammaglobulinemia, the remaining features resemble autoimmune lymphoproliferative syndrome (ALPS). Here, we report the case of a 14-year-old boy with the ALPS phenotype, eventually diagnosed with LRBA deficiency. He presented with lymphadenopathy and hepatosplenomegaly, along with autoimmune cytopenia. Due to recurrent infections and worsening gastrointestinal symptoms, whole-exome sequencing was conducted and revealed a novel homozygous pathogenic variant in the LRBA gene (c.534del; p.9Asp179IIef*16). The patient recently suffered from clinical deterioration due to SARS-COV-2 which appears to have triggered an acute worsening of his existing Cytomegalovirus colitis leading to an eventual demise. A literature search for reported LRBA deficient patients with ALPS-like phenotype revealed 11 patients. The most common clinical presentations in LRBA patients with ALPS-like phenotype included autoimmunity (100%), splenomegaly (91%), lymphadenopathy (36.4%), and respiratory tract infections (63.6%). LRBA deficiency is unique in the fact that it encompasses immune deficiency, autoimmunity, and lymphoproliferation. In children with multiple symptoms related to these domains, a genetic diagnosis is necessary to ensure tailored and precise medical therapy.

Autoantibodies Against Proteins Previously Associated With Autoimmunity in Adult and Pediatric Patients With COVID-19 and Children With MIS-C.

The antibody profile against autoantigens previously associated with autoimmune diseases and other human proteins in patients with COVID-19 or multisystem inflammatory syndrome in children (MIS-C) remains poorly defined. Here we show that 30% of adults with COVID-19 had autoantibodies against the lung antigen KCNRG, and 34% had antibodies to the SLE-associated Smith-D3 protein. Children with COVID-19 rarely had autoantibodies; one of 59 children had GAD65 autoantibodies associated with acute onset of insulin-dependent diabetes. While autoantibodies associated with SLE/Sjögren's syndrome (Ro52, Ro60, and La) and/or autoimmune gastritis (gastric ATPase) were detected in 74% (40/54) of MIS-C patients, further analysis of these patients and of children with Kawasaki disease (KD), showed that the administration of intravenous immunoglobulin (IVIG) was largely responsible for detection of these autoantibodies in both groups of patients. Monitoring in vivo decay of the autoantibodies in MIS-C children showed that the IVIG-derived Ro52, Ro60, and La autoantibodies declined to undetectable levels by 45-60 days, but gastric ATPase autoantibodies declined more slowly requiring >100 days until undetectable. Further testing of IgG and/or IgA antibodies against a subset of potential targets identified by published autoantigen array studies of MIS-C failed to detect autoantibodies against most (16/18) of these proteins in patients with MIS-C who had not received IVIG. However, Troponin C2 and KLHL12 autoantibodies were detected in 2 of 20 and 1 of 20 patients with MIS-C, respectively. Overall, these results suggest that IVIG therapy may be a confounding factor in autoantibody measurements in MIS-C and that antibodies against antigens associated with autoimmune diseases or other human proteins are uncommon in MIS-C.

Risk-focused differences in molecular processes implicated in SARS-CoV-2 infection: corollaries in DNA methylation and gene expression.

BACKGROUND: Understanding the molecular basis of susceptibility factors to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global health imperative. It is well-established that males are more likely to acquire SARS-CoV-2 infection and exhibit more severe outcomes. Similarly, exposure to air pollutants and pre-existing respiratory chronic conditions, such as asthma and chronic obstructive respiratory disease (COPD) confer an increased risk to coronavirus disease 2019 (COVID-19). METHODS: We investigated molecular patterns associated with risk factors in 398 candidate genes relevant to COVID-19 biology. To accomplish this, we downloaded DNA methylation and gene expression data sets from publicly available repositories (GEO and GTEx Portal) and utilized data from an empirical controlled human exposure study conducted by our team. RESULTS: First, we observed sex-biased DNA methylation patterns in autosomal immune genes, such as NLRP2, TLE1, GPX1, and ARRB2 (FDR < 0.05, magnitude of DNA methylation difference Δß > 0.05). Second, our analysis on the X-linked genes identified sex associated DNA methylation profiles in genes, such as ACE2, CA5B, and HS6ST2 (FDR < 0.05, Δß > 0.05). These associations were observed across multiple respiratory tissues (lung, nasal epithelia, airway epithelia, and bronchoalveolar lavage) and in whole blood. Some of these genes, such as NLRP2 and CA5B, also exhibited sex-biased gene expression patterns. In addition, we found differential DNA methylation patterns by COVID-19 status for genes, such as NLRP2 and ACE2 in an exploratory analysis of an empirical data set reporting on human COVID-9 infections. Third, we identified modest DNA methylation changes in CpGs associated with PRIM2 and TATDN1 (FDR < 0.1, Δß > 0.05) in response to particle-depleted diesel exhaust in bronchoalveolar lavage. Finally, we captured a DNA methylation signature associated with COPD diagnosis in a gene involved in nicotine dependence (COMT) (FDR < 0.1, Δß > 0.05). CONCLUSION: Our findings on sex differences might be of clinical relevance given that they revealed molecular associations of sex-biased differences in COVID-19. Specifically, our results hinted at a potentially exaggerated immune response in males linked to autosomal genes, such as NLRP2. In contrast, our findings at X-linked loci such as ACE2 suggested a potentially distinct DNA methylation pattern in females that may interact with its mRNA expression and inactivation status. We also found tissue-specific DNA methylation differences in response to particulate exposure potentially capturing a nitrogen dioxide (NO2) effect-a contributor to COVID-19 susceptibility. While we identified a molecular signature associated with COPD, all COPD-affected individuals were smokers, which may either reflect an association with the disease, smoking, or may highlight a compounded effect of these two risk factors in COVID-19. Overall, our findings point towards a molecular basis of variation in susceptibility factors that may partly explain disparities in the risk for SARS-CoV-2 infection.

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.