A virus‐derived microRNA targets immune response genes during SARS‐CoV‐2 infection

SARS‐CoV‐2 infection results in impaired interferon response in patients with severe COVID‐19. However, how SARS‐CoV‐2 interferes with host immune responses is incompletely understood. Here, we sequence small RNAs from SARS‐CoV‐2‐infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus‐derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3′UTR of interferon‐stimulated genes and represses their expression in a miRNA‐like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID‐19 patients. We propose that SARS‐CoV‐2 can potentially employ a virus‐derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon‐mediated immune response.     

TMPRSS2 expression dictates the entry route used by SARS‐CoV‐2 to infect host cells

SARS‐CoV‐2 is a newly emerged coronavirus that caused the global COVID‐19 outbreak in early 2020. COVID‐19 is primarily associated with lung injury, but many other clinical symptoms such as loss of smell and taste demonstrated broad tissue tropism of the virus. Early SARS‐CoV‐2–host cell interactions and entry mechanisms remain poorly understood. Investigating SARS‐CoV‐2 infection in tissue culture, we found that the protease TMPRSS2 determines the entry pathway used by the virus. In the presence of TMPRSS2, the proteolytic process of SARS‐CoV‐2 was completed at the plasma membrane, and the virus rapidly entered the cells within 10 min in a pH‐independent manner. When target cells lacked TMPRSS2 expression, the virus was endocytosed and sorted into endolysosomes, from which SARS‐CoV‐2 entered the cytosol via acid‐activated cathepsin L protease 40–60 min post‐infection. Overexpression of TMPRSS2 in non‐TMPRSS2 expressing cells abolished the dependence of infection on the cathepsin L pathway and restored sensitivity to the TMPRSS2 inhibitors. Together, our results indicate that SARS‐CoV‐2 infects cells through distinct, mutually exclusive entry routes and highlight the importance of TMPRSS2 for SARS‐CoV‐2 sorting into either pathway.     

SARS‐CoV‐2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage

SARS‐CoV‐2 is an emerging coronavirus that causes dysfunctions in multiple human cells and tissues. Studies have looked at the entry of SARS‐CoV‐2 into host cells mediated by the viral spike protein and human receptor ACE2. However, less is known about the cellular immune responses triggered by SARS‐CoV‐2 viral proteins. Here, we show that the nucleocapsid of SARS‐CoV‐2 inhibits host pyroptosis by blocking Gasdermin D (GSDMD) cleavage. SARS‐CoV‐2‐infected monocytes show enhanced cellular interleukin‐1β (IL‐1β) expression, but reduced IL‐1β secretion. While SARS‐CoV‐2 infection promotes activation of the NLRP3 inflammasome and caspase‐1, GSDMD cleavage and pyroptosis are inhibited in infected human monocytes. SARS‐CoV‐2 nucleocapsid protein associates with GSDMD in cells and inhibits GSDMD cleavage in vitro and in vivo. The nucleocapsid binds the GSDMD linker region and hinders GSDMD processing by caspase‐1. These insights into how SARS‐CoV‐2 antagonizes cellular inflammatory responses may open new avenues for treating COVID‐19 in the future.     

Molecular basis of cross‐species ACE2 interactions with SARS‐CoV‐2‐like viruses of pangolin origin

Pangolins have been suggested as potential reservoir of zoonotic viruses, including SARS‐CoV‐2 causing the global COVID‐19 outbreak. Here, we study the binding of two SARS‐CoV‐2‐like viruses isolated from pangolins, GX/P2V/2017 and GD/1/2019, to human angiotensin‐converting enzyme 2 (hACE2), the receptor of SARS‐CoV‐2. We find that the spike protein receptor‐binding domain (RBD) of pangolin CoVs binds to hACE2 as efficiently as the SARS‐CoV‐2 RBD in vitro. Furthermore, incorporation of pangolin CoV RBDs allows entry of pseudotyped VSV particles into hACE2‐expressing cells. A screen for binding of pangolin CoV RBDs to ACE2 orthologs from various species suggests a broader host range than that of SARS‐CoV‐2. Additionally, cryo‐EM structures of GX/P2V/2017 and GD/1/2019 RBDs in complex with hACE2 show their molecular binding in modes similar to SARS‐CoV‐2 RBD. Introducing the Q498H substitution found in pangolin CoVs into the SARS‐CoV‐2 RBD expands its binding capacity to ACE2 homologs of mouse, rat, and European hedgehog. These findings suggest that these two pangolin CoVs may infect humans, highlighting the necessity of further surveillance of pangolin CoVs.     

Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and outbreaks of new variants highlight the need for preventive treatments. Here, we identified heparan sulfate proteoglycans as attachment receptors for SARS‐CoV‐2. Notably, neutralizing antibodies against SARS‐CoV‐2 isolated from COVID‐19 patients interfered with SARS‐CoV‐2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS‐CoV‐2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS‐CoV‐2, both DC subsets efficiently captured SARS‐CoV‐2 via heparan sulfate proteoglycans and transmitted the virus to ACE2‐positive cells. Notably, human primary nasal cells were infected by SARS‐CoV‐2, and infection was blocked by pre‐treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS‐CoV‐2 infection.     

Genome‐scale metabolic modeling reveals SARS‐CoV‐2‐induced metabolic changes and antiviral targets

Tremendous progress has been made to control the COVID‐19 pandemic caused by the SARS‐CoV‐2 virus. However, effective therapeutic options are still rare. Drug repurposing and combination represent practical strategies to address this urgent unmet medical need. Viruses, including coronaviruses, are known to hijack host metabolism to facilitate viral proliferation, making targeting host metabolism a promising antiviral approach. Here, we describe an integrated analysis of 12 published in vitro and human patient gene expression datasets on SARS‐CoV‐2 infection using genome‐scale metabolic modeling (GEM), revealing complicated host metabolism reprogramming during SARS‐CoV‐2 infection. We next applied the GEM‐based metabolic transformation algorithm to predict anti‐SARS‐CoV‐2 targets that counteract the virus‐induced metabolic changes. We successfully validated these targets using published drug and genetic screen data and by performing an siRNA assay in Caco‐2 cells. Further generating and analyzing RNA‐sequencing data of remdesivir‐treated Vero E6 cell samples, we predicted metabolic targets acting in combination with remdesivir, an approved anti‐SARS‐CoV‐2 drug. Our study provides clinical data‐supported candidate anti‐SARS‐CoV‐2 targets for future evaluation, demonstrating host metabolism targeting as a promising antiviral strategy.     

Characterization of the SARS‐CoV‐2 E Protein: Sequence,Structure, Viroporin,and Inhibitors

The COVID‐19 epidemic is one of the most influential epidemics in history. Understanding the impact of coronaviruses (CoVs) on host cells is very important for disease treatment. The SARS‐CoV‐2 envelope (E) protein is a small structural protein involved in many aspects of the viral life cycle. The E protein promotes the packaging and reproduction of the virus, and deletion of this protein weakens or even abolishes the virulence. This review aims to establish new knowledge by combining recent advances in the study of the SARS‐CoV‐2 E protein and by comparing it with the SARS‐CoV E protein. The E protein amino acid sequence, structure, self‐assembly characteristics, viroporin mechanisms and inhibitors are summarized and analyzed herein. Although the mechanisms of the SARS‐CoV‐2 and SARS‐CoV E proteins are similar in many respects, specific studies on the SARS‐CoV‐2 E protein, for both monomers and oligomers, are still lacking. A comprehensive understanding of this protein should prompt further studies on the design and characterization of effective targeted therapeutic measures.     

An organoid‐derived bronchioalveolar model for SARS‐CoV‐2 infection of human alveolar type II‐like cells

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causes coronavirus disease 2019 (COVID‐19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air–liquid interface culture system which was characterized by confocal and electron microscopy and single‐cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self‐renewing fetal lung bud tip organoids. These cultures were readily infected by SARS‐CoV‐2 with mainly surfactant protein C‐positive alveolar type II‐like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS‐CoV‐2 infection and can be applied for drug screens.     

Vitamin D and COVID‐19: A review on the role of vitamin D in preventing and reducing the severity of COVID‐19 infection

Severe Acute Respiratory Syndrome Coronavirus‐2 (SARS‐CoV‐2) is a pathogenic coronavirus causing COVID‐19 infection. The interaction between the SARS‐CoV‐2 spike protein and the human receptor angiotensin‐converting enzyme 2, both of which contain several cysteine residues, is impacted by the disulfide‐thiol balance in the host cell. The host cell redox status is affected by oxidative stress due to the imbalance between the reactive oxygen/nitrogen species and antioxidants. Recent studies have shown that Vitamin D supplementation could reduce oxidative stress. It has also been proposed that vitamin D at physiological concentration has preventive effects on many viral infections, including COVID‐19. However, the molecular‐level picture of the interplay of vitamin D deficiency, oxidative stress, and the severity of COVID‐19 has remained unclear. Herein, we present a thorough review focusing on the possible molecular mechanism by which vitamin D could alter host cell redox status and block viral entry, thereby preventing COVID‐19 infection or reducing the severity of the disease.     

Interactomes of SARS‐CoV‐2 and human coronaviruses reveal host factors potentially affecting pathogenesis

Host–virus protein–protein interactions play key roles in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity‐labeling strategies and identified 437 human proteins as the high‐confidence interacting proteins. Further characterization of these interactions and comparison to other large‐scale study of cellular responses to SARS‐CoV‐2 infection elucidated how distinct SARS‐CoV‐2 viral proteins participate in its life cycle. With these data mining, we discovered potential drug targets for the treatment of COVID‐19. The interactomes of two key SARS‐CoV‐2‐encoded viral proteins, NSP1 and N, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein–protein interactions that may explain differences in disease pathology. This comprehensive interactome of SARS‐CoV‐2 provides valuable resources for the understanding and treating of this disease.     

SARS‐CoV‐2 sensing by RIG‐I and MDA5 links epithelial infection to macrophage inflammation

SARS‐CoV‐2 infection causes broad‐spectrum immunopathological disease, exacerbated by inflammatory co‐morbidities. A better understanding of mechanisms underpinning virus‐associated inflammation is required to develop effective therapeutics. Here, we discover that SARS‐CoV‐2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through the activation of cytoplasmic RNA sensors RIG‐I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing or by inhibiting downstream signalling pathways. SARS‐CoV‐2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS‐CoV‐2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation‐associated COVID‐19.     

Biparatopic nanobodies protect mice from lethal challenge with SARS‐CoV‐2 variants of concern

The ongoing COVID‐19 pandemic and the emergence of new SARS‐CoV‐2 variants of concern (VOCs) requires continued development of effective therapeutics. Recently, we identified high‐affinity neutralizing nanobodies (Nbs) specific for the receptor‐binding domain (RBD) of SARS‐CoV‐2. Taking advantage of detailed epitope mapping, we generate two biparatopic Nbs (bipNbs) targeting a conserved epitope outside and two different epitopes inside the RBD:ACE2 interface. Both bipNbs bind all currently circulating VOCs with high affinities and are capable to neutralize cellular infection with VOC B.1.351 (Beta) and B.1.617.2 (Delta) in vitro. To assess if the bipNbs NM1267 and NM1268 confer protection against SARS‐CoV‐2 infection in vivo, human ACE2 transgenic mice are treated intranasally before infection with a lethal dose of SARS‐CoV‐2 B.1, B.1.351 (Beta) or B.1.617.2 (Delta). Nb‐treated mice show significantly reduced disease progression and increased survival rates. Histopathological analyses further reveal a drastically reduced viral load and inflammatory response in lungs. These data suggest that both bipNbs are broadly active against a variety of emerging SARS‐CoV‐2 VOCs and represent easily applicable drug candidates.     

Machine learning identifies molecular regulators and therapeutics for targeting SARS‐CoV2‐induced cytokine release

Although 15–20% of COVID‐19 patients experience hyper‐inflammation induced by massive cytokine production, cellular triggers of this process and strategies to target them remain poorly understood. Here, we show that the N‐terminal domain (NTD) of the SARS‐CoV‐2 spike protein substantially induces multiple inflammatory molecules in myeloid cells and human PBMCs. Using a combination of phenotypic screening with machine learning‐based modeling, we identified and experimentally validated several protein kinases, including JAK1, EPHA7, IRAK1, MAPK12, and MAP3K8, as essential downstream mediators of NTD‐induced cytokine production, implicating the role of multiple signaling pathways in cytokine release. Further, we found several FDA‐approved drugs, including ponatinib, and cobimetinib as potent inhibitors of the NTD‐mediated cytokine release. Treatment with ponatinib outperforms other drugs, including dexamethasone and baricitinib, inhibiting all cytokines in response to the NTD from SARS‐CoV‐2 and emerging variants. Finally, ponatinib treatment inhibits lipopolysaccharide‐mediated cytokine release in myeloid cells in vitro and lung inflammation mouse model. Together, we propose that agents targeting multiple kinases required for SARS‐CoV‐2‐mediated cytokine release, such as ponatinib, may represent an attractive therapeutic option for treating moderate to severe COVID‐19.     

CyDisCo production of functional recombinant SARS‐CoV‐2 spike receptor binding domain

The COVID‐19 pandemic caused by SARS‐CoV‐2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of a SARS‐CoV‐2 spike protein receptor binding domain (RBD) that includes the SD1 domain. Bacterial cytoplasm is a reductive environment, which is problematic when the recombinant protein of interest requires complicated folding and/or processing. The use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) bypasses this issue by pre‐expressing a sulfhydryl oxidase and a disulfide isomerase, allowing the recombinant protein to be correctly folded with disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce an active RBD in bacteria that is capable of recognizing and binding to the ACE2 (angiotensin‐converting enzyme) receptor as well as antibodies in COVID‐19 patient sera.     

Aging‐related cell type‐specific pathophysiologic immune responses that exacerbate disease severity in aged COVID‐19 patients

Coronavirus disease 2019 (COVID‐19) is especially severe in aged patients, defined as 65 years or older, for reasons that are currently unknown. To investigate the underlying basis for this vulnerability, we performed multimodal data analyses on immunity, inflammation, and COVID‐19 incidence and severity as a function of age. Our analysis leveraged age‐specific COVID‐19 mortality and laboratory testing from a large COVID‐19 registry, along with epidemiological data of ~3.4 million individuals, large‐scale deep immune cell profiling data, and single‐cell RNA‐sequencing data from aged COVID‐19 patients across diverse populations. We found that decreased lymphocyte count and elevated inflammatory markers (C‐reactive protein, D‐dimer, and neutrophil–lymphocyte ratio) are significantly associated with age‐specific COVID‐19 severities. We identified the reduced abundance of naïve CD8 T cells with decreased expression of antiviral defense genes (i.e., IFITM3 and TRIM22) in aged severe COVID‐19 patients. Older individuals with severe COVID‐19 displayed type I and II interferon deficiencies, which is correlated with SARS‐CoV‐2 viral load. Elevated expression of SARS‐CoV‐2 entry factors and reduced expression of antiviral defense genes (LY6E and IFNAR1) in the secretory cells are associated with critical COVID‐19 in aged individuals. Mechanistically, we identified strong TGF‐beta‐mediated immune–epithelial cell interactions (i.e., secretory‐non‐resident macrophages) in aged individuals with critical COVID‐19. Taken together, our findings point to immuno‐inflammatory factors that could be targeted therapeutically to reduce morbidity and mortality in aged COVID‐19 patients.     

SARS‐CoV‐2–host proteome interactions for antiviral drug discovery

Treatment options for COVID‐19, caused by SARS‐CoV‐2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have explored SARS‐CoV‐2–host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS‐CoV‐2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP‐MS) and the complementary proximity‐based labeling MS method (BioID‐MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS‐CoV‐2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image‐based drug screen with infectious SARS‐CoV‐2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein–protein interactions.     

Dynamics of NK,CD8 and Tfh cell mediated the production of cytokines and antiviral antibodies in Chinese patients with moderate COVID‐19

Recent studies have demonstrated a marked decrease in peripheral lymphocyte levels in patients with coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Few studies have focused on the changes of NK, T‐ and B‐cell subsets, inflammatory cytokines and virus‐specific antibodies in patients with moderate COVID‐19. A total of 11 RT‐PCR‐confirmed convalescent patients with COVID‐19 and 11 patients with non‐SARS‐CoV‐2 pneumonia (control patients) were enrolled in this study. NK, CD8⁺ T, CD4⁺ T, Tfh‐like and B‐cell subsets were analysed using flow cytometry. Cytokines and SARS‐CoV‐2‐specific antibodies were analysed using an electrochemiluminescence immunoassay. NK cell counts were significantly higher in patients with COVID‐19 than in control patients (P = 0.017). Effector memory CD8⁺ T‐cell counts significantly increased in patients with COVID‐19 during a convalescent period of 1 week (P = 0.041). TIM‐3⁺ Tfh‐like cell and CD226⁺ Tfh‐like cell counts significantly increased (P = 0.027) and decreased (P = 0.022), respectively, during the same period. Moreover, ICOS⁺ Tfh‐like cell counts tended to decrease (P = 0.074). No abnormal increase in cytokine levels was observed. The high expression of NK cells is important in innate immune response against SARS‐CoV‐2. The increase in effector memory CD8⁺ T‐cell counts, the up‐regulation of inhibitory molecules and the down‐regulation of active molecules on CD4⁺ T cells and Tfh‐like cells in patients with COVID‐19 would benefit the maintenance of balanced cellular and humoural immune responses, may prevent the development of severe cases and contribute to the recovery of patients with COVID‐19.