Predicting the angiotensin converting enzyme 2 (ACE2) utilizing capability as the receptor of SARS-CoV-2

SARS-CoV-2, the newly identified human coronavirus causing severe pneumonia pandemic, was probably originated from Chinese horseshoe bats. However, direct transmission of the virus from bats to humans is unlikely due to lack of direct contact, implying the existence of unknown intermediate hosts. Angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2, but only ACE2s of certain species can be utilized by SARS-CoV-2. Here, we evaluated and ranked the receptor-utilizing capability of ACE2s from various species by phylogenetic clustering and sequence alignment with the currently known ACE2s utilized by SARS-CoV-2. As a result, we predicted that SARS-CoV-2 tends to utilize ACE2s of various mammals, except murines, and some birds, such as pigeon. This prediction may help to screen the intermediate hosts of SARS-CoV-2.     

A diabetic milieu increases ACE2 expression and cellular susceptibility to SARS-CoV-2 infections in human kidney organoids and patient cells

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Antibody Cocktail Exhibits Broad Neutralization Activity Against SARS-CoV-2 and SARS-CoV-2 Variants

Virologica Sinica - Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated multiple variants resistant to therapeutic antibodies. In this study, 12 high-affinity antibodies...     

Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

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Impact of temperature on the affinity of SARS-CoV-2 Spike glycoprotein for host ACE2

The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect the effect of temperature on the receptor–Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike glycoprotein with the ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.     

Structural insights into the binding of SARS-CoV-2, SARS-CoV,and hCoV-NL63 spike receptor-binding domain to horse ACE2

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Analysis of SARS-CoV-2 infection associated cell entry proteins ACE2, CD147, PPIA,and PPIB in datasets from non SARS-CoV-2 infected neuroblastoma patients,as potential prognostic and infection biomarkers in neuroblastoma

SARS-CoV-2 viral contagion has given rise to a worldwide pandemic. Although most children experience minor symptoms from SARS-CoV-2 infection, some have severe complications including Multisystem Inflammatory Syndrome in Children. Neuroblastoma patients may be at higher risk of severe infection as treatment requires immunocompromising chemotherapy and SARS-CoV-2 has demonstrated tropism for nervous cells. To date, there is no sufficient epidemiological data on neuroblastoma patients with SARS-CoV-2. Therefore, we evaluated datasets of non-SARS-CoV-2 infected neuroblastoma patients to assess for key genes involved with SARS-CoV-2 infection as possible neuroblastoma prognostic and infection biomarkers. We hypothesized that ACE2, CD147, PPIA and PPIB, which are associated with viral-cell entry, are potential biomarkers for poor prognosis neuroblastoma and SARS-CoV-2 infection.We have analysed three publicly available neuroblastoma gene expression datasets to understand the specific molecular susceptibilities that high-risk neuroblastoma patients have to the virus. Gene Expression Omnibus (GEO) GSE49711 and GEO GSE62564 are the microarray and RNA-Seq data, respectively, from 498 neuroblastoma samples published as part of the Sequencing Quality Control initiative. TARGET, contains microarray data from 249 samples and is part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. ACE2, CD147, PPIA and PPIB were identified through their involvement in both SARS-CoV-2 infection and cancer pathogenesis.In-depth statistical analysis using Kaplan-Meier, differential gene expression, and Cox multivariate regression analysis, demonstrated that overexpression of ACE2, CD147, PPIA and PPIB is significantly associated with poor-prognosis neuroblastoma samples. These results were seen in the presence of amplified MYCN, unfavourable tumour histology and in patients older than 18 months of age. Previously, we have shown that high levels of the nerve growth factor receptor NTRK1 together with low levels of the phosphatase PTPN6 and TP53 are associated with increased relapse-free survival of neuroblastoma patients. Interestingly, low levels of expression of ACE2, CD147, PPIA and PPIB are associated with this NTRK1-PTPN6-TP53 module, suggesting that low expression levels of these genes are associated with good prognosis. These findings have implications for clinical care and therapeutic treatment. The upregulation of ACE2, CD147, PPIA and PPIB in poor-prognosis neuroblastoma samples suggests that these patients may be at higher risk of severe SARS-CoV-2 infection. Importantly, our findings reveal ACE2, CD147, PPIA and PPIB as potential biomarkers and therapeutic targets for neuroblastoma.     

低氧对肺内小动脉平滑肌细胞血管紧张素转化酶活性和基因表达的影响

实验采用分离培养兔肺内小动脉平滑细胞,观察低氧对ＰＡＳＭＣ的血管紧张素转化酶活性和基因表达的影响。     

SARS病毒受体ACE2的克隆、原核表达及其功能区鉴定

ACE2(angiotensin-converting enzyme 2,ACE2)是SARS冠状病毒(severe acute respiratory syndrome associatedcoronavirus,SARS-CoV)的主要受体。此研究旨在鉴定ACE2的SARS-CoV受体功能区,为进一步阐明SARS-CoV与细胞间的相互作用机制及研制抗病毒药物等提供理论依据。利用RT-PCR从Vero-E6细胞的mRNA中分两段扩增ACE2基因,其中N端片段ACE2A1-367(102~1 210nt)不包括ACE2的酶活性位点(1 223~1 237nt,或374~378aa),而C端片段ACE2B335-805(1 101~2 524nt)包括ACE2的酶活性位点。扩增片段克隆入pMD-18T,并进行测序鉴定。进一步构建与GST基因融合表达的原核表达质粒pGEX-ACE2A与pGEX-ACE2B,IPTG诱导表达。表达的融合蛋白分子量为65kD和77kD,主要以包涵体形式存在。Western blot证实表达产物具有免疫学活性。将纯化的包涵体蛋白质复性后进行Western blot分析,证实pGEX-ACE2A表达的蛋白(~65kD)能与SARS-CoV S1蛋白特异结合,而pGEX-ACE2B表达的蛋白(~77kD)不能与S1蛋白结合。结果表明,ACE2的受体活性与酶活性位点无关,受体功能区在ACE2 N端367个氨基酸内。     

新冠病毒抗体药物研发进展及展望分析

抗体药物以其独特的作用机制和靶向性强、特异性好等优点,在恶性肿瘤、自身免疫性疾病、感染类疾病的诊断和治疗中发挥着越来越重要的作用,成为国际创新药物研发的热点。新冠肺炎(COVID-19)疫情发生以来,国内外多家研究机构和企业正在加快推进新冠病毒(SARS-CoV-2)抗体药物的开发。在此情势下,认真分析抗体药物现状和趋势,梳理国内外新冠病毒抗体药物研究进展,明确我国当前抗体药物创新的机遇、挑战和建议,对加快我国药物自主创新研发具有重要意义。     

Peroxiredoxin 6 phosphorylation and subsequent phospholipase A2 activity are required for agonist-mediated activation of NADPH oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA(2)) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA(2) activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47(phox), cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA(2) activity, blocked agonist-induced PLA(2) activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA(2)s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA(2) active site (S32A, H26A, and D140A), "rescued" Ang II-induced PLA(2) activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA(2) activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA(2) activity facilitates assembly of the NOX2 complex and activation of the oxidase.     

ANXA2 Facilitates Enterovirus 71 Infection by Interacting with 3D Polymerase and PI4KB to Assist the Assembly of Replication Organelles

Virologica Sinica - Similar to that of other enteroviruses, the replication of enterovirus 71 (EV71) occurs on rearranged membranous structures called replication organelles (ROs)....     

Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia

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Angiotensin converting enzyme (kininase II) mRNA production and enzymatic activity in human peripheral blood monocytes are induced by GM-CSF but not other cytokines

Peripheral blood monocytes (PBM) do not possess angiotensin converting enzyme (ACE) activity in the inactive state. However, measurable PBM ACE activity is found in patients with certain inflammatory disease. We have examined the effect of cytokines likely to be present during granulomatous inflammation on the regulation of ACE mRNA in PBM. The presence of ACE mRNA in human PBM cultured in vitri with various cytokines for up to 6 days was analyzed using polymerase chain reaction. PBM not exposed to cytokines did not express ACE mRNA, while incubation of PBM with recombinant human GM-CSF resulted in high levels of ACE mRNA expression after 72 h of cell culture, which persisted through day six. Increased ACE mRNA expression occurred concommitantly with phenotypic changes in cell size and shape consistent with cell activation. A 5-fold increase in ACE enzymatic activity also occurred. Incubation of PBM with all other cytokines tested failed to induce ACE mRNA expression. Alveolar macrophages expressed ACE mRNA immediately following their isolation, but mRNA expression decreased markedly during a 24-h period of incubation and was only partially reversed with exogenous GM-CSF. We conclude that GM-CSF enhances ACE mRNA levels in human PBM, but not in alveolar macrophages.     

基于密码子优化策略的新型冠状病毒主蛋白酶在大肠杆菌中的表达条件优化与活性鉴定

新型冠状病毒主蛋白酶(Main protease,Mpro)在调控新冠病毒RNA复制中具有重要的生物学功能,且Mpro在冠状病毒中的进化高度保守并不易突变,已成为新型广谱抗冠状病毒药物开发的理想靶标之一.为了制备高纯度、高活性的Mpro,根据密码子偏爱性原则,将优化的Mpro基因分别连接到pET-21a与pET-28a...     

Caveolin-1 and dynamin-2 are essential for removal of the complement C5b-9 complex via endocytosis

The complement system, an important element of both innate and adaptive immunity, is executing complement-dependent cytotoxicity (CDC) with its C5b-9 protein complex that is assembled on cell surfaces and transmits to the cell death signals. In turn, cells, and in particular cancer cells, protect themselves from CDC in various ways. Thus, cells actively remove the C5b-9 complexes from their plasma membrane by endocytosis. Inhibition of clathrin by transfection with shRNA or of EPS-15 with a dominant negative plasmid had no effect on C5b-9 endocytosis and on cell death. In contrast, inhibition of caveolin-1 (Cav-1) by transfection with an shRNA or a dominant negative plasmid sensitized cells to CDC and inhibited C5b-9 endocytosis. Similarly, both inhibition of dynamin-2 by transfection with a dominant negative plasmid or by treatment with Dynasore reduced C5b-9 endocytosis and enhanced CDC. C5b-9 endocytosis was also disrupted by pretreatment of the cells with methyl-β-cyclodextrin or Filipin III, hence implicating membrane cholesterol in the process. Analyses by confocal microscopy demonstrated co-localization of Cav-1-EGFP with C5b-9 at the plasma membrane, in early endosomes, at the endocytic recycling compartment and in secreted vesicles. Further investigation of the process of C5b-9 removal by exo-vesiculation demonstrated that inhibition of Cav-1 and cholesterol depletion abrogated C5b-9 exo-vesiculation, whereas, over-expression of Cav-1 increased C5b-9 exo-vesiculation. Our results show that Cav-1 and dynamin-2 (but not clathrin) support cell resistance to CDC, probably by facilitating purging of the C5b-9 complexes by endocytosis and exo-vesiculation.     

Summary of the Detection Kits for SARS-CoV-2 Approved by the National Medical Products Administration of China and Their Application for Diagnosis of COVID-19

Virologica Sinica - The on-going global pandemic of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been...     

S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection,in vitro reconstitution and role in viral infectivity

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of β-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.     

Angiomotin-like2 gene (amotl2) is required for migration and proliferation of endothelial cells during angiogenesis

Angiogenesis involves sprouting, migration, and proliferation of endothelial cells. The angiomotin-like2 gene (amotl2) has been found in blood vessels in zebrafish embryos, but its function in angiogenesis and underlying mechanisms remain unknown. In this study, we demonstrate that knockdown of amotl2 in zebrafish Tg(fli1:EGFP)(y1) and Tg(fli1:nEGFP)(y7) transgenic embryos impairs the intersegmental vessel growth and suppresses proliferation of endothelial cells. Transplantation experiments indicate that function of amotl2 in intersegmental vessel growth is cell-autonomous. AMOTL2 knockdown in cultured human umbilical vein endothelial cells also inhibits cell proliferation and migration and disrupts cell polarity, ultimately interrupting the formation of vascular tube-like structures. Amotl2 promotes MAPK/ERK activation via c-Src, which is dependent on phosphorylation of tyrosine residue at position 103 but independent of the C-terminal PDZ-binding domain. Taking together, our data indicate that Amotl2 plays a pivotal role in polarity, migration and proliferation of angiogenic endothelial cells.