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1.
Front Biosci (Landmark Ed) ; 26(10): 740-751, 2021 10 30.
Article in English | MEDLINE | ID: covidwho-1498507

ABSTRACT

Objectives: To quantify the integrated levels of ACE2 and TMPRSS2, the two well-recognized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry-related genes, and to further identify key factors contributing to SARS-CoV-2 susceptibility in head and neck squamous cell carcinoma (HNSC). Methods: We developed a metric of the potential for tissue infected with SARS-CoV-2 ("TPSI") based on ACE2 and TMPRSS2 transcript levels and compared TPSI levels between tumor and matched normal tissues across 11 tumor types. For further analysis of HNSC, weighted gene co-expression network analysis (WGCNA), functional analysis, and single sample gene set enrichment analysis (ssGSEA) were conducted to investigate TPSI-relevant biological processes and their relationship with the immune landscape. TPSI-related factors were identified from clinical and mutational domains, followed by lasso regression to determine their relative effects on TPSI levels. Results: TPSI levels in tumors were generally lower than in the normal tissues. In HNSC, the genes highly associated with TPSI were enriched in viral entry-related processes, and TPSI levels were positively correlated with both eosinophils and T helper 17 (Th17) cell infiltration. Furthermore, the site of onset, human papillomaviruses (HPV) status, and nuclear receptor binding SET domain protein 1 (NSD1) mutations were identified as the most important factors shaping TPSI levels. Conclusions: This study identified the infection risk of SARS-CoV-2 between tumor and normal tissues, and provided evidence for the risk stratification of HNSC.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Carcinoma, Squamous Cell/genetics , Head and Neck Neoplasms/genetics , Serine Endopeptidases/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , COVID-19/virology , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/virology , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/virology , Humans , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Virus Internalization
2.
Proc Natl Acad Sci U S A ; 118(43)2021 10 26.
Article in English | MEDLINE | ID: covidwho-1493345

ABSTRACT

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.


Subject(s)
Benzothiazoles/pharmacology , COVID-19/drug therapy , Oligopeptides/pharmacology , SARS-CoV-2/drug effects , Serine Endopeptidases/genetics , Animals , Benzamidines/chemistry , Benzothiazoles/pharmacokinetics , COVID-19/genetics , COVID-19/virology , Cell Line , Drug Design , Epithelial Cells/drug effects , Epithelial Cells/virology , Esters/chemistry , Guanidines/chemistry , Humans , Lung/drug effects , Lung/virology , Mice , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Oligopeptides/pharmacokinetics , SARS-CoV-2/pathogenicity , Serine Endopeptidases/drug effects , Serine Endopeptidases/ultrastructure , Small Molecule Libraries/pharmacology , Substrate Specificity/drug effects , Virus Internalization/drug effects
3.
J Virol ; 95(19): e0086121, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1486519

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. No effective treatment for COVID-19 has been established yet. The serine protease transmembrane protease serine 2 (TMPRSS2) is essential for viral spread and pathogenicity by facilitating the entry of SARS-CoV-2 into host cells. The protease inhibitor camostat, an anticoagulant used in the clinic, has potential anti-inflammatory and antiviral activities against COVID-19. However, the potential mechanisms of viral resistance and antiviral activity of camostat are unclear. Herein, we demonstrate high inhibitory potencies of camostat for a panel of serine proteases, indicating that camostat is a broad-spectrum inhibitor of serine proteases. In addition, we determined the crystal structure of camostat in complex with a serine protease (uPA [urokinase-type plasminogen activator]), which reveals that camostat is inserted in the S1 pocket of uPA but is hydrolyzed by uPA, and the cleaved camostat covalently binds to Ser195. We also generated a homology model of the structure of the TMPRSS2 serine protease domain. The model shows that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2, subsequently preventing SARS-CoV-2 spread. IMPORTANCE Serine proteases are a large family of enzymes critical for multiple physiological processes and proven diagnostic and therapeutic targets in several clinical indications. The serine protease transmembrane protease serine 2 (TMPRSS2) was recently found to mediate SARS-CoV-2 entry into the host. Camostat mesylate (FOY 305), a serine protease inhibitor active against TMPRSS2 and used for the treatment of oral squamous cell carcinoma and chronic pancreatitis, inhibits SARS-CoV-2 infection of human lung cells. However, the direct inhibition mechanism of camostat mesylate for TMPRSS2 is unclear. Herein, we demonstrate that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2 as uPA, subsequently preventing SARS-CoV-2 spread.


Subject(s)
Antiviral Agents/pharmacology , Esters/pharmacology , Guanidines/pharmacology , SARS-CoV-2/drug effects , Serine Endopeptidases/chemistry , Serine Endopeptidases/pharmacology , Serine Proteases/pharmacology , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/prevention & control , Carcinoma, Squamous Cell , Esters/chemistry , Esters/metabolism , Guanidines/chemistry , Guanidines/metabolism , Humans , Molecular Dynamics Simulation , Mouth Neoplasms , Protein Domains , Sequence Alignment , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Serine Proteases/chemistry , Serine Proteases/metabolism , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Virus Internalization/drug effects
4.
Cell Transplant ; 30: 9636897211049814, 2021.
Article in English | MEDLINE | ID: covidwho-1484237

ABSTRACT

During the past 18 months as the world dealt with the COVID-19 pandemic, articles published in Cell Transplantation (CT) voiced unique perspectives on the disease which have since been supported by additional research. Intrigued by the variability in COVID-19 severity, CT authors explored the influence of variants in angiotensin-converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2) genes, as well as the role of androgen receptors on disease development. Mesenchymal stem cells (MSC) were offered up as a potential COVID-19 therapy because of their immune modulating characteristics and successful use in other acute respiratory diseases. Two CT author groups gave proof of principle when hospitalized COVID-19 patients were infused with MSC after no other interventions seemed to work. MSC treatment reduced disease severity and shortened hospitalization stays. Lastly, CT authors speculated why we are still in the midst of a pandemic and the consequences of disillusioned comfort as we face new emerging variants that may undermine all we have accomplished thus far.


Subject(s)
COVID-19/immunology , COVID-19/therapy , Mesenchymal Stem Cells/cytology , Serine Endopeptidases/genetics , Angiotensin-Converting Enzyme 2/metabolism , Biomedical Research , Cell Transplantation , Cytokines/metabolism , Hospitalization , Humans , Immune System , Mesenchymal Stem Cells/metabolism , Peptidyl-Dipeptidase A/genetics , Publications , Receptors, Androgen/metabolism , Retrospective Studies , SARS-CoV-2 , Severity of Illness Index
5.
Biomed Res Int ; 2021: 9982729, 2021.
Article in English | MEDLINE | ID: covidwho-1476892

ABSTRACT

The human transmembrane protease serine 2 (TMPRSS2) protein plays an important role in prostate cancer progression. It also facilitates viral entry into target cells by proteolytically cleaving and activating the S protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the current study, we used different available tools like SIFT, PolyPhen2.0, PROVEAN, SNAP2, PMut, MutPred2, I-Mutant Suite, MUpro, iStable, ConSurf, ModPred, SwissModel, PROCHECK, Verify3D, and TM-align to identify the most deleterious variants and to explore possible effects on the TMPRSS2 stability, structure, and function. The six missense variants tested were evaluated to have deleterious effects on the protein by SIFT, PolyPhen2.0, PROVEAN, SNAP2, and PMut. Additionally, V160M, G181R, R240C, P335L, G432A, and D435Y variants showed a decrease in stability by at least 2 servers; G181R, G432A, and D435Y are highly conserved and identified posttranslational modifications sites (PTMs) for proteolytic cleavage and ADP-ribosylation using ConSurf and ModPred servers. The 3D structure of TMPRSS2 native and mutants was generated using 7 meq as a template from the SwissModeller group, refined by ModRefiner, and validated using the Ramachandran plot. Hence, this paper can be advantageous to understand the association between these missense variants rs12329760, rs781089181, rs762108701, rs1185182900, rs570454392, and rs867186402 and susceptibility to SARS-CoV-2.


Subject(s)
COVID-19/genetics , Mutation, Missense , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Binding Sites , Computational Biology/methods , Evolution, Molecular , Genetic Predisposition to Disease , Humans , Models, Molecular , Phylogeny , Polymorphism, Single Nucleotide , Protein Conformation , Protein Stability , Serine Endopeptidases/metabolism
7.
Microvasc Res ; 138: 104232, 2021 11.
Article in English | MEDLINE | ID: covidwho-1446976

ABSTRACT

The mechanisms by which the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) induces neurological complications remain to be elucidated. We aimed to identify possible effects of hypoxia on the expression of SARS-CoV-2 cell entry mediators, angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane protease serine 2 (TMPRSS2) protein, in human brain endothelial cells, in vitro. hCMEC/D3 cells were exposed to different oxygen tensions: 20% (Control group), 8% or 2% O2 (Hypoxia groups). Cells were harvested 6-, 24- and 48 h following hypoxic challenge for assessment of mRNA and protein, using qPCR and Western Blot. The response of the brain endothelial cells to hypoxia was replicated using modular incubator chambers. We observed an acute increase (6 h, p < 0.05), followed by a longer-term decrease (48 h, p < 0.05) in ACE2 mRNA and protein expression, accompanied by reduced expression of TMPRSS2 protein levels (48 h, p < 0.05) under the more severe hypoxic condition (2% O2). No changes in levels of von Willebrand Factor (vWF - an endothelial cell damage marker) or interleukin 6 (IL-6 - a pro-inflammatory cytokine) mRNA were observed. We conclude that hypoxia regulates brain endothelial cell ACE2 and TMPRSS2 expression in vitro, which may indicate human brain endothelial susceptibility to SARS-CoV-2 infection and subsequent brain sequelae.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Brain/blood supply , COVID-19/virology , Endothelial Cells/virology , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Virus Internalization , Angiotensin-Converting Enzyme 2/genetics , COVID-19/enzymology , Cell Hypoxia , Cell Line , Endothelial Cells/enzymology , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Serine Endopeptidases/genetics
8.
Mol Cells ; 44(9): 680-687, 2021 Sep 30.
Article in English | MEDLINE | ID: covidwho-1444539

ABSTRACT

Coronavirus disease, COVID-19 (coronavirus disease 2019), caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), has a higher case fatality rate in European countries than in others, especially East Asian ones. One potential explanation for this regional difference is the diversity of the viral infection efficiency. Here, we analyzed the allele frequencies of a nonsynonymous variant rs12329760 (V197M) in the TMPRSS2 gene, a key enzyme essential for viral infection and found a significant association between the COVID-19 case fatality rate and the V197M allele frequencies, using over 200,000 present-day and ancient genomic samples. East Asian countries have higher V197M allele frequencies than other regions, including European countries which correlates to their lower case fatality rates. Structural and energy calculation analysis of the V197M amino acid change showed that it destabilizes the TMPRSS2 protein, possibly negatively affecting its ACE2 and viral spike protein processing.


Subject(s)
COVID-19/genetics , COVID-19/mortality , Serine Endopeptidases/genetics , Asian Continental Ancestry Group , COVID-19/ethnology , European Continental Ancestry Group , Gene Frequency , Humans , Models, Molecular , Mortality , Polymorphism, Single Nucleotide , Republic of Korea , Serine Endopeptidases/chemistry
9.
Sci Rep ; 11(1): 19140, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440481

ABSTRACT

Despite the reported low expression of the primary SARS-CoV-2 receptor ACE2 in distinct ocular tissues, some clinical evidence suggests that SARS-CoV-2 can infect the eye. In this study, we explored potential entry sites for SARS-CoV-2 by viral S protein histochemistry on various ocular tissues and compared the staining patterns with RNA and protein expression of TMPRSS2 and ACE2. Potential viral entry sites were investigated by histochemistry using tagged recombinant viral S protein on 52 ocular tissue samples including specimens of the cornea, conjunctiva, lid margin, lacrimal gland tissue, retina, choroid, and RPE. In addition, ACE2 and TMPRSS2 immunohistochemistry were performed on the same ocular tissue, each with distinct antibodies binding to different epitopes. Lung tissue samples were used as positive controls. Finally, bulk RNA sequencing (RNA-Seq) was used to determine the expression of ACE2 and its auxiliary factors in the tissues mentioned above. S protein histochemistry revealed a positive staining in lung tissue but absent staining in the cornea, the conjunctiva, eye lid samples, the lacrimal glands, the retina and the optic nerve which was supported by hardly any immunoreactivity for ACE2 and TMPRSS2 and scarce ACE2 and TMPRSS2 RNA expression. Negligible staining with antibodies targeting ACE2 or TMPRSS2 was seen in the main and accessory lacrimal glands. In contrast, ocular staining (S protein, ACE2, TMPRSS2) was distinctly present in pigmented cells of the RPE and choroid, as well as in the ciliary body and the iris stroma. S protein histochemistry revealed hardly any SARS-CoV-2 entry sites in all ocular tissues examined. Similarly, no significant ACE2 or TMPRSS2 expression was found in extra- and intraocular tissue. While this study suggest a rather low risk of ocular infection with SARS-CoV-2, it should be noted, that potential viral entry sites may increase in response to inflammation or in certain disease states.


Subject(s)
COVID-19/prevention & control , Conjunctiva/metabolism , Cornea/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Conjunctiva/virology , Cornea/virology , Gene Expression Profiling/methods , Humans , Immunohistochemistry/methods , RNA-Seq/methods , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Virus Internalization
10.
Nutrients ; 13(10)2021 Sep 25.
Article in English | MEDLINE | ID: covidwho-1438684

ABSTRACT

BACKGROUND: Obesity increases the severity of SARS-CoV-2 outcomes. Thus, this study tested whether obesogenic and ketogenic diets distinctly affect SARS-CoV-2 entry proteins and the renin-angiotensin system (RAS) in rat pulmonary and cardiac tissues. METHODS: Male Sprague-Dawley rats were fed either standard chow (SC), a high-fat sucrose-enriched diet (HFS), or a ketogenic diet (KD) for 16 weeks. Afterwards, levels of angiotensin converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), RAS components, and inflammatory genes were measured in the lungs and hearts of these animals. RESULTS: In the lungs, HFS elevated ACE2 and TMPRSS2 levels relative to SC diet, whereas the KD lowered the levels of these proteins and the gene expressions of toll-like receptor 4 and interleukin-6 receptor relative to HFS. The diets did not alter ACE2 and TMPRSS2 in the heart, although ACE2 was more abundant in heart than lung tissues. CONCLUSION: Diet-induced obesity increased the levels of viral entry proteins in the lungs, providing a mechanism whereby SARS-CoV-2 infectivity can be enhanced in obese individuals. Conversely, by maintaining low levels of ACE2 and TMPRSS2 and by exerting an anti-inflammatory effect, the KD can potentially attenuate the severity of infection and migration of SARS-CoV-2 to other ACE2-expressing tissues.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , Diet, High-Fat/adverse effects , Diet, Ketogenic/methods , Lung/metabolism , Myocardium/metabolism , Serine Endopeptidases/metabolism , Angiotensin-Converting Enzyme 2/genetics , Animals , Biomarkers/metabolism , COVID-19/complications , COVID-19/metabolism , Disease Models, Animal , Male , Obesity/complications , Obesity/metabolism , Rats , Rats, Sprague-Dawley , Renin-Angiotensin System , SARS-CoV-2 , Serine Endopeptidases/genetics , Virus Internalization
11.
Nat Commun ; 12(1): 5498, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1428814

ABSTRACT

Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Biological Specimen Banks , CRISPR-Cas Systems , Coronavirus , Dipeptidyl Peptidase 4/genetics , Organoids/metabolism , Serine Endopeptidases/genetics , COVID-19 , Cell Line , Humans , Middle East Respiratory Syndrome Coronavirus , SARS-CoV-2 , Transcriptome , Virus Replication
12.
Signal Transduct Target Ther ; 6(1): 344, 2021 09 20.
Article in English | MEDLINE | ID: covidwho-1428800

ABSTRACT

Coronavirus disease 2019 (COVID-19) caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in considerable morbidity and mortality worldwide. COVID-19 incidence, severity, and mortality rates differ greatly between populations, genders, ABO blood groups, human leukocyte antigen (HLA) genotypes, ethnic groups, and geographic backgrounds. This highly heterogeneous SARS-CoV-2 infection is multifactorial. Host genetic factors such as variants in the angiotensin-converting enzyme gene (ACE), the angiotensin-converting enzyme 2 gene (ACE2), the transmembrane protease serine 2 gene (TMPRSS2), along with HLA genotype, and ABO blood group help to explain individual susceptibility, severity, and outcomes of COVID-19. This review is focused on COVID-19 clinical and viral characteristics, pathogenesis, and genetic findings, with particular attention on genetic diversity and variants. The human genetic basis could provide scientific bases for disease prediction and targeted therapy to address the COVID-19 scourge.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Peptidyl-Dipeptidase A/genetics , SARS-CoV-2/genetics , ABO Blood-Group System/genetics , COVID-19/epidemiology , COVID-19/virology , Ethnic Groups/genetics , Female , Genotype , HLA Antigens/genetics , Humans , Male , Risk Factors , SARS-CoV-2/pathogenicity , Serine Endopeptidases/genetics
13.
Cancer Res Treat ; 53(3): 650-656, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1403959

ABSTRACT

PURPOSE: Coronavirus disease 2019 (COVID-19) pandemic has spread worldwide rapidly and patients with cancer have been considered as a vulnerable group for this infection. This study aimed to examine the expressions of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) in tumor tissues of six common cancer types. MATERIALS AND METHODS: The expression levels of ACE2 and TMPRSS2 in tumors and control samples were obtained from online databases. Survival prognosis and biological functions of these genes were investigated for each tumor type. RESULTS: There was the overexpression of ACE2 in colon and stomach adenocarcinomas compared to controls, meanwhile colon and prostate adenocarcinomas showed a significantly higher expression of TMPRSS2. Additionally, survival prognosis analysis has demonstrated that upregulation of ACE2 in liver hepatocellular carcinoma was associated with higher overall survival (hazard ratio, 0.65; p=0.016) and disease-free survival (hazard ratio, 0.66; p=0.007), while overexpression of TMPRSS2 was associated with a 26% reduced risk of death in lung adenocarcinoma (p=0.047) but 50% increased risk of death in breast invasive carcinoma (p=0.015). CONCLUSION: There is a need to take extra precautions for COVID-19 in patients with colorectal cancer, stomach cancer, and lung cancer. Further information on other types of cancer at different stages should be investigated.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/diagnosis , Neoplasms/diagnosis , Neoplasms/genetics , Serine Endopeptidases/genetics , Adenocarcinoma/complications , Adenocarcinoma/diagnosis , Adenocarcinoma/epidemiology , Adenocarcinoma/genetics , Breast Neoplasms/complications , Breast Neoplasms/diagnosis , Breast Neoplasms/epidemiology , Breast Neoplasms/genetics , COVID-19/complications , COVID-19/epidemiology , COVID-19/genetics , Case-Control Studies , Databases as Topic , Female , Gastrointestinal Neoplasms/complications , Gastrointestinal Neoplasms/diagnosis , Gastrointestinal Neoplasms/epidemiology , Gastrointestinal Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Genetic Predisposition to Disease , Humans , Liver Neoplasms/complications , Liver Neoplasms/diagnosis , Liver Neoplasms/epidemiology , Liver Neoplasms/genetics , Lung Neoplasms/complications , Lung Neoplasms/diagnosis , Lung Neoplasms/epidemiology , Lung Neoplasms/genetics , Male , Mutation , Neoplasms/complications , Neoplasms/epidemiology , Pandemics , Prognosis , Prostatic Neoplasms/complications , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/epidemiology , Prostatic Neoplasms/genetics , Retrospective Studies , SARS-CoV-2/physiology , Survival Analysis
14.
Biochem Biophys Res Commun ; 577: 146-151, 2021 11 05.
Article in English | MEDLINE | ID: covidwho-1401239

ABSTRACT

The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI culture yielded a layer of mucus on their apical surface, exhibited decreased expression levels of the proliferation marker KI-67 and intriguingly became susceptible to SARS-CoV-2 infection. We found that A549 cells increased the endogenous expression levels of ACE2 and TMPRSS2 following adaptation to ALI culture conditions. Camostat, a TMPRSS2 inhibitor, reduced SARS-CoV-2 infection in ALI-cultured A549 cells. These findings indicate that ALI culture switches the phenotype of A549 cells from resistance to susceptibility to SARS-CoV-2 infection through upregulation of ACE2 and TMPRSS2.


Subject(s)
Alveolar Epithelial Cells/virology , COVID-19/virology , Cell Culture Techniques/methods , SARS-CoV-2/physiology , A549 Cells , Alveolar Epithelial Cells/pathology , Cells, Cultured , Disease Susceptibility , Gene Expression Regulation, Neoplastic , Humans , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Up-Regulation/genetics
15.
PLoS One ; 16(8): e0255622, 2021.
Article in English | MEDLINE | ID: covidwho-1388954

ABSTRACT

The SARS-CoV-2 pandemic is a major global threat that sparked global research efforts. Pre-clinical and biochemical SARS-CoV-2 studies firstly rely on cell culture experiments where the importance of choosing an appropriate cell culture model is often underestimated. We here present a bottom-up approach to identify suitable permissive cancer cell lines for drug screening and virus research. Human cancer cell lines were screened for the SARS-CoV-2 cellular entry factors ACE2 and TMPRSS2 based on RNA-seq data of the Cancer Cell Line Encyclopedia (CCLE). However, experimentally testing permissiveness towards SARS-CoV-2 infection, we found limited correlation between receptor expression and permissiveness. This underlines that permissiveness of cells towards viral infection is determined not only by the presence of entry receptors but is defined by the availability of cellular resources, intrinsic immunity, and apoptosis. Aside from established cell culture infection models CACO-2 and CALU-3, three highly permissive human cell lines, colon cancer cell lines CL-14 and CL-40 and the breast cancer cell line CAL-51 and several low permissive cell lines were identified. Cell lines were characterised in more detail offering a broader choice of non-overexpression in vitro infection models to the scientific community. For some cell lines a truncated ACE2 mRNA and missense variants in TMPRSS2 might hint at disturbed host susceptibility towards viral entry.


Subject(s)
COVID-19/virology , Receptors, Virus , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Cell Line, Tumor , Humans , Receptors, Virus/genetics , Receptors, Virus/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism
18.
Viruses ; 12(10)2020 10 16.
Article in English | MEDLINE | ID: covidwho-1389518

ABSTRACT

To address the expression pattern of the SARS-CoV-2 receptor ACE2 and the viral priming protease TMPRSS2 in the respiratory tract, this study investigated RNA sequencing transcriptome profiling of samples of airway and oral mucosa. As shown, ACE2 has medium levels of expression in both small airway epithelium and masticatory mucosa, and high levels of expression in nasal epithelium. The expression of ACE2 is low in mucosal-associated invariant T (MAIT) cells and cannot be detected in alveolar macrophages. TMPRSS2 is highly expressed in small airway epithelium and nasal epithelium and has lower expression in masticatory mucosa. Our results provide the molecular basis that the nasal mucosa is the most susceptible locus in the respiratory tract for SARS-CoV-2 infection and consequently for subsequent droplet transmission and should be the focus for protection against SARS-CoV-2 infection.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/genetics , Peptidyl-Dipeptidase A/biosynthesis , Pneumonia, Viral/genetics , Serine Endopeptidases/biosynthesis , Virus Internalization , Angiotensin-Converting Enzyme 2 , COVID-19 , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Epithelium/metabolism , Epithelium/virology , Gene Expression , Gene Expression Profiling , Humans , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Pandemics , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , Respiratory System/metabolism , Respiratory System/virology , SARS-CoV-2 , Serine Endopeptidases/genetics
19.
BMC Pulm Med ; 21(1): 275, 2021 Aug 23.
Article in English | MEDLINE | ID: covidwho-1370938

ABSTRACT

BACKGROUND: How cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection and severity is controversial. We investigated the effects of COPD and CS on the expression of SARS-CoV-2 entry receptor ACE2 in vivo in COPD patients and controls and in CS-exposed mice, and the effects of CS on SARS-CoV-2 infection in human bronchial epithelial cells in vitro. METHODS: We quantified: (1) pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and/or TMPRSS2 mRNA levels by RT-qPCR in two independent human cohorts; and (2) pulmonary ACE2 protein levels by immunostaining and ELISA in C57BL/6 WT mice exposed to air or CS for up to 6 months. The effects of CS exposure on SARS-CoV-2 infection were evaluated after in vitro infection of Calu-3 cells and differentiated human bronchial epithelial cells (HBECs), respectively. RESULTS: ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus controls but similar in central airways. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice. CS treatment decreased viral replication in Calu-3 cells, as determined by immunofluorescence staining for replicative double-stranded RNA (dsRNA) and western blot for viral N protein. Acute CS exposure decreased in vitro SARS-CoV-2 replication in HBECs, as determined by plaque assay and RT-qPCR. CONCLUSIONS: ACE2 levels were decreased in both bronchial and alveolar epithelial cells from COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-exposure potently inhibited SARS-CoV-2 replication in vitro. These findings urge to investigate further the controversial effects of CS and COPD on SARS-CoV-2 infection.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/enzymology , Cigarette Smoking/metabolism , Pulmonary Disease, Chronic Obstructive/enzymology , SARS-CoV-2/physiology , Smoke , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/genetics , Animals , Bronchi , Cell Line, Tumor , Female , Humans , Male , Mice , Middle Aged , Patient Acuity , Pulmonary Alveoli , RNA, Messenger/metabolism , Respiratory Mucosa/metabolism , Serine Endopeptidases/genetics , Tobacco , Virus Replication
20.
Immunobiology ; 226(5): 152130, 2021 09.
Article in English | MEDLINE | ID: covidwho-1368671

ABSTRACT

In this review, we highlight the interaction of SARS-CoV-2 virus and host genomes, reporting the current studies on the sequence analysis of SARS-CoV-2 isolates and host genomes from diverse world populations. The main genetic variants that are present in both the virus and host genomes were particularly focused on the ACE2 and TMPRSS2 genes, and their impact on the patients' susceptibility to the virus infection and severity of the disease. Finally, the interaction of the virus and host non-coding RNAs is described in relation to their regulatory roles in target genes and/or signaling pathways critically associated with SARS-CoV-2 infection. Altogether, these studies provide a significant contribution to the knowledge of SARS-CoV-2 mechanisms of infection and COVID-19 pathogenesis. The described genetic variants and molecular factors involved in host/virus genome interactions have significantly contributed to defining patient risk groups, beyond those based on patients' age and comorbidities, and they are promising candidates to be potentially targeted in treatment strategies for COVID-19 and other viral infectious diseases.


Subject(s)
COVID-19/genetics , Genome , Host-Pathogen Interactions/genetics , RNA, Untranslated , SARS-CoV-2/genetics , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/therapy , Genes, Viral , Humans , Serine Endopeptidases/genetics
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