SARS-CoV-2 Omicron Specific Mutations Affecting Infectivity, Fusogenicity, and Partial TMPRSS2-Independency.

The COVID-19 pandemic resulted from the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since its first appearance in 2019, new SARS-CoV-2 variants of concern (VOCs) have emerged frequently, changing the infection's dynamic. SARS-CoV-2 infects cells via two distinct entry routes; receptor-mediated endocytosis or membrane fusion, depending on the absence or presence of transmembrane serine protease 2 (TMPRSS2), respectively. In laboratory conditions, the Omicron SARS-CoV-2 strain inefficiently infects cells predominantly via endocytosis and is phenotypically characterized by decreased syncytia formation compared to the earlier Delta variant. Thus, it is important to characterize Omicron's unique mutations and their phenotypic manifestations. Here, by utilizing SARS-CoV-2 pseudovirions, we report that the specific Omicron Spike F375 residue decreases infectivity, and its conversion to the Delta S375 sequence significantly increases Omicron infectivity. Further, we identified that residue Y655 decreases Omicron's TMPRSS2 dependency and entry via membrane fusion. The Y655H, K764N, K856N and K969N Omicron revertant mutations, bearing the Delta variant sequence, increased the cytopathic effect of cell-cell fusion, suggesting these Omicron-specific residues reduced the severity of SARS-CoV-2. This study of the correlation of the mutational profile with the phenotypic outcome should sensitize our alertness towards emerging VOCs.

Analyzing the role of ACE2, AR, MX1 and TMPRSS2 genetic markers for COVID-19 severity.

BACKGROUND: The use of molecular biomarkers for COVID-19 remains unconclusive. The application of a molecular biomarker in combination with clinical ones that could help classifying aggressive patients in first steps of the disease could help clinician and sanitary system a better management of the disease. Here we characterize the role of ACE2, AR, MX1, ERG, ETV5 and TMPRSS2 for trying a better classification of COVID-19 through knowledge of the disease mechanisms. METHODS: A total of 329 blood samples were genotyped in ACE2, MX1 and TMPRSS2. RNA analyses were also performed from 258 available samples using quantitative polymerase chain reaction for genes: ERG, ETV5, AR, MX1, ACE2, and TMPRSS2. Moreover, in silico analysis variant effect predictor, ClinVar, IPA, DAVID, GTEx, STRING and miRDB database was also performed. Clinical and demographic data were recruited from all participants following WHO classification criteria. RESULTS: We confirm the use of ferritin (p < 0.001), D-dimer (p < 0.010), CRP (p < 0.001) and LDH (p < 0.001) as markers for distinguishing mild and severe cohorts. Expression studies showed that MX1 and AR are significantly higher expressed in mild vs severe patients (p < 0.05). ACE2 and TMPRSS2 are involved in the same molecular process of membrane fusion (p = 4.4 × 10-3), acting as proteases (p = 0.047). CONCLUSIONS: In addition to the key role of TMPSRSS2, we reported for the first time that higher expression levels of AR are related with a decreased risk of severe COVID-19 disease in females. Moreover, functional analysis demonstrates that ACE2, MX1 and TMPRSS2 are relevant markers in this disease.

ACE-2, TMPRSS2, and Neuropilin-1 Receptor Expression on Human Brain Astrocytes and Pericytes and SARS-CoV-2 Infection Kinetics.

Angiotensin Converting Enzyme 2 (ACE-2), Transmembrane Serine Protease 2 (TMPRSS-2) and Neuropilin-1 cellular receptors support the entry of SARS-CoV-2 into susceptible human target cells and are characterized at the molecular level. Some evidence on the expression of entry receptors at mRNA and protein levels in brain cells is available, but co-expression of these receptors and confirmatory evidence on brain cells is lacking. SARS-CoV-2 infects some brain cell types, but infection susceptibility, multiple entry receptor density, and infection kinetics are rarely reported in specific brain cell types. Highly sensitive Taqman ddPCR, flow-cytometry and immunocytochemistry assays were used to quantitate the expression of ACE-2, TMPRSS-2 and Neuropilin-1 at mRNA and protein levels on human brain-extracted pericytes and astrocytes, which are an integral part of the Blood-Brain-Barrier (BBB). Astrocytes showed moderate ACE-2 (15.9 ± 1.3%, Mean ± SD, n = 2) and TMPRSS-2 (17.6%) positive cells, and in contrast show high Neuropilin-1 (56.4 ± 39.8%, n = 4) protein expression. Whereas pericytes showed variable ACE-2 (23.1 ± 20.7%, n = 2), Neuropilin-1 (30.3 ± 7.5%, n = 4) protein expression and higher TMPRSS-2 mRNA (667.2 ± 232.3, n = 3) expression. Co-expression of multiple entry receptors on astrocytes and pericytes allows entry of SARS-CoV-2 and progression of infection. Astrocytes showed roughly four-fold more virus in culture supernatants than pericytes. SARS-CoV-2 cellular entry receptor expression and "in vitro" viral kinetics in astrocytes and pericytes may improve our understanding of viral infection "in vivo". In addition, this study may facilitate the development of novel strategies to counter the effects of SARS-CoV-2 and inhibit viral infection in brain tissues to prevent the spread and interference in neuronal functions.

Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2.

SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus' evolution.

Expression of TMPRSS2 is up-regulated by bacterial flagellin, LPS, and Pam3Cys in human airway cells.

Many viruses require proteolytic activation of their envelope proteins for infectivity, and relevant host proteases provide promising drug targets. The transmembrane serine protease 2 (TMPRSS2) has been identified as a major activating protease of influenza A virus (IAV) and various coronaviruses (CoV). Increased TMPRSS2 expression has been associated with a higher risk of severe influenza infection and enhanced susceptibility to SARS-CoV-2. Here, we found that Legionella pneumophila stimulates the increased expression of TMPRSS2-mRNA in Calu-3 human airway cells. We identified flagellin as the dominant structural component inducing TMPRSS2 expression. The flagellin-induced increase was not observed at this magnitude for other virus-activating host proteases. TMPRSS2-mRNA expression was also significantly increased by LPS, Pam3Cys, and Streptococcus pneumoniae, although less pronounced. Multicycle replication of H1N1pdm and H3N2 IAV but not SARS-CoV-2 and SARS-CoV was enhanced by flagellin treatment. Our data suggest that bacteria, particularly flagellated bacteria, up-regulate the expression of TMPRSS2 in human airway cells and, thereby, may support enhanced activation and replication of IAV upon co-infections. In addition, our data indicate a physiological role of TMPRSS2 in antimicrobial host response.

ACE2 Receptor and TMPRSS2 Protein Expression Patterns in the Human Brainstem Reveal Anatomical Regions Potentially Vulnerable to SARS-CoV-2 Infection.

Angiotensin-converting enzyme 2 receptor (ACE2R) is a transmembrane protein expressed in various tissues throughout the body that plays a key role in the regulation of blood pressure. Recently, ACE2R has gained significant attention due to its involvement in the pathogenesis of COVID-19, the disease caused by the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2). While ACE2 receptors serve as entry points for the novel coronavirus, Transmembrane Serine Protease 2 (TMPRSS2), an enzyme located on the cell membrane, is required for SARS-CoV-2 S protein priming. Even though numerous studies have assessed the effects of COVID-19 on the brain, very little information is available concerning the distribution of ACE2R and TMPRSS2 in the human brain, with particular regard to their topographical expression in the brainstem. In this study, we investigated the expression of ACE2R and TMPRSS2 in the brainstem of 18 adult subjects who died due to pneumonia/respiratory insufficiency. Our findings indicate that ACE2R and TMPRSS2 are expressed in neuronal and glial cells of the brainstem, particularly at the level of the vagal nuclei of the medulla and the midbrain tegmentum, thus confirming the expression and anatomical localization of these proteins within specific human brainstem nuclei. Furthermore, our findings help to define anatomically susceptible regions to SARS-CoV-2 infection in the brainstem, advancing knowledge on the neuropathological underpinnings of neurological manifestations in COVID-19.

Identification of Potential TMPRSS2 Inhibitors for COVID-19 Treatment in Chinese Medicine by Computational Approaches and Surface Plasmon Resonance Technology.

BACKGROUND: Coronavirus disease-19 (COVID-19) pneumonia continues to spread in the entire globe with limited medication available. In this study, the active compounds in Chinese medicine (CM) recipes targeting the transmembrane serine protease 2 (TMPRSS2) protein for the treatment of COVID-19 were explored. METHODS: The conformational structure of TMPRSS2 protein (TMPS2) was built through homology modeling. A training set covering TMPS2 inhibitors and decoy molecules was docked to TMPS2, and their docking poses were re-scored with scoring schemes. A receiver operating characteristic (ROC) curve was applied to select the best scoring function. Virtual screening of the candidate compounds (CCDs) in the six highly effective CM recipes against TMPS2 was conducted based on the validated docking protocol. The potential CCDs after docking were subject to molecular dynamics (MD) simulations and surface plasmon resonance (SPR) experiment. RESULTS: A training set of 65 molecules were docked with modeled TMPS2 and LigScore2 with the highest area under the curve, AUC, value (0.886) after ROC analysis selected to best differentiate inhibitors from decoys. A total of 421 CCDs in the six recipes were successfully docked into TMPS2, and the top 16 CCDs with LigScore2 higher than the cutoff (4.995) were screened out. MD simulations revealed a stable binding between these CCDs and TMPS2 due to the negative binding free energy. Lastly, SPR experiments validated the direct combination of narirutin, saikosaponin B1, and rutin with TMPS2. CONCLUSIONS: Specific active compounds including narirutin, saikosaponin B1, and rutin in CM recipes potentially target and inhibit TMPS2, probably exerting a therapeutic effect on COVID-19.

Association between tissue stress reaction and ACE2/TMPRSS2 expression in endometria of reproductive aged women before and during Covid-19 pandemic.

BACKGROUND: A potential concern has been raised regarding fertility and reproductive outcome during the Covid-19 pandemic with growing stress and anxiety. However, information on the association between tissue stress reaction and expression profiles of SARS-CoV-2 viral entry proteins, ACE2 and TMPRSS2, in endometria collected from women before (pre-pandemic) and during the Covid-19 pandemic (in-pandemic) is unknown. We aim to investigate the relationship between the expression of stress-reactive proteins and of ACE2 and TMPRSS2 in endometria collected from women during these two different time frames. METHODS: We retrospectively retrieved tissue blocks of endometrial samples from 25 women in 2019 (pre-pandemic) and 25 women in 2020 (in-pandemic) who underwent hysterectomy for different gynecological indications. Immunohistochemical analysis was performed with endometrial tissue samples that were collected before and during the pandemic, using respective antibodies targeting ACE2/TMPRSS2, ADRB2 and NK1R (stress and anxiety receptor markers, respectively). The quantification of immunoreactive cells for each marker was calculated by the immunoreactive score (IRS) analysis. This retrospective cohort study was limited to small sample size. RESULTS: No significant differences in the IRS of ACE2 and TMPRSS2 were found between the endometria that were collected before and during the pandemic with a lack of correlation between ACE2 and TMPRSS2 expression in respective endometria (r = 0.11, pre-pandemic; r = 0.04, in-pandemic). The immunostaining levels of stress marker, ADRB2 were significantly higher in the endometria of in-pandemic group (p = 0.015) comparing to that of pre-pandemic group. Pearson's correlation coefficient analysis showed a significant correlation in the expression between ADRB2 and TMPRSS2 (r = 0.41, p = 0.042) in the endometria of in-pandemic group but not in the pre-pandemic group. CONCLUSION: The rise in stress and anxiety among women during current pandemic may elicit substantial amount of tissue stress reaction with consequent increase in the expression of SARS-CoV-2 viral entry proteins in their endometria. A lack of correlation between ACE2 and TMPRSS2 expression in endometria may reassure women in their reproductive age that they are not more susceptible to infection by SARS-CoV-2 and suggest that stressful women during this pandemic can safely decide to conceive naturally or by artificial reproductive technology.

Relevance of TMPRSS2, CD163/CD206, and CD33 in clinical severity stratification of COVID-19.

Background: Approximately 13.8% and 6.1% of coronavirus disease 2019 (COVID-19) patients require hospitalization and sometimes intensive care unit (ICU) admission, respectively. There is no biomarker to predict which of these patients will develop an aggressive stage that we could improve their quality of life and healthcare management. Our main goal is to include new markers for the classification of COVID-19 patients. Methods: Two tubes of peripheral blood were collected from a total of 66 (n = 34 mild and n = 32 severe) samples (mean age 52 years). Cytometry analysis was performed using a 15-parameter panel included in the Maxpar® Human Monocyte/Macrophage Phenotyping Panel Kit. Cytometry by time-of-flight mass spectrometry (CyTOF) panel was performed in combination with genetic analysis using TaqMan® probes for ACE2 (rs2285666), MX1 (rs469390), and TMPRSS2 (rs2070788) variants. GemStone™ and OMIQ software were used for cytometry analysis. Results: The frequency of CD163+/CD206- population of transitional monocytes (T-Mo) was decreased in the mild group compared to that of the severe one, while T-Mo CD163-/CD206- were increased in the mild group compared to that of the severe one. In addition, we also found differences in CD11b expression in CD14dim monocytes in the severe group, with decreased levels in the female group (p = 0.0412). When comparing mild and severe disease, we also found that CD45- [p = 0.014; odds ratio (OR) = 0.286, 95% CI 0.104-0.787] and CD14dim/CD33+ (p = 0.014; OR = 0.286, 95% CI 0.104-0.787) monocytes were the best options as biomarkers to discriminate between these patient groups. CD33 was also indicated as a good biomarker for patient stratification by the analysis of GemStone™ software. Among genetic markers, we found that G carriers of TMPRSS2 (rs2070788) have an increased risk (p = 0.02; OR = 3.37, 95% CI 1.18-9.60) of severe COVID-19 compared to those with A/A genotype. This strength is further increased when combined with CD45-, T-Mo CD163+/CD206-, and C14dim/CD33+. Conclusions: Here, we report the interesting role of TMPRSS2, CD45-, CD163/CD206, and CD33 in COVID-19 aggressiveness. This strength is reinforced for aggressiveness biomarkers when TMPRSS2 and CD45-, TMPRSS2 and CD163/CD206, and TMPRSS2 and CD14dim/CD33+ are combined.

Omicsynin B4 potently blocks coronavirus infection by inhibiting host proteases cathepsin L and TMPRSS2.

The emergence of SARS-CoV-2 variants represents a major threat to public health and requires identification of novel therapeutic agents to address the unmet medical needs. Small molecules impeding viral entry through inhibition of spike protein priming proteases could have potent antiviral effects against SARS-CoV-2 infection. Omicsynin B4, a pseudo-tetrapeptides identified from Streptomyces sp. 1647, has potent antiviral activity against influenza A viruses in our previous study. Here, we found omicsynin B4 exhibited broad-spectrum anti-coronavirus activity against HCoV-229E, HCoV-OC43 and SARS-CoV-2 prototype and its variants in multiple cell lines. Further investigations revealed omicsynin B4 blocked the viral entry and might be related to the inhibition of host proteases. SARS-CoV-2 spike protein mediated pseudovirus assay supported the inhibitory activity on viral entry of omicsynin B4 with a more potent inhibition of Omicron variant, especially when overexpression of human TMPRSS2. Moreover, omicsynin B4 exhibited superior inhibitory activity in the sub-nanomolar range against CTSL, and a sub-micromolar inhibition against TMPRSS2 in biochemical assays. The molecular docking analysis confirmed that omicsynin B4 fits well in the substrate binding sites and forms a covalent bond to Cys25 and Ser441 in CTSL and TMPRSS2, respectively. In conclusion, we found that omicsynin B4 may serve as a natural protease inhibitor for CTSL and TMPRSS2, blocking various coronavirus S protein-driven entry into cells. These results further highlight the potential of omicsynin B4 as an attractive candidate for broad-spectrum antiviral therapy that could rapidly respond to emerging variants of SARS-CoV-2.

Polymorphism of tmprss2 (rs12329760) and severity of COVID-19 in the Ukrainian population: Comment.

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Polymorphism of tmprss2 (rs12329760) and severity of COVID-19 in the Ukrainian population: Response.

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ACE2 and TMPRSS2 SNPs as Determinants of Susceptibility to, and Severity of, a COVID-19 Infection.

Background: Genetic risk factors may be related to the infectivity and severity of SARS-CoV-2 infection. Angiotensin-converting enzyme 2 (ACE2) and host transmembrane serine protease (TMPRSS2) have key role in viral cell entrance and priming. Methods: This case-control study on 147 healthy controls and 299 COVID-19 patients identified potential determinants and risk factors, including gene polymorphism involved in the severity (mild, moderate, severe) of COVID-19 disease defined by CORAD radiological criteria. Results: The ACE2 s2285666 and TMPRSS2 rs12329760 SNPs were significantly linked with COVID-19 disease severity, as were certain co-morbidities (hypertension, heart disease) and laboratory parameters. Both SNPs were amongst the highest predictors of disease severity: TMPRSS2 rs12329760 CT + TT [odds ratio (95% CI) 17.6 (5.1-61.10), ACE2 rs2285666 CT + TT 9.9 (3.2-30.9), both p < 0.001]. There was an increase in the expression of genotype frequencies of ACE2 rs2285666 and TMPRSS2 rs1232976 (TT), (CT + TT), and (T) allele in severe COVID-19 group compared to control and mild groups. Disease severity was also linked to elevated CRP, ferritin and D-dimer, and lower lymphocytes and platelet count (all p < 0.001). Conclusion: ACE2 rs2285666 and TMPRSS2 rs12329760 SNPs, in addition to lymphocyte count, CRP, D-dimers, ferritin, and hypertension, are predictors of COVID-19 disease severity.

TMPRSS2 polymorphism (rs12329760) and the severity of the COVID-19 in Iranian population.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been responsible for the recent pandemic since early 2020. Due to the wide range of clinical symptoms of this disease, from asymptomatic to severe and critical forms, it seems that genetic differences among patients, along with other factors (such as gender, age, and underlying diseases), can explain part of the variation in disease symptoms. The TMPRSS2 enzyme plays a vital role in the early stages of the interaction of the SARS-CoV-2 with the host cells by facilitating viral entry. There is a polymorphism in the TMPRSS2 gene, called rs12329760(C to T) as a missense variant, which causes the replacement of valine to methionine in the TMPRSS2 protein at position 160. The present study investigated the association between the TMPRSS2 genotype and the severity of the Coronavirus disease 2019 (COVID-19) in Iranian patients. The TMPRSS2 genotype of 251 COVID-19 patients (151 patients with asymptomatic to mild and 100 patients with severe to critical symptoms) was detected on genomic DNA extracted from patients' peripheral blood via the ARMS-PCR method. Our results showed a significant association between the minor T allele and the severity of the COVID-19 (P-value = 0.043) under the dominant and additive inheritance model. In conclusion, the results of this study showed that the T allele of the rs12329760 in the TMPRSS2 gene is a risk allele for severe form of COVID-19 in Iranian patients in contrast to most previous studies on this variant in European ancestry populations which suggested this variant as a protective allele. Our results reiterate to the ethnic-specific risk alleles and hidden unknown complexity behind the host genetic susceptibility. However, further studies are needed to address the complex mechanisms behind the interaction of the TMPRSS2 protein and the SARS-CoV-2 and the role of rs12329760 polymorphism in determining the disease severity.

Correlation of Expression of MMP-2, ACE2, and TMPRSS2 Genes with Lymphopenia for Mild and Severity of COVID-19.

Some risk causes may be associated with the severity of COVID-19. The central host-pathogen factors might affect infection are human receptor angiotensin-converting enzyme 2 (ACE2), trans-membrane protease serine 2 (TMPRSS2), and SARS-CoV-2 surface spike (S)-protein. The main purpose of this study was to determine the differences in the expression the metalloproteinases-2  (MMP-2), MMP-9, ACE2, and TMPRSS2 genes and their correlation with lymphopenia in the mild and severe types of the COVID-19 patients. Eighty-eight patients, aged 36 to 60 years old with the mild (n=44) and severe (n=44) types of COVID-19 were enrolled. Total RNA was isolated from the peripheral blood mononuclear cells (PBMCs). The changes of MMP-2, MMP-9, ACE2 and TMPRSS2 gene expression in PBMCs from mild and severe COVID-19 patients were examined by the real time-quantitative polymerase chain reaction (RT-qPCR) assay and, compared between the groups. Data were collected from May 2021 to March 2022. The mean age of the patients in both groups was 48 (interquartile range, 36-60), and there were no appreciable differences in age or gender distribution between the two groups. The present study showed that a significant increase in the expression of ACE2, TMPRSS2, MMP-2, and MMP-9 genes in the severe type of the COVID-19 patients compared, to the mild type of the COVID-19 patients. Overall, it suggests the expression levels of these genes on the PBMC surface in the immune system are susceptible to infection by SARS-COV-2 and therefore could potentially predict the patients' outcome.

Impact of ACE2 and TMPRSS2 Expression in Patients With Preeclampsia.

BACKGROUND/AIM: As maternal morbidity and mortality during pregnancy have increased during the COVID-19 pandemic, studies on pregnancy-related complications from SARS-CoV-2 infection are being actively conducted. Considering that pregnant women with COVID-19 may develop a preeclampsia (PE)-like syndrome, it is necessary to differentiate it from PE because true PE can result in an unfavorable perinatal outcome during a hasty delivery. MATERIALS AND METHODS: We investigated the protein expression of transmembrane serine protease 2 (TMPRSS2) and angiotensin-converting enzyme 2 (ACE2) in placental samples from 42 normotensive (n=9) and PE (n=33) patients without SARS-CoV-2 infection. We isolated placental trophoblast cells from normotensive and PE patients without evidence of SARS-CoV-2 infection to determine the mRNA and protein expression levels of TMPRSS2 and ACE2. RESULTS: High ACE2 cytoplasmic expression in extravillous trophoblasts (EVTs) was correlated with lower fibrin deposition (p=0.017). In comparison with high nuclear TMPRSS2 expression, low nuclearTMPRSS2 expression in endothelial cells (ECs) was positively correlated with PE (p=0.005), significantly higher systolic blood pressure (p=0.006), and higher urine protein-to-creatinine ratio (p=0.022). In contrast, high cytoplasmic TMPRSS2 expression in fibroblasts (FBs) was correlated with higher urine protein-to-creatinine ratio (p=0.018). Trophoblast cells extracted from PE placental tissue showed lower mRNA levels for both ACE2 and TMPRSS2. CONCLUSION: TMPRSS2 nuclear expression in ECs and cytoplasmic expression in FBs of the placenta may be related to a trophoblast-independent PE mechanism, and TMPRSS2 could be a new biomarker to discriminate actual PE from a PE-like syndrome associated with COVID-19.

Protease-Responsive Potential-Tunable AIEgens for Cell Selective Imaging of TMPRSS2 and Accurate Inhibitor Screening.

Transmembrane protease serine 2 (TMPRSS2) is a plasma membrane protease that activates both spike protein of coronaviruses for cell entry and oncogenic signaling pathways for tumor progression. TMPRSS2 inhibition can reduce cancer invasion and metastasis and partially prevent the entry of SARS-CoV-2 into host cells. Thus, there is an urgent need for both TMPRSS2-selective imaging and precise screening of TMPRSS2 inhibitors. Here, we report a TMPRSS2-responsive surface-potential-tunable peptide-conjugated probe (EGTP) with aggregation-induced emission (AIE) features for TMPRSS2 selective imaging and accurate inhibitor screening. The amphiphilic EGTP was constructed with tunable surface potential and responsive efficiency with TMPRSS2 and its inhibitor. The rational construction of AIE luminogens (AIEgens) with modular peptides indicated that the cleavage of EGTP led to a gradual aggregation with bright fluorescence in high TMPRSS2-expressing cells. This strategy may have value for selective detection of cancer cells, SARS-CoV-2-target cells, and screening of protease inhibitors.

TMPRSS2 Is Essential for SARS-CoV-2 Beta and Omicron Infection.

The COVID-19 pandemic remains a global health threat and novel antiviral strategies are urgently needed. SARS-CoV-2 employs the cellular serine protease TMPRSS2 for entry into lung cells, and TMPRSS2 inhibitors are being developed for COVID-19 therapy. However, the SARS-CoV-2 Omicron variant, which currently dominates the pandemic, prefers the endo/lysosomal cysteine protease cathepsin L over TMPRSS2 for cell entry, raising doubts as to whether TMPRSS2 inhibitors would be suitable for the treatment of patients infected with the Omicron variant. Nevertheless, the contribution of TMPRSS2 to the spread of SARS-CoV-2 in the infected host is largely unclear. In this study, we show that the loss of TMPRSS2 strongly reduced the replication of the Beta variant in the nose, trachea and lung of C57BL/6 mice, and protected the animals from weight loss and disease. The infection of mice with the Omicron variant did not cause disease, as expected, but again, TMPRSS2 was essential for efficient viral spread in the upper and lower respiratory tract. These results identify the key role of TMPRSS2 in SARS-CoV-2 Beta and Omicron infection, and highlight TMPRSS2 as an attractive target for antiviral intervention.

Role of Vitamin D, ACE2 and the Proteases as TMPRSS2 and Furin on SARS-CoV-2 Pathogenesis and COVID-19 Severity.

BACKGROUND: COVID-19, the 21st century pandemic disease caused by SARS-CoV-2, has shown a wide clinical spectrum ranging from asymptomatic to deadly serious pneumonia. OBJECTIVE: In our study, the relationship between the pathogenesis and clinical severity of COVID-19 and vitamin D, ACE2, Furin and TMPRSS2 was investigated. METHODS: Serum 25(OH)D, 1,25(OH)2D and ACE2 protein were measured in 85 COVID-19 cases, divided into 5 groups, according to disease severity, from asymptomatic to severe and including a healthy control group. Expression levels of ACE2, VDR, TMPRSS2 and Furin mRNAs in PBMC were also measured. The relationship of the parameters within each group, the severity of the disease and the effect on the patients' fate were investigated. RESULTS: Statistically significant differences were found between the severity of COVID-19 and all study parameters, except for serum 25(OH)D. A strong negative correlation was found between serum ACE2 protein, 1,25(OH)2D, and ACE2 mRNA, and disease severity, length of hospital stay and death/survival rate. Vitamin D deficiency increased the death risk by 5.6-fold (95% CI 0.75-41.47), and the levels of 1,25(OH)2D lower than 1 ng/mL increased the risk of death by 3.8-fold (95% CI 1.07-13.30). CONCLUSION: This study suggests that vitamin D supplementation could be beneficial in the treatment and/or prevention of COVID-19.