ACE2-enriched extracellular vesicles enhance infectivity of live SARS-CoV-2 virus.

The importance of activated TMPRSS2 in the proviral role of small extracellular vesicles in SARS-CoV-2 infection.

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

Phosphatidylinositol kinases (PI kinases) play an important role in the life cycle of several viruses after infection. Using gene knockdown technology, we demonstrate that phosphatidylinositol 4-kinase IIIβ (PI4KB) is required for cellular entry by pseudoviruses bearing the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike protein and that the cell entry mediated by SARS-CoV spike protein is strongly inhibited by knockdown of PI4KB. Consistent with this observation, pharmacological inhibitors of PI4KB blocked entry of SARS pseudovirions. Further research suggested that PI4P plays an essential role in SARS-CoV spike-mediated entry, which is regulated by the PI4P lipid microenvironment. We further demonstrate that PI4KB does not affect virus entry at the SARS-CoV S-ACE2 binding interface or at the stage of virus internalization but rather at or before virus fusion. Taken together, these results indicate a new function for PI4KB and suggest a new drug target for preventing SARS-CoV infection.

Mouse-Passaged Severe Acute Respiratory Syndrome-Associated Coronavirus Leads to Lethal Pulmonary Edema and Diffuse Alveolar Damage in Adult but Not Young Mice

Open AccessCCS ChemistryCOMMUNICATION1 Jan 2022Potential Antiviral Target for SARS-CoV-2: A Key Early Responsive Kinase during Viral Entry Siwen Liu†, Lin Zhu†, Guangshan Xie†, Bobo Wing-Yee Mok, Zhu Yang, Shaofeng Deng, Siu-Ying Lau, Pin Chen, Pui Wang, Honglin Chen and Zongwei Cai Siwen Liu† State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Lin Zhu† State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 HKBU Shenzhen Institute of Research and Continuing Education, Shenzhen 518000 , Guangshan Xie† State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 , Bobo Wing-Yee Mok State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Zhu Yang State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 , Shaofeng Deng State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Siu-Ying Lau State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Pin Chen State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Pui Wang State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Honglin Chen *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 and Zongwei Cai *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 Beijing Normal University-Hong Kong Baptist University United International College, Zhuhai 519087 https://doi.org/10.31635/ccschem.021.202000603 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Currently, there is no effective antiviral medication for coronavirus disease 2019 (COVID-19) and the knowledge on the potential therapeutic target is in great need. Guided by a time-course transmission electron microscope (TEM) imaging, we analyzed early phosphorylation dynamics within the first 15 min during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry. Based on alterations in the phosphorylation events, we found that kinase activities such as protein kinase C (PKC), interleukin-1 receptor-associated kinase 4 (IRAK4), MAP/microtubule affinity-regulating kinase 3 (MARK3), and TANK-binding kinase 1 (TBK1) were affected within 15 min of infection. Application of the corresponding kinase inhibitors of PKC, IRAK4, and p38 showed significant inhibition of SARS-CoV-2 replication. Additionally, proinflammatory cytokine production was reduced by applying PKC and p38 inhibitors. By an acquisition of a combined image data using positive- and negative-sense RNA probes, as well as pseudovirus entry assay, we demonstrated that PKC contributed to viral entry into the host cell, and therefore, could be a potential COVID-19 therapeutic target. Download figure Download PowerPoint Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has been identified to be the cause of coronavirus disease 2019 (COVID-19) since December 2019,1–3 posing huge challenges on health, social, and economic systems globally. SARS-CoV-2 is a zoonotic betacoronavirus; its exact origin or reservoir has not been defined.4–6 Besides, several betacoronaviruses have infected humans, causing respiratory diseases.7 SARS-CoV-2 infection could induce asymptomatic, mild to severe disease, characterized by a range of symptoms, including fever, dry cough, extreme tiredness associated with acute respiratory distress syndrome (ARDS), and lung injury.3,8 Presently, there are no clinically approved antiviral drugs that could effectively inhibit the replication of the SARS-CoV-2. It has been shown that SARS-CoV-2 has a structure similar to receptor-binding domain (RBD) like SARS-CoV or cellular receptor angiotensin-converting enzyme 2 (ACE2), critical for its entry into the host cell.9 Therefore, targeting the viral entry process could be a useful approach to antiviral strategy for COVID-19.10 Enfuvirtide was the first approved viral entry inhibitor that obstructed HIV fusion to host cells.11 Fusion inhibitors, blocking the fusion process of multiple viruses have been developed and shown antiviral activity in vivo.12,13 Consequently, gaining knowledge on SARS-CoV-2 viral entry stage is an urgent requirement for valuable drug development. It is widely reported that coordinated kinase activities are crucial during viral entry.14,15 For example, an activation of protein kinase C (PKC) contributed to influenza viral entry through late endosomes.14,15 Profiling of kinase activity post influenza virus infection showed G protein-coupled receptor kinase 2 was activated within 5 min of influenza infection.16 A comprehensive analysis on SARS-CoV-2 phosphorylation networks during viral uncoating to replication phase (2–24 h postinfection [PI]) was performed by Bouhaddou et al.,17 which demonstrated that SARS-CoV-2 infection promoted multiple kinases' activation, including casein kinase II and p38. However, information on how phosphorylation dynamics changes during the SARS-CoV-2 entry process is still limited. Results and Discussion To determine the appropriate time points to examine changes in the host phosphorylation network, transmission electron microscopy (TEM) was employed to monitor the cell entry process of SARS-CoV-2 within the first 30 min of host infection (Figure 1a). A fetal rhesus monkey kidney Vero E6 cell line was used as a model of infection, as it is highly susceptible to SARS-CoV-2. A high multiplicity of infection (MOI) of 25 was used to ensure universal infection of the Vero E6 cells. Viral particles were found to attach onto the cell surface at 5 min post viral infection, while a thickened membrane was observed at 15 min PI (Figure 1a), denoting the areas where the viral envelope was fusing with the plasma membrane. Within the first 30 min, whole virion was no longer observed. Instead, viral cores of consistent size (40–50 nm in diameter) without envelope were observed in large vacuoles (Figure 1a, 30 min), indicative of the endosome, as coronavirus entered the cells by endocytosis.18 Hence, we selected 5 and 15 min PI as time points to examine the phosphorylation dynamics during viral infection. Figure 1 | Phosphorylation network response during the early phase of SARS-CoV-2 infection. (a) TEM analysis of Vero E6 infected with SARS-CoV-2 for 5, 15, 30 min. Viral particles attaching and fusing with cell membrane (5 and 15 min, black arrow). Some viral particles were observed in the large vacuoles (30 min, white arrowhead). (b) Workflow of phosphopeptides' enrichment. (c and d) Regulation of phosphopeptides identified in 5 (c) and 15 min (d) PI with SARS-CoV-2. Download figure Download PowerPoint Vero E6 cells were harvested at 5 or 15 min PI after SARS-CoV-2 or mock infection (Figure 1b) in four independent biological experiments. At 5 min PI, 115 phosphorylation sites were upregulated and 106 were downregulated, while 37 were upregulated and 65 were downregulated at 15 min PI (Figures 1c and 1d). The altered phosphopeptides were then used for further functional analysis and kinase prediction. At both study time-points, PKC activity was the highest enriched molecular function by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis ( Supporting Information Figure S1a). Enrichment map analysis also showed significant overlaps in PKC related pathways ( Supporting Information Figure S1c), suggesting that kinase activities were perturbed during the viral entry process. We then established a kinase prediction pipeline using altered phosphosites (both up- and down-altered) identified. Kinase prediction was obtained initially by the Group-based Prediction System 5.0,19,20 followed by highly stringent cutoff adopted from previous publication.16 Four kinases were consistently predicted at both 5 and 15 min PI (Figure 2a), and consequently selected for further validation, as follows: PKC-gamma (PKCγ; p = 0.0109, which was also identified directly in phosphoproteomics analysis), interleukin-1 receptor-associated kinase 4 (IRAK4; p = 0.0203), MAP/microtubule affinity-regulating kinase 3 (MARK3; p < 0.0001), and TANK-binding kinase 1 (TBK1; p = 0.0003). Subsequently, amino acid sequences flanking hyperphosphorylation sites were retrieved to reveal the phosphorylation motifs (Figure 2b). Basophilic motif of arginine (R) at position-3 was significantly enriched, compared with background, a specific feature of conventional PKCs, including PKCγ,21 which was in agreement with our prediction. Intriguingly, recent studies showed that MARK3 and TBK1 could directly interact with SARS-CoV-2 viral proteins.22 Furthermore, TBK1 was targeted by SARS-CoV-2 proteins to antagonize type I interferon (IFN-I) response.23 Collectively, these lines of evidence confirmed our kinase prediction pipeline was able to identify crucial kinase for SARS-CoV-2 replication (Figure 2c). Figure 2 | Predicted early responsive kinase activity of SARS-CoV-2 infection. (a) Top kinases predicted to regulate differential phosphorylation at 5- and 15-min PI are marked in red. All enriched kinases passed the stringent score filter. (b) Enriched phosphorylation motifs from hyperphosphorylated peptides in both 5 and 15 min PI, phosphorylated sites (S/T) set as position 0. Size of the letter represents the enrichment degree. (c) Protein–protein interactions (PPI) map of SARS-CoV-2 viral protein with predicted host kinase-substrates network. Download figure Download PowerPoint Further, we used kinase inhibitors targeting the predicted kinases to evaluate their effects on viral replication. Inhibitors of PKC (Bisindolylmaleimide IX), TBK1 (Amlexanox & MRT67307 HCL), and IRAK (IRAK-1-4 Inhibitor I) were used. p38 kinase MAPK12 and cyclin-dependent kinase 6 (CDK6) were top-predicted kinases before a stringent filter was applied, and the hyperphosphorylated motifs suggested the involvement of CDK and MAPK kinases (Figure 2b). In addition, p38 was reported to affect SARS-CoV replication.24 Therefore, we included CDK inhibitor (Palbociclib HCI) and p38 inhibitor (SB203580) as well. Cytotoxicity of these inhibitors were determined ( Supporting Information Figure S2). Then inhibitors were used at concentrations without major cytotoxicity. Bafilomycin A1 (BafA1), reported to block SARS-CoV-2 viral entry,25,26 was used as positive control. As shown in Figure 3a, p38 inhibitor efficiently blocked virus replication in all three cell lines, as expected. Inhibition of either IRAK or PKC leads to suppression of viral replication and viral mRNA synthesis in a dose-dependent manner in both Calu3 (non-small-cell lung cancer) and Caco2 (human colorectal adenocarcinoma) cell lines (Figure 3b), confirming our kinase prediction. Importantly, PKC the inhibitor showed the most pronounced inhibition of viral replication and mRNA synthesis, consistent with both KEGG (Kyoto Encyclopedia of Genes and Genomes, a database resource for functional studies) analysis and kinase prediction. As kinase inhibitors might have off-target effect, two additional PKC inhibitors (Sotrastaurin and Enzastaumn) were used to evaluate their effects on viral replication ( Supporting Information Figure S5). All three PKC inhibitors demonstrated inhibitory effects on SARS-CoV-2 replication in a dose-dependent manner, confirming the critical role of PKC activity in viral replication. The discrepancy of inhibitory effects for kinase inhibitors were observed between Vero E6 and two human cell lines, which should be caused by the absence of IFN-I in cells ( Supporting Information Figure S3). The lack of IFN-I would only affect the overall viral replication but not the phosphodynamics we observed, as we focused on the viral entry process, which was before the participation of IFN-I. This observation also confirmed the role of interferon, as reported previously.27 Inhibition of CDK led to a slight increase in terms of viral replication, suggesting that alteration of CDK kinase activity might be an adversary for SARS-CoV-2 replication. Figure 3 | Effect of different inhibitor treatment on viral mRNA level and viral titer. Cells were pretreated with different inhibitors at the indicated dose, followed by SARS-CoV-2 infection. Indicated three different relative viral mRNA levels cells were measured by normalizing to control (a). Corresponding viral titers by plaque assay were shown in (b). For all panels, *p < 0.05, **p < 0.005, ***p < 0.0005, nonsignificant (ns) for two-tail Student's t-test. Error bars indicate SD (n = 3). Download figure Download PowerPoint Furthermore, we used RNA fluorescence in situ hybridization (RNA-FISH) to visualize the viral replication process. SARS-CoV-2 generates negative-strand RNA template to synthesize new genomic RNAs; therefore, the distribution of negative-strand RNA refers to the location of replicative-intermediate in replication-transcription complex.28 An A549 cell line expressing human ACE2 was generated (Figure 4c) and pretreated with inhibitors before SARS-CoV-2 infection. In the control group, viral genomic RNA and mRNA were widely distributed and accumulated in the perinuclear area. Replicative-intermediate RNAs, indicative of ongoing viral replication, were also clearly detected (Figure 4). In contrast, p38 or PKC inhibitors significantly repressed viral infection rate. Particularly, effect of PKC inhibitor was more significant than BafA1-positive control (Figure 4a). We found that inhibitor of p38 kinase did not change signal intensity of negative-sense viral RNA, suggesting the inhibition probably occurred in the late stage of viral cycle. However, perinuclear dots of negative-sense viral RNA were significantly decreased after the treatment of PKC inhibitor (Figure 4b), which signified the lack of ongoing replication events. The observation indicated that PKC activity was crucial during viral entry as we predicted. We then validated the role of PKC during the early stage of viral replication by SARS-CoV-2 pseudovirus entry assay. A home-made SARS-CoV-2 pseudovirus was constructed and used to infect 293T-ACE2 cells pretreated with PKC inhibitors. As shown in Figure 4d, an inhibition of PKC activity diminished pseudovirus signals, confirming the inhibitory role of PKC inhibitor during the early stage of SARS-CoV-2 replication. PKC is known to regulate PKC-dependent endocytosis and involve in influenza viral entry by regulating late endosomes.14,15 Alternatively, coronaviruses, including Middle East respiratory syndrome (MERS)- and SARS-CoV, are known to rely on endocytic pathway for entry.18,29 Consequently, we proposed that PKC was required by SARS-CoV-2 as an early responsive kinase for viral entry via an endocytic pathway, making it a potential therapeutic target for COVID-19. Figure 4 | Confocal images suggested that PKC inhibitor block SARS-CoV-2 entry. A549-Ace2 cells were pretreated with the indicated inhibitor, followed by SARS-CoV-2 infection. (a) FISH and IFA imaging of infected cells using positive-sense RNA probe (purple) and antibody against viral N protein (green). (b) Fixed cells were processed for FISH assay using positive- (purple) and negative-sense RNA probe (green). Merge images also include 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). (c) Whole cell lysates were analyzed for Ace2 and tubulin expression by Western blot using their respective antibodies. (d) Pseudovirus entry assay showed that the inhibition of PKC activity prevented viral entry signals. Download figure Download PowerPoint It has been reported that poor prognosis outcomes of patients with COVID-19 were associated with cytokine storm, generated by innate immune response, while several cytokines have been reported as potential biomarkers for disease progression.30–32 Additionally, emerging pieces of evidence have shown that SARS-CoV-2 infection induces low types I and III IFNs' levels and limited interferon-stimulated genes' (ISG) response, but high level of chemokine expression.8,33 We showed that the inhibition of cytokine mRNA levels caused by kinase inhibitors correlated with that of viral titer. A significant and dose-dependent reduction of cytokine levels were observed when treated with PKC and p38 inhibitors across all three cell lines ( Supporting Information Figure S4). Also, these cytokines' expression were inhibited by IRAK inhibitor, but at a relatively moderate level. Finally, to validate the essential role of PKC activities in viral replication, three commercially available small interfering RNAs (siRNAs) targeting PKC-alpha (PKCα), PKC-beta (PKCβ), and PKC-epsilon (PKCɛ) were ordered to knock down the corresponding PKC isoforms. PKCα siRNA failed to achieve effective knock down, so only PKCβ and PKCɛ siRNAs were used in viral inhibition assay (Figure 5a). As shown in Figure 5b, only PCKβ knock down showed significant inhibitory effect on viral replication. A siRNA knock down of PKCɛ led to an increase in PKCβ activity, suggesting a potential compensation effect, which might explain the inefficiency of viral inhibition of PKCɛ siRNA. These results further confirmed with our earlier PKC inhibitors' data, as all three PKC inhibitors we tested in the study are efficient PKCβ inhibitors. Consequently, PKC activity, particularly PKCβ, might play a vital role in optimum replication of SARS-CoV-2. Figure 5 | siRNA knock down of PKCβ inhibits SARS-CoV-2 replication. (a) Transcriptional levels of PKC isoforms in siRNA knock down Calu-3 cells were examined using q-PCR. (b) Viral mRNA levels in siRNA knock down Calu-3 cells infected with SARS-CoV-2 were measured by normalizing to control. **p < 0.005, ***p < 0.0005, ****p < 0.00005, nonsignificant (ns) for two-tail Student's t-test. Error bars indicate SD (n = 3). Download figure Download PowerPoint Conclusion Using a time-course TEM imaging, we identified key time points of viral attachment and fusion of SARS-CoV-2 infection. By combining phosphoproteomics and kinase prediction pipeline, we found that PKC and IRAK4 activities were activated at the first 5–15 min of viral entry. We showed that the inhibition of PKC, IRAK4, and p38 could suppress optimal replication of the SARS-CoV-2 virus, among which IRAK4 activity initially associated with SARS-CoV-2 replication. We further demonstrated that inhibition of PKC activity, particularly PKCβ, would inhibit viral replication at early stage, probably via blockage of specific endocytosis phosphorylation events required for viral entry. Therefore, PKC might be required for SARS-CoV-2 entry, and thus, could serve as a potential therapeutic target for COVID-19. Data Availability The raw MS data from this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with accession number PXD021610. Supporting Information Supporting Information is available and includes detailed material and methods, as well as Figures S1–S5. Conflict of Interest There is no conflict of interest to report. Funding Information This research was made possible because of a generous grant from the National Key R&D Program, Ministry of Science and Technology, China (no. 2017YFC1600500), the National Natural Science Foundation of China (no. 21705137), the Theme-Based Research Scheme (no. T11/707/15) and General Research Fund (no. 17107019) of the Research Grants Council, Hong Kong Special Administrative Region, and the Sanming-Project of Medicine in Shenzhen, China (nos. SZSM201911014 and SZSM201811070).

Coronavirus disease 2019 (COVID-19) is caused by a novel strain of coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused a global pandemic rapidly s...

Severe acute respiratory syndrome (SARS) first emerged in Guangdong province, China in November 2002. During the following 3 months, it spread rapidly across the world, resulting in approximately 800 deaths. In 2004, subsequent sporadic cases emerged in Singapore and China. A novel coronavirus, SARS-CoV, was identified as the etiological agent of SARS.1,2 This virus belongs to a family of large, positive, single-stranded RNA viruses. Nevertheless, genomic characterization shows that the SARS-CoV is only moderately related to other known coronaviruses.3 In contrast with previously described coronaviruses, SARS-CoV infection typically causes severe symptoms related to the lower respiratory tract. The SARS-CoV genome includes 14 putative open reading frames encoding 28 potential proteins, and the functions of many of these proteins are not known.4 A number of complete and partial autopsies of SARS patients have been reported since the first outbreak in 2003. The predominant pathological finding in these cases was diffuse alveolar damage (DAD). This severe pulmonary injury of SARS patients is caused both by direct viral effects and immunopathogenetic factors.5 Many important aspects of the pathogenesis of SARS have not yet been fully clarified. In this article, we summarize the most important mechanisms involved in the complex pathogenesis of SARS, including clinical characters, host and receptors, immune system response and genetic factors. CLINICAL PATHOLOGY CHARACTERS OF SARS Patients with SARS-CoV infection have a wide spectrum of disease, varying from a self limiting illness to a fatal outcome.6,7 This disease consists of two phases, including prodromal influenza-like symptoms characterized by myalgia, malaise, chills and fever, and the onset of respiratory and gastrointestinal symptoms.8 Fever was the most common and the earliest symptom.9 The clinical picture is characterized by pulmonary inflammation and respiratory failure, resembling that of acute respiratory distress syndrome (ARDS). Upper-respiratory-tract symptoms are not prominent9 but gastrointestinal symptoms were common.10,11 A majority of the patients admitted to the hospital showed pulmonary X-ray abnormalities varying from bilateral interstitial infiltrates to focal consolidation.6,12 Autopsies of SARS cases indicated that the lungs were edematous and increased in weight.13,14 In some SARS cases organizing features, like dense septal and alveolar fibrosis, were demonstrated.15,16 The longer the disease persists, the more extensive becomes the fibrous organization of the lung tissue.17 Fibrin and collagen were found deposited in the alveolar space.18 Morphological changes identified were bronchial epithelial denudation, loss of cilia and squamous metaplasia. DAD is the most consistent finding in the lungs of SARS patients in the terminal stage.9 In SARS postmortem samples viral RNA has been localized by in situ hybridization to cells of the conducting airways and alveoli.19 The infection and release of virus was close to the pulmonary capillary bed, which might allow systemic spread of virus to distant organs, especially in the context of inflammation and alveolar capillary leak.20 In many cases, cellular infiltration has been observed, including macrophages, neutrophils and CD8+ T cells. Macrophages are a prominent component of the cellular exudates in the alveoli and lung interstitium.9,21 In addition, immunohistochemistry, in situ hybridization and electron microscopy examination of tissue upon autopsy or tissue biopsy showed that SARS-CoV replicates in pneumocytes and macrophages.22 The replication of SARS in macrophages suggests a passive role for macrophages as scavengers, rather than being the primary target.23 A disproportionate scarcity of inflammatory cells has been noted.5,13 Mononuclear infiltrates increased in the interstitium. Large multinucleated cells have been frequently observed in the lungs of SARS patients.5,14 The presence of hemophagocytosis supports the contention that cytokine dysregulation may account for the severity of the clinical disease. The lack of a prominent inflammatory response is also distinctive. Such changes reflect the combined effects of primary infection, host immune responses and therapeutic interventions. A substantial number of SARS patients have diarrhea.11,24 In the intestine, little pathology is observed at the light microscopy level, either in biopsies taken during early phases11 or in autopsy specimens.13,19 Severe depletion of mucosal lymphoid tissue in the small intestines and appendix has been described.25 Both extensive necrosis of the spleen and atrophy of the white pulp with severe lymphocyte depletion have also been found.14,26,27 A sharp decrease in the number of periarterial sheaths in the spleen have been demonstrated. CD4+ lymphocytes, CD8+ lymphocytes, CD20+ lymphocytes, dendritic cells, macrophages, and natural killer cells in the spleen showed a decrease of 78, 83, 90, 80, 39 and 48%, respectively. The average size of macrophages was found to be increased by more than 100%. T lymphocytes and macrophages in the spleen have been detected to be infected by SARS-CoV.27,28 Lymph nodes usually show atrophy and reduction of lymphocytes with loss of germinal centers.28 Focal necrotic inflammation of hilar lymph nodes has been found in some cases.29 Evidence of hemophagocytosis in lymph nodes was observed in a limited number of cases.30 High viral loads have been detected in lymph nodes, whereas viral isolation was negative.26 T lymphocytes and macrophages in lymph nodes have also confirmed SARS-CoV infection.28 Several observations suggest that SARS-CoV is also capable of causing an infection of the central nervous system. Cerebrospinal fluid, brain tissue specimens and neurons were detected to have SARS-CoV infection.28,31 Kidneys of autopsied SARS patients have shown focal necrosis and vasculitis of small veins in the renal interstitial tissue.14 High viral loads have been detected in the renal tissue specimens and the distal convoluted tubules, which suggest that urine may be an additional source of sewage contamination.32 In addition, monocytic infiltration, acute tubular necrosis and other nonspecific changes, such as glomerular fibrosis and nephrosclerosis, have been all observed.33 A high proliferative index has been demonstrated in hepatocytes in the liver in some cases. But viral particles are not detected by electron microscopy (EM).34 In both the liver and the kidney signals for SARS-CoV were detected by both immunohistochemical (IHC) and in situ hybridization (ISH),35 yet EM failed to reveal recognizable viral particles. This raises the question that whether the virus exists in a non-packaged form.25 Destruction of epithelial cells with significant changes in the follicular architecture was present in the thyroid glands. Both myofiber necrosis and atrophy were observed in the limited number of skeletal muscle tissue specimens of SARS autopsies examined.36 Edema of the walls of small veins and arteries has also been reported. The few available studies on adrenal glands described the presence of necrosis and vasculitis of the medulla with monocytic and lymphocytic infiltration. Edema of both myocardial stroma, as well as vascular walls, and atrophy of cardiac muscle fibers all have been demonstrated.14,22 SARS-CoV genomic sequences and antigens have also been detected in sweat glands and pancreatic islet cells.35,37 The presence of virus in the sweat glands suggests that SARS may be spread via contact with the skin.25 In some cases, evidence of reactive hemophagocytosis or bone marrow hypoplasia was present.29 Raised creatine kinase, thrombocytopenia, an increase in lactate dehydrogenase and a decrease in absolute lymphocyte counts are the most common laboratory findings. The majority of SARS patients showed a transient increase in serum alanine aminotransferase levels during the course of their disease.38 In some autopsy cases fatty degeneration were observed. These findings suggest that SARS is a systemic disease with widespread extrapulmonary dissemination, resulting in viral shedding in respiratory secretions, stools, urine and possibly even in sweat. The organ damage in patients with SARS could be due to both local viral replication and the immunopathologic consequences of the host response, hence it is important to delineate what human cells the SARS-CoV can infect and replicate in as well as the subsequent host immune response.35,39 HOST AND RECEPTORS SARS-CoV was isolated from Himalayan palm civets found in a live-animal market in Guangdong, China in 2003. The full-length genome sequences had 99.8% homology to the SARS-CoV genomic found in humans. So primarily, palm civets were suspected as the origin of the SARS outbreak in 2003. Subsequently, many other animals have also been found to be a host or to be infected by the virus.40 Bats and swine were also reported as natural carriers of SARS virus.41,42 Recently, horseshoe bats were designated as the natural reservoir for SARS-CoV-like virus and civets were identified as the amplification host. This highlights the importance of wildlife and biosecurity in farms and wet markets, which can serve as the source and amplification centers for emerging infections.43 SARS-CoV spreads via droplet and contact transmission and via the fecal-oral route.44 Through these routes, SARS-CoV can be transmitted from animal to human or from human to human. The primary target of SARS-CoV is epithelial cells in the respiratory and intestinal tract.18 Epithelia are primary barrier to infection by microorganisms entering their host via body cavities. Epithelial cells are organized in a polarized fashion that involves the separation of the plasma membrane into an apical and a basolateral domain. The polarity of these cells affects both the early and late stages of infection, i.e. viruses may enter into and exit from a cell either via the apical membrane facing the external environment or via the basolateral membrane directed to the internal milieu of the organism. An important determinant of the virus infection is the presence of suitable receptors on the cell surface that allow attachment to and penetration through the plasma membrane.20 Angiotensin-converting enzyme 2 (ACE2), a protector of lung damage, has been identified as the primary functional receptor for SARS-CoV.45 ACE2 is a membrane-associated aminopeptidase.46 A region of the extracellular portion of ACE2 that includes the first α-helix and lysine 353 and proximal residues of the N terminus of β-sheet 5 interacts with high affinity to the receptor binding domain of the SARS-CoV S glycoprotein.47 The N terminal half of the S protein (S1) contains the receptor binding domain whereas the C-terminal half (S2) is the membrane-anchored membrane-fusion subunit, which contains two heptad repeat regions (HR1 and HR2).48 After binding to ACE2 on the target cells, the transmembrane S protein changes conformation by association between the HR1 and HR2 regions to form a six helix oligomeric complex, leading to fusion between the viral and target-cell membranes. Apart from direct membrane fusion at the target cell surface, SARS-CoV might gain cell entry via pH-dependent endocytosis, which is also mediated by the S protein.49 In addition to being a cellular receptor, ACE2 may contribute to the pathogenesis of DAD in SARS through its role in the tissue rennin-angiotensin system (RAS).8 ACE2 is a negative regulator of the RAS and has a negative effect on the formation of angiotensin II. Angiotensin II appears to be one of the elements of the RAS that contributes to exacerbation of acute lung injury.50 With respect to SARS-related lung injury, binding of SARS-CoV Spike proteins to ACE2 has been found to reduce ACE2 expression, thus inducing acute lung edema.51 Based on animal experiments, ACE2 may protect against respiratory failure and down-regulation of ACE2 may cause acute lung injury. The insert/deletion genotype of the ACE gene was associated with DAD after SARS-CoV infection in a small cohort of 44 patients.52 ACE2 protein is reportedly present in type 1 and type 2 pneumocytes, enterocytes in all parts of the small intestine, the brush border of the proximal tubular cells of the kidney, as well as the endothelial cells of small and large arteries and veins and arterial smooth muscle cells.46 This localization of ACE2 explains the tissue tropism of SARS-CoV for the lung, small intestine and kidney. Theoretically, all tissues and cell types expressing ACE2 may be potential targets of SARS-CoV infection. However, notable discrepancies were found including virus replication in colonic epithelium, which has no ACE2, and no virus infection in endothelial cells, which have ACE2. Despite the fact that SARS-CoV can infect the lung and intestine the tissue responses in these two organs are different. Furthermore, studies in a new human cell culture model have indicated that the presence of ACE2 alone is not sufficient for maintaining viral infection.39,53 Other findings indicate that ACE2 expression positively correlated with the differentiation state of the epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected.18 It is apparent that the effect of SARS-CoV infection is different in different cell types and it is possible that the virus may utilize different receptors, or involve various co-receptors, in these different cells. C-type lectins, including CD209 and CD209L, were identified as alternative SARS-COV receptors.53 CD209, also known as dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN), is mainly expressed in certain types of dendritic cells (DCs) and alveolar macrophages.54 However, in the lung tissue of SARS autopsies, CD209 has been localized to pneumocytes.55In vitro, CD209 was also inducible in lung epithelial and monocytic cells after SARS-CoV infection,55 which confirmed that SARS infection is capable of inducing CD209 expression. The glycosylated S protein has been shown to bind to the CD209 expressed on the DCs; these cells then mediate SARS-CoV infection in trans of cells that express human ACE2. CD209L, also known as L-SIGN or DC-SIGNR, is generally found in lymph nodes and liver sinusoidal cells. By IHC it has been demonstrated that CD209L is also expressed on type II pneumocytes and endothelial cells. CD209L can also bind to S protein and mediate virus entry.56,57 Although SARS-CoV does not replicate in DCs, these cells may act as a reservoir and distribute the virus to other cell types.58 This is an attractive concept and similar biological behaviours have been proposed for human immunodeficiency virus I (HIV I).59In vitro experiments have demonstrated that cells expressing CD209 or CD209L without ACE-2 are not, or are only partially, susceptible to SARS-CoV infection. This would imply that these molecules are much less efficient receptors than ACE2 as specific receptors and may therefore merely enhance infection of permissive cells.49,56,57 SARS-CoV infection of ACE2-expressing cells also seems to be dependent on the proteolytic enzyme cathepsin L. Cathepsin L is poorly expressed in endothelial cells which may explain the low infection rate of these cells despite the high expression of ACE2. SARS-CoV infection seems to be pH-dependent because the activation of cathepsin L is pH sensitive. Differential expression of cathepsin L in various cell types may explain the differences in viral distribution in relation to the ACE2 expression pattern.60,61 CYTOKINES AND CHEMOKINES Both cytokines and chemokines are soluble proteins with a key function in the innate immune system. Dysregulation of these proteins may result in immunemediated injury.5 High levels of cytokines and chemokines, triggered by the host immune response to SARS coronavirus (SARS-CoV), are believed to contribute to the progressive pulmonary infiltration of macrophages,9 polymorphonuclear leukocytes, T cells,62 eventual DAD and fibrosis.6 This assumption is supported by the clinical deterioration of many patients in the second week of the disease's course, despite decreasing viral loads.9,63 Increased serum levels of several cytokines were found in major SARS patients.51,57 Most cytokines showed only transient and short-lived activation in patients after SARS-CoV infection.64 Even in patients who developed DAD, most cytokine concentrations were not significantly increased.65 In contrast, circulating concentrations of several chemokines, including chemokine C-X-C motif ligand 9 or monokine induced by γ-interferon (CXCL9), chemokine C-X-C motif ligand 10 or interferoninducible protein-10 (CXCL10) and C-C motif ligand 2 or monocyte chemoattractant protein-1 (CCL2), were markedly increased in SARS patients.62,64,66 Recent studies have focused on the role of chemokines rather than cytokines in SARS infection.5 The chemokines are a family of small-molecule proteins that important in intercellular and Based on their protein are into with a common C-X-C of residues the which interacts with and with a C-C with In the lung tissues from SARS patients who chemokines and were markedly and a chemokine C-X-C motif ligand were also markedly A number of chemokines, including and were increased 1 after to the Most for the receptor for and was in the lungs of of gene expression changes in cells from in vitro with show an early activation of the innate in the first including expression of receptor 9 chemokines and their receptors with and and macrophages in the lungs express In in vitro cells chemoattractant protein 1 and after with SARS-CoV and expressed and vascular cell adhesion SARS-CoV induced cells to express and which and T cells in a The showed low expression of a and of cytokines necrosis and but significant of inflammatory chemokine It activation of T cell to express and and also to be significantly increased in lung tissue and lymphoid tissue of autopsied SARS which was confirmed by IHC with An increased has been found to be an of SARS-CoV, through a with lung epithelial cells and monocytic cells, an environment to immune cell and that to lung The lack of cytokine response against a of chemokine could a of immune by A model to explain cellular infiltration may result in SARS was pulmonary epithelial cells infected by SARS-CoV express adhesion molecules and high levels of and which macrophages and and macrophages by with SARS-CoV and a of chemokines that more and neutrophils as well as T viral effects are also to contribute to the pulmonary injury resulting from SARS-CoV infection. In during the first 10 of the disease, virus replication is viral effects to an important The presence of multinucleated cells in SARS lungs may be the result of viral The virus is also capable of causing effects in both renal epithelial cells and epithelial cells in and formation are in infected studies have reported evidence of in cells of the thyroid cells, epithelial cells, pneumocytes, lymphocytes and vitro experiments indicate that expression of certain proteins may in several cell expression of a protein by SARS-CoV, can via a dependent in cell from different organs, including lung, liver and kidney. of may be one of the mechanisms for the pathogenesis of SARS-CoV SARS protein also appears to be important in in some cell It is into the and may also act as one of the Through an host cells SARS have increased expression of Furthermore, SARS-CoV proteins also have the to in SARS-CoV proteins may be involved in through of proteins Increased expression of has been detected in infected alveolar and bronchial epithelial cells. is an of cell of this cytokine may also account for of such cells. OF SARS and viral in the first 10 of SARS immune by The innate immune system the first of the immune against viruses and involves several cellular and soluble factors. T lymphocytes and are the key immune cells that are infected by vitro infected cells have shown viral replication for to In from SARS SARS-CoV has also been found to infect and replication was Both T lymphocytes and were found in the circulating lymph nodes, lungs and in SARS These findings may a partial for the and the widespread of spleen and lymphoid tissue in the majority SARS immune cells may cause widespread to various and T cells are involved in both the innate and immune the of such cells may result in a immune with a decrease in both CD4+ and CD8+ T cells is common during the acute of SARS and may be associated with an Several other viral such as virus and respiratory virus are also associated with severe However, in these direct infection and subsequent of lymphocytes are generally as to account for the severe lymphocyte In for only a small of the are infected during acute with the high infection of in SARS immune syndrome with respect to the fact that both are viral that result in However, SARS various immune cells and rapidly whereas human immunodeficiency virus mainly CD4+ lymphocytes and is In addition, SARS-CoV seems to the of macrophages, which may SARS patients to pulmonary SARS-CoV also causes and functional of dendritic cells in vitro, resulting in a of cytokines and an T cells may in the to pulmonary injury. In viral in are and by infected cells. These cells cause cells to that to viral to other SARS-CoV is not capable of inducing significant or gene expression in infected macrophages, or in infected dendritic Furthermore, in contrast to patients with was a lack of expression of for the and in cells of patients with studies have found that from SARS patients could be an of of serum and against SARS-CoV and by against the that most patients after onset of can be detected as early as after the onset of The for and most of the is to In a of SARS the levels at and were for on the of 14 patients showed that the was in for a from developed a and and of the not The indicated that specific and could be in the could the SARS-CoV and protect cells from SARS-CoV The low response of SARS-CoV has been identified in which proposed a question that whether the serve as a host for were SARS cases that in Guangdong in In contrast to in these cases with clinical and caused a lower and transient immune The of the cases to a at at and then rapidly a of This that SARS-CoV may have to during the These can the particles the S protein from different SARS-CoV that these are and that the S protein is the S the other proteins, such as or is the only significant SARS-CoV and with as the major The on the S was also developed may also be involved in the pathogenesis of Both and immune responses to animal have been identified to be capable of the disease or causing new are with and coronavirus a of by with the SARS-CoV of were to the specific of the while of the could of the or These may have some of the that were to the and characterized the binding of SARS-CoV by an detected specific for the of an human serum In cohort of SARS patients immune against antigens from lung epithelial cell and endothelial cell was found in some approximately 1 after high levels of these in the were shown to be to lung epithelial cells and endothelial cells in may be to the of against specific SARS-CoV against the domain 2 of the protein have been found to with pulmonary epithelial that possibly explains is the of caused by organ has been a the of since some antigens in the may that enhance viral infection rather than also to a role in the pathogenesis of a of the was associated with severity of SARS This association has not been for certain Nevertheless, in the a association was demonstrated between and and an increased to SARS infection. is a serum protein that can bind to the of various for immune of a specific is capable of binding to the glycosylated SARS-CoV S protein and SARS-CoV in In a of SARS patients and have been shown to be associated with increased of In contrast, CD209L to have a significantly lower of SARS infection. The in genetic between is for by In the context of to after at the of were associated with high concentrations of in the plasma and a In addition to other genetic such as enzyme are also associated with severity and of Although ACE2 as its receptor and ACE2 is known to be an important protector of lung damage in no association between of the two ACE and and the severity of after SARS infection was is in the of different and the of in clinical is studies are to fully the genetic for both to infection and the after infection with the The pathogenesis of SARS appears to be and The most and possible appears to of a direct injury to the target cells by the virus and an injury mediated by subsequent immune system By droplet SARS-CoV the respiratory and the epithelial cells of the and infection and replication in target cells causes direct damage to the respiratory tract. inflammatory changes the of the barrier and increase the of the capillary of in the formation of membranes. The infection and associated inflammation acute injury of type II alveolar cells, decreasing the of alveolar resulting in alveolar the SARS-CoV and circulating immune cells. The infected immune cells mainly macrophages and T cells. immune cells the virus to other organs, including the spleen and the lymph The of immune cells with extensive damage to the white pulp in A immune the infection and replication of the virus in the lungs and viral damage to the respiratory resulting in respiratory The proposed mechanisms of SARS have significant for the and on this emerged disease. Although much has been of SARS since its many with respect to the pathogenesis of SARS is a that SARS for the of at 2 known animal in civets and horseshoe bats that are found in the as well as and in China. are

SNX27-mediated endocytic recycling of GLUT1 is suppressed by SARS-CoV-2 spike, possibly explaining neuromuscular disorders in patients with COVID-19

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

Viruses require specific cellular receptors to infect their target cells. Angiotensin-converting enzyme 2 (ACE2) is a cellular receptor for two divergent coronaviruses, SARS coronavirus (SARS-CoV) and human coronavirus NL63 (HCoV-NL63). In addition to hostcell receptors, lysosomal cysteine proteases are required for productive infection by some viruses. Here we show that SARS-CoV, but not HCoV-NL63, utilizes the enzymatic activity of the cysteine protease cathepsin L to infect ACE2-expressing cells. Inhibitors of cathepsin L blocked infection by SARS-CoV and by a retrovirus pseudotyped with the SARS-CoV spike (S) protein but not infection by HCoV-NL63 or a retrovirus pseudotyped with the HCoV-NL63 S protein. Expression of exogenous cathepsin L substantially enhanced infection mediated by the SARS-CoV S protein and by filovirus GP proteins but not by the HCoV-NL63 S protein or the vesicular stomatitis virus G protein. Finally, an inhibitor of endosomal acidification had substantially less effect on infection mediated by the HCoV-NL63 S protein than on that mediated by the SARS-CoV S protein. Our data indicate that two coronaviruses that utilize a common receptor nonetheless enter cells through distinct mechanisms.

SARS-CoV-2 infection and ACE2 inhibition.

A potential inhibitory role for integrin in the receptor targeting of SARS-CoV-2

he prevalence of coronavirus disease 2019 (COVID-19) has posed a great threat to people's health worldwide, bringing a great challenges to the public healthcare systems. A recent study has confirmed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses severe acute respiratory syndrome coronavirus (SARS-CoV) receptor angiotensin-converting enzyme 2 (ACE2) for host cell entry. 1 ACE2 expression was previously found to correlate with susceptibility to SARS-CoV infection in vitro.

Infection with severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) leads to coronavirus disease 2019 (COVID‐19), which poses an unprecedented worldwide health crisis, and has been declared a pandemic by the World Health Organization (WHO) on March 11, 2020. The angiotensin converting enzyme 2 (ACE2) has been suggested to be the key protein used by SARS‐CoV‐2 for host cell entry. In their recent work, Lindskog and colleagues (Hikmet et al, 2020) report that ACE2 is expressed at very low protein levels—if at all—in respiratory epithelial cells. Severe COVID‐19, however, is characterized by acute respiratory distress syndrome and extensive damage to the alveoli in the lung parenchyma. Then, what is the role of the airway epithelium in the early stages of COVID‐19, and which cells need to be studied to characterize the biological mechanisms responsible for the progression to severe disease after initial infection by the novel coronavirus?

Extracellular vesicles: A promising therapy against SARS-CoV-2 infection