A potential peptide inhibitor of SARS-CoV-2 S and human ACE2 complex

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.

SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs.

Human angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS-CoV). Here we identify the SARS-CoV spike (S)-protein-binding site on ACE2. We also compare S proteins of SARS-CoV isolated during the 2002–2003 SARS outbreak and during the much less severe 2003–2004 outbreak, and from palm civets, a possible source of SARS-CoV found in humans. All three S proteins bound to and utilized palm-civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S-protein-binding site of human ACE2 to those of civet ACE2, or by altering S-protein residues 479 and 487 to residues conserved during the 2002–2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS-CoV to human cells, and provide insight into the severity of the 2002–2003 SARS epidemic.

SARS-CoV-2 infection and ACE2 inhibition.

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...

SARS-CoV-2 mRNA vaccine induces robust specific and cross-reactive IgG and unequal neutralizing antibodies in naive and previously infected people

The outbreak of the coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 triggered a global pandemic where control is needed through therapeutic and preventive interventions. This study aims to identify natural compounds that could affect the fusion between the viral membrane (receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 spike protein) and the human cell receptor angiotensin-converting enzyme 2. Accordingly, we performed the enzyme-linked immunosorbent assay-based screening of 10 phytochemicals that already showed numerous positive effects on human health in several epidemiological studies and clinical trials. Among these phytochemicals, epigallocatechin gallate, a polyphenol and a major component of green tea, could effectively inhibit the interaction between the receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 spike protein and the human cell receptor angiotensin-converting enzyme 2. Alternately, in silico molecular docking studies of epigallocatechin gallate and angiotensin-converting enzyme 2 indicated a binding score of −7.8 kcal/mol and identified a hydrogen bond between R393 and angiotensin-converting enzyme 2, which is considered as a key interacting residue involved in binding with the severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain, suggesting the possible blocking of interaction between receptor-binding domain and angiotensin-converting enzyme 2. Furthermore, epigallocatechin gallate could attenuate severe acute respiratory syndrome coronavirus 2 infection and replication in Caco-2 cells. These results shed insight into identification and validation of severe acute respiratory syndrome coronavirus 2 entry inhibitors.

Tackling COVID19 by Exploiting Pre-existing Cross-Reacting Spike-Specific Immunity

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

Facing the challenge of viral mutations in the age of pandemic: Developing highly potent, broad-spectrum, and safe COVID-19 vaccines and therapeutics.

The rapidly spreading Omicron variant is highly resistant to vaccines, convalescent sera, and neutralizing antibodies (nAbs), highlighting the urgent need for potent therapeutic nAbs. Here, a panel of human nAbs from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent patients show diverse neutralization against Omicron, of which XMA01 and XMA04 maintain nanomolar affinities and excellent neutralization (half maximal inhibitory concentration [IC50]: ∼20 ng/mL). nAb XMA09 shows weak but unattenuated neutralization against all variants of concern (VOCs) as well as SARS-CoV. Structural analysis reveals that the above three antibodies could synergistically bind to the receptor-binding domains (RBDs) of both wild-type and Omicron spikes and defines the critical determinants for nAb-mediated broad neutralizations. Three nAbs confer synergistic neutralization against Omicron, resulting from the inter-antibody interaction between XMA04 and XMA01(or XMA09). Furthermore, the XMA01/XMA04 cocktail provides synergistic protection against Beta and Omicron variant infections in hamsters. In summary, our results provide insights for the rational design of antibody cocktail therapeutics or universal vaccines against Omicron.

SARS-CoV-2 Vaccines: The Mucosal Immunity Imperative

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of infections and deaths worldwide since its emergence in Wuhan, China, in late 2019. Natural product inhibitors targeting the interaction between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and human angiotensin-converting enzyme 2 (ACE2), crucial for viral attachment and cellular entry, are of significant interest as potential antiviral agents. In this study a library of nitrile- and sulfur-containing natural product derived compounds were used for virtual drug screening against the RBD of the SARS-CoV-2 spike protein. The top 18 compounds from docking were tested for their efficacy to inhibit virus entry. In vitro experiments revealed that compounds 9, 14, and 15 inhibited SARS-CoV-2 pseudovirus and live virus entry in HEK-ACE2 and Vero E6 host cells at low micromolar IC50 values. Cell viability assays showed these compounds exerted low cytotoxicity towards MRC5, Vero E6, and HEK-ACE2 cell lines. Microscale thermophoresis revealed all three compounds strongly bound to the RBDs of SARS-CoV-2, SARS-CoV-2 XBB, SARS-CoV-1, MERS-CoV, and HCoV-HKU1, with their Kd values increasing as RBD sequence similarity decreased. Molecular docking studies indicated compounds 9, 14, and 15 bound to the SARS-CoV-2 spike protein RBD and interacted with hotspot amino acid residues required for the RBD-ACE2 interaction and cellular infection. These three nitrile-containing candidates, particularly compound 15, should be considered for further development as potential pan-coronavirus entry inhibitors.

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).

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), similar to SARS-CoV and Middle East respiratory syndrome (MERS-CoV), which cause acute respiratory distress syndrome and case fatalities. COVID-19 disease severity is worse in older obese patients with comorbidities such as diabetes, hypertension, cardiovascular disease, and chronic lung disease. Cell binding and entry of betacoronaviruses is via their surface spike glycoprotein; SARS-CoV binds to the metalloprotease angiotensin-converting enzyme 2 (ACE2), MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4), and recent modeling of the structure of SARS-CoV-2 spike glycoprotein predicts that it can interact with human DPP4 in addition to ACE2. DPP4 is a ubiquitous membrane-bound aminopeptidase that circulates in plasma; it is multifunctional with roles in nutrition, metabolism, and immune and endocrine systems. DPP4 activity differentially regulates glucose homeostasis and inflammation via its enzymatic activity and nonenzymatic immunomodulatory effects. The importance of DPP4 for the medical community has been highlighted by the approval of DPP4 inhibitors, or gliptins, for the treatment of type 2 diabetes mellitus. This review discusses the dysregulation of DPP4 in COVID-19 comorbid conditions; DPP4 activity is higher in older individuals and increased plasma DPP4 is a predictor of the onset of metabolic syndrome. DPP4 upregulation may be a determinant of COVID-19 disease severity, which creates interest regarding the use of gliptins in management of COVID-19. Also, knowledge of the chemistry and biology of DPP4 could be utilized to develop novel therapies to block viral entry of some betacoronaviruses, potentially including SARS-CoV-2.