In Silico,In Vitro and In Cellulo Models for Monitoring SARS-CoV-2 Spike/Human ACE2 Complex,Viral Entry and Cell Fusion

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a computational model of the SARS-CoV-2 S/ACE2 complex, as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research, aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step.     

Target-Centered Drug Repurposing Predictions of Human Angiotensin-Converting Enzyme 2 (ACE2) and Transmembrane Protease Serine Subtype 2 (TMPRSS2) Interacting Approved Drugs for Coronavirus Disease 2019 (COVID-19) Treatment through a Drug-Target Interaction Deep Learning Model

Previously, our group predicted commercially available Food and Drug Administration (FDA) approved drugs that can inhibit each step of the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using a deep learning-based drug-target interaction model called Molecule Transformer-Drug Target Interaction (MT-DTI). Unfortunately, additional clinically significant treatment options since the approval of remdesivir are scarce. To overcome the current coronavirus disease 2019 (COVID-19) more efficiently, a treatment strategy that controls not only SARS-CoV-2 replication but also the host entry step should be considered. In this study, we used MT-DTI to predict FDA approved drugs that may have strong affinities for the angiotensin-converting enzyme 2 (ACE2) receptor and the transmembrane protease serine 2 (TMPRSS2) which are essential for viral entry to the host cell. Of the 460 drugs with K_d of less than 100 nM for the ACE2 receptor, 17 drugs overlapped with drugs that inhibit the interaction of ACE2 and SARS-CoV-2 spike reported in the NCATS OpenData portal. Among them, enalaprilat, an ACE inhibitor, showed a K_d value of 1.5 nM against the ACE2. Furthermore, three of the top 30 drugs with strong affinity prediction for the TMPRSS2 are anti-hepatitis C virus (HCV) drugs, including ombitasvir, daclatasvir, and paritaprevir. Notably, of the top 30 drugs, AT1R blocker eprosartan and neuropsychiatric drug lisuride showed similar gene expression profiles to potential TMPRSS2 inhibitors. Collectively, we suggest that drugs predicted to have strong inhibitory potencies to ACE2 and TMPRSS2 through the DTI model should be considered as potential drug repurposing candidates for COVID-19.     

COVID-19 and diabetes; Possible role of polymorphism and rise of telemedicine

BackgroundDiabetes has been found to be one of the leading comorbidities associated with fatality in COVID-19 patients. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is facilitated by interaction with Angiotensin Converting Enzyme-2 (ACE2) and possible polymorphisms in ACE2 can be a determining factor in host-viral protein interaction. A significant shift of healthcare towards ‘Telemedicine’ is also on the rise. In this review, the possible effects of ACE2 polymorphisms on SARS-CoV-2 entry along with the escalation of ‘telemedicine’ is discussed.MethodAn expansive literature search using keywords: “COVID-19”, “SARS-CoV-2”, “diabetes”, “type 2 diabetes’’, “type 1 diabetes”, “ACE2”, “polymorphism”, “DPP4” and “telemedicine” was conducted on Pubmed and EMBASE till 7th August 2020.ResultPossible polymorphisms in ACE2 gene can play a role in influencing the virus entry in host body. Telemedicine can bring a new revolution for medical sector.ConclusionCOVID-19 severity is more heinous among diabetic population. So far, the in-silico studies involving human ACE2-viral Spike (S) interaction showed inconsistent predictions regarding some SNPs. But without actual in-vivo studies, a holistic understanding can’t be established.     

ACE2-Independent Alternative Receptors for SARS-CoV-2

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is highly contagious and remains a major public health challenge despite the availability of effective vaccines. SARS-CoV-2 enters cells through the binding of its spike receptor-binding domain (RBD) to the human angiotensin-converting enzyme 2 (ACE2) receptor in concert with accessory receptors/molecules that facilitate viral attachment, internalization, and fusion. Although ACE2 plays a critical role in SARS-CoV-2 replication, its expression profiles are not completely associated with infection patterns, immune responses, and clinical manifestations. Additionally, SARS-CoV-2 infects cells that lack ACE2, and the infection is resistant to monoclonal antibodies against spike RBD in vitro, indicating that some human cells possess ACE2-independent alternative receptors, which can mediate SARS-CoV-2 entry. Here, we discuss these alternative receptors and their interactions with SARS-CoV-2 components for ACE2-independent viral entry. These receptors include CD147, AXL, CD209L/L-SIGN/CLEC4M, CD209/DC-SIGN/CLEC4L, CLEC4G/LSECtin, ASGR1/CLEC4H1, LDLRAD3, TMEM30A, and KREMEN1. Most of these receptors are known to be involved in the entry of other viruses and to modulate cellular functions and immune responses. The SARS-CoV-2 omicron variant exhibits altered cell tropism and an associated change in the cell entry pathway, indicating that emerging variants may use alternative receptors to escape the immune pressure against ACE2-dependent viral entry provided by vaccination against RBD. Understanding the role of ACE2-independent alternative receptors in SARS-CoV-2 viral entry and pathogenesis may provide avenues for the prevention of infection by SARS-CoV-2 variants and for the treatment of COVID-19.     

Human ACE2 Polymorphisms from Different Human Populations Modulate SARS-CoV-2 Infection

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 6 million deaths worldwide. The high variability in COVID-19 symptoms remains one of the most interesting mysteries of the pandemic. Genetic and environmental factors are likely to be key determinants of COVID-19 symptomatology. Here, we explored ACE2 as a genetic determinant for SARS-CoV-2 infection and COVID-19 symptomatology. Each human genome encodes two alleles of ACE2, which encodes the cell entry receptor for SARS-CoV-2. Here, we determined whether naturally occurring human ACE2 (hACE2) polymorphisms in the human population affect SARS-CoV-2 infection and the severity of COVID-19 symptoms. ACE2 variants S19P, I21V, E23K, K26R, K31R, N33I, H34R, E35K, and T92I showed increased virus infection compared to wild-type ACE2; thus, these variants could increase the risk for COVID-19. In contrast, variants D38V, Y83H, I468V, and N638S showed reduced infection, indicating a potential protective effect. hACE2 variants K26R and T92I increased infection by three-fold without changing the levels of ACE2 on the surface of the cells, suggesting that these variants may increase the risk of severe COVID-19. On the contrary, hACE2 variants D38V and Y83H decreased SARS-CoV-2 infection by four- and ten-fold, respectively, without changing surface expression, suggesting that these variants may protect against severe COVID-19. Remarkably, all protective hACE2 Polymorphisms were found almost exclusively in Asian populations, which may provide a partial explanation for the low COVID-19 mortality rates in Asian countries. Thus, hACE2 polymorphisms may modulate susceptibility to SARS-CoV-2 in the host and partially account for the differences in severity of COVID-19 among different ethnic groups.     

Cross-species recognition of SARS-CoV-2 to bat ACE2

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a major threat to global health. Although varied SARS-CoV-2–related coronaviruses have been isolated from bats and SARS-CoV-2 may infect bat, the structural basis for SARS-CoV-2 to utilize the human receptor counterpart bat angiotensin-converting enzyme 2 (bACE2) for virus infection remains less understood. Here, we report that the SARS-CoV-2 spike protein receptor binding domain (RBD) could bind to bACE2 from Rhinolophus macrotis (bACE2-Rm) with substantially lower affinity compared with that to the human ACE2 (hACE2), and its infectivity to host cells expressing bACE2-Rm was confirmed with pseudotyped SARS-CoV-2 virus and SARS-CoV-2 wild virus. The structure of the SARS-CoV-2 RBD with the bACE2-Rm complex was determined, revealing a binding mode similar to that of hACE2. The analysis of binding details between SARS-CoV-2 RBD and bACE2-Rm revealed that the interacting network involving Y41 and E42 of bACE2-Rm showed substantial differences with that to hACE2. Bats have extensive species diversity and the residues for RBD binding in bACE2 receptor varied substantially among different bat species. Notably, the Y41H mutant, which exists in many bats, attenuates the binding capacity of bACE2-Rm, indicating the central roles of Y41 in the interaction network. These findings would benefit our understanding of the potential infection of SARS-CoV-2 in varied species of bats.

The coronavirus disease 2019 (COVID-19), caused by infection with the novel coronavirus (CoV) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a major threat to global health with an increasing number of infected cases globally, and the end to this pandemic is still full of uncertainties (1–3). Seven CoVs have been reported to infect humans: SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), NL-63, OC43, 229E, HKU1, and the newly emerging SARS-CoV-2. Among the SARS-CoV-2 proteins, the spike (S) protein, which consists of an N-terminal S1 subunit and a C-terminal S2 subunit, is critical for the recognition of host cell receptors and serves as the key determinant of host specificity for CoVs (4). The C-terminal domain of the S1 subunit, also known as the receptor binding domain (RBD), binds to the human angiotensin-converting enzyme 2 (hACE2), the receptor for SARS-CoV and the human coronavirus NL-63 (5–9). Monoclonal antibodies (mAbs) that block the binding between SARS-CoV-2 RBD and ACE2 could efficiently inhibit virus infection in host cells expressing ACE2 (10, 11). Animal studies revealed that a single dose of these neutralizing mAbs showed promising therapeutic efficacy in reducing both viral load and pathological lung damage in hACE2 transgenic mice or rhesus macaques (10, 11). Recently, the structures of the complex between SARS-CoV-2 S protein (or RBD) with hACE2 have been determined, showing similar binding mode to that of SARS-CoV but with enhanced affinity (5–7).Bats are considered as the reservoir host animals of SARS-CoV-2, and several SARS-CoV-2-related CoVs have been identified from bats (12, 13). The binding assay showed that bat ACE2 (bACE2) from Rhinolophus macrotis (bACE2-Rm) bound to SARS-CoV-2 RBD efficiently (14). The entry capacity of SARS-CoV-2 through ACE2 orthologs from 46 bat species was evaluated by virus-host receptor binding and infection assays. The results indicated that although some bACE2 receptors could mediate SARS-CoV-2 entry, there are many bACE2 receptors that do not yet support SARS-CoV-2 entry (15). To date, the genome most closely related to SARS-CoV-2 is RaTG13, which was identified from a Rhinolophus affinis sampled from Yunnan Province in 2013 and had a 92.9% amino acid identity in the S gene (13). RmYN02 was also a coronavirus identified from bat with 93.3% nucleotide identity with SARS-CoV-2 at the scale of the complete virus genome (12). Yongyi Shen and colleagues (16) reported a coronavirus isolated from Malayan pangolin and shared 90.7% amino acid identity with SARS-CoV-2 in the S proteins. In addition, a coronavirus that showed 97.4% amino acid identity with SARS-CoV-2 in the RBD region was identified from another batch of Malayan pangolin (17). Bioinformatics analysis indicated that bats are the primary reservoir for the SARS-CoV-2 lineage (18). However, the origin of SARS-CoV-2 is full of uncertainties and whether or not there is an intermediate host is still unknown. Moreover, more than 1,400 species of bats have been identified and extensive species diversity among different species of bats has resulted in varied susceptibility for CoVs. Research into the mechanisms of viral entry and virus–host interaction would not only benefit our understanding of this virus but is also important for the design of antivirals and vaccines. However, the structure of bACE2 has not been determined, and the molecular basis of the binding between SARS-CoV-2 S protein and bACE2 has not been well studied.Here, we report that SARS-CoV-2 RBD can bind to bACE2-Rm with substantially lower affinity than that to hACE2, and infection of host cells carrying bACE2-Rm was also investigated with pseudotyped or wild SARS-CoV-2. Interaction mechanisms between SARS-CoV-2 RBD and bACE2-Rm were elucidated by determining the structure of the SARS-CoV-2 RBD and bACE2-Rm complex. The results of this study would broaden our understanding of the receptor binding mechanisms for SARS-CoV-2.     

Predictive Models of within- and between-Species SARS-CoV-2 Transmissibility

Viruses from the Coronaviridae family have been reported to infect a large range of hosts, including humans. The latest human-infecting coronavirus, SARS-CoV-2, turned into a pandemic and subtypes with different transmissibility have appeared since then. The SARS-CoV-2 Spike (S) protein interacts with the angiotensin-converting enzyme 2 (ACE2) host receptor, and thus, in silico models, based on the structural features of the SARS-CoV-2 S protein–ACE2 receptor complex, as well as ACE2 amino acid patterns, may be used to predict the within- and between-species transmissibility of SARS-CoV-2 subtypes. Here, it is shown that at the beginning of the pandemic, the SARS-CoV-2 S protein was, as expected for a virus that just jumped the species barrier, ill-adapted to the human ACE2 receptor, and that the replacement of one SARS-CoV-2 variant by another is partially due to a better fitting of the S protein–human ACE2 complex. Moreover, it is shown that mutations that are predicted to lead to a better fit have increased in the population due to positive selection. It is also shown that the number of ACE2-interfacing residues is positively correlated with the transmissibility rate of SARS-CoV-2 variants. Finally, it is shown that the number of species that are susceptible to infection by SARS-CoV-2, and that could be a reservoir for this virus, is likely higher than previously thought.     

Extracellular vimentin is an attachment factor that facilitates SARS-CoV-2 entry into human endothelial cells

SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography–tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) (1). SARS-CoV-2 binds to host receptors and attachment factors through its spike (S) glycoprotein and mediates membrane fusion and viral entry (2, 3). Although infection of cells along the respiratory tract was almost immediately defined as an important hallmark of the disease, the SARS-CoV-2 virus has also been detected not only in the lungs, but also in the cardiovascular system, brain, liver, kidneys, and intestine (4–7). Angiotensin-converting enzyme 2 (ACE2) is recognized as an important receptor for SARS-CoV-2 (1, 2). However, recent single-cell sequencing studies have demonstrated that ACE2 expression is relatively high in upper respiratory cells but low in the lower respiratory tract (8–12), and this result coupled with the identification of multiple other factors—including AXL receptor tyrosine kinase (11), Neuropilin-1 (13), CD209L/L-SIGN, CD209/DC-SIGN (14), and heparin sulfate (15)—as additional potential receptors or coreceptors for SARS-CoV-2, suggest that multiple receptors/coreceptors may facilitate SARS-CoV-2 entry in a cell type-dependent manner. Therefore, variations in the mechanisms of SARS-CoV-2 entry could account for its robust tropism, transmission, and pathogenesis.To search for unidentified receptors involved in SARS-CoV-2 entry into endothelial cells, we used SARS-CoV-2 S-receptor binding domain (S-RBD) as bait and whole-cell lysate (WCL) of human umbilical vein endothelial cells (HUVEC-TERT) as a source for prey proteins followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of proteins that showed an affinity for the SARS-CoV-2 S-RBD. Our analysis identified vimentin (VIM) as a SARS-CoV-2 binding protein. Further biochemical and cell culture studies using pseudotyped viruses expressing SARS-CoV-2 S-protein or infectious SARS-CoV-2 demonstrated that VIM interacts with both SARS-CoV-2 S and ACE2 and acts as a coreceptor for SARS-CoV-2.VIM is a type III intermediate filament protein and is widely expressed in cells of mesenchymal origin, such as endothelial cells, fibroblasts, and monocytes (16). In addition to its key role in intermediate filament formation, VIM is also present at the extracellular surface of endothelial cells and macrophages (17–19). Previous studies have found that extracellular VIM functions as an attachment factor or coreceptor for various viruses, including, SARS-CoV-1 (20), cowpea mosaic virus (21), Japanese encephalitis virus (22), dengue virus (23), and human papillomavirus (24). VIM could play an important role in promoting SARS-CoV-2 entry into the human vascular system, as well as other organs and tissues via cis- and trans-infection mechanisms.     

Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.     

Nonmuscle myosin heavy chain IIA facilitates SARS-CoV-2 infection in human pulmonary cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), binds to host receptor angiotensin-converting enzyme 2 (ACE2) through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. However, the expression of ACE2 is extremely low in a variety of human tissues, especially in the airways. Thus, other coreceptors and/or cofactors on the surface of host cells may contribute to SARS-CoV-2 infection. Here, we identified nonmuscle myosin heavy chain IIA (MYH9) as an important host factor for SARS-CoV-2 infection of human pulmonary cells by using APEX2 proximity-labeling techniques. Genetic ablation of MYH9 significantly reduced SARS-CoV-2 pseudovirus infection in wild type (WT) A549 and Calu-3 cells, and overexpression of MYH9 enhanced the pseudovirus infection in WT A549 and H1299 cells. MYH9 was colocalized with the SARS-CoV-2 S and directly interacted with SARS-CoV-2 S through the S2 subunit and S1-NTD (N-terminal domain) by its C-terminal domain (designated as PRA). Further experiments suggested that endosomal or myosin inhibitors effectively block the viral entry of SARS-CoV-2 into PRA-A549 cells, while transmembrane protease serine 2 (TMPRSS2) and cathepsin B and L (CatB/L) inhibitors do not, indicating that MYH9 promotes SARS-CoV-2 endocytosis and bypasses TMPRSS2 and CatB/L pathway. Finally, we demonstrated that loss of MYH9 reduces authentic SARS-CoV-2 infection in Calu-3, ACE2-A549, and ACE2-H1299 cells. Together, our results suggest that MYH9 is a candidate host factor for SARS-CoV-2, which mediates the virus entering host cells by endocytosis in an ACE2-dependent manner, and may serve as a potential target for future clinical intervention strategies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a pandemic coronavirus disease (COVID-19) (1–3), resulting in more than 209 million infections worldwide since December 2019. SARS-CoV-2 is the seventh coronavirus (CoV) infecting humans beyond endemic human CoVs (HCoV-HKU1, -NL63, -OC43, and -229E), SARS-CoV, and Middle East respiratory syndrome CoV (MERS-CoV). SARS-CoV-2 preferentially infects the airway epithelial cells (1, 4), although viral infection has been detected in a variety of human organs, including the lungs, pharynx, heart, liver, brain, kidneys, and digestive system organs (5–7), causing upper respiratory diseases, fever, and severe pneumonia in humans.CoVs were not considered to be highly pathogenic to humans until the outbreak of SARS-CoV started in 2002 in Guangdong province, China (8–10). The primary determinant of CoV tropism is their spike (S) glycoproteins, which mediate the viral infection by binding to membrane receptors on the host cells and could be cleaved to the N-terminal S1 and C-terminal S2 subunit by the host proteases such as transmembrane protease serine 2 (TMPRSS2) and Furin (11). Studies have shown that ACE2, a cellular receptor for SARS-CoV, also binds SARS-CoV-2 S and serves as the entry point for SARS-CoV-2 (3, 12). However, ACE2 cannot fully explain the tissue tropism of SARS-CoV-2 because of the virus detection in tissues with little ACE2 expression, such as the liver, brain, and blood, and even in the lung, only a small subset of cells expresses ACE2 (SI Appendix, Fig. 1B) (13–16). Recently, neuropilin-1 (NRP1) (17, 18) and heparan sulfate (19, 20) have been identified as cofactors implicated in enhancing ACE2-dependent SARS-CoV-2 infection, while tyrosine-protein kinase receptor (AXL) (21) and CD147 (22) were identified as receptors involved in SARS-CoV-2 cell entry independently of ACE2. These highlight the multiple routes of SARS-CoV-2 infection, likely contributing to high infectivity and spread of COVID-19. Hence, SARS-CoV-2 is assumed to be dependent on other potential receptors or coreceptors to facilitate its infection in humans.Here, we applied engineered ascorbate peroxidase APEX2-based subcellular proteomics (23–25) to capture spatiotemporal membrane protein complexes interacting with SARS-CoV-2 S in living cells. We found that the nonmuscle myosin heavy chain IIA (MYH9) specifically interacts with SARS-CoV-2 S protein. The C-terminal domain (designated as PRA) of MYH9 interacts with the S2 subunit and the N-terminal domain (NTD) of the S1 subunit, and PRA overexpression facilitates a pan-CoV entry into host cells. In addition, depletion of MYH9 significantly reduced authentic SARS-CoV-2 infection in human lung cell lines. Mechanistically, a myosin inhibitor but not TMPRSS2 and CatB/L inhibitors blocked SARS-CoV-2 infection in PRA-A549 cells, and the presence of ACE2 is required for MYH9 mediated SARS-CoV-2 entry. Collectively, our results suggest that MYH9 is a coreceptor of ACE2 for SARS-CoV-2 infection in human pulmonary cells.     

Functional and genetic analysis of viral receptor ACE2 orthologs reveals a broad potential host range of SARS-CoV-2

The pandemic of COVID-19, caused by SARS-CoV-2, is a major global health threat. Epidemiological studies suggest that bats (Rhinolophus affinis) are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. By analyzing the conservation of five residues in two virus-binding hotspots of ACE2 (hotspot 31Lys and hotspot 353Lys), we predicted 80 ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 ACE2 orthologs among them for functional analysis, and showed that 44 of these orthologs—including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria—could bind the SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind the SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.

Coronaviruses are a group of positive-stranded, enveloped RNA viruses that circulate broadly among humans, other mammals, and birds, causing respiratory, enteric, or hepatic diseases (1). In the last two decades, coronaviruses have caused three major outbreaks: severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the recent coronavirus disease 2019 (COVID-19) (2, 3). As of December 7, 2020, COVID-19 has already caused 50 million infections, leading to 1 million deaths globally. The pathogen responsible is a novel coronavirus-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (4, 5). Phylogenetic and epidemiological analyses suggest that SARS-CoV, MERS-CoV, and SARS-CoV-2 likely originated from bats, with SARS-CoV spreading from bats to palm civets to humans, and MERS-CoV spreading from bats to camel to humans (6). However, the intermediate host of SARS-CoV-2, fueling spillover to humans, remains unknown.The SARS-CoV-2 genome encodes a spike (S) protein, the receptor-binding domain (RBD) of which binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) to mediate viral entry (5, 7). Following binding of ACE2, the S protein is subsequently cleaved by the host transmembrane serine protease 2 (TMPRSS2) to release the spike fusion peptide, promoting virus entry into target cells (7). It has been demonstrated that the interaction of a virus with species-specific receptors is a primary determinant of host tropism and therefore constitutes a major interspecies barrier at the level of viral entry (8). For example, murine ACE2 does not efficiently bind the SARS-CoV or SARS-CoV-2 S protein, hindering viral entry into murine cells; consequently, a human ACE2 transgenic mouse was developed as an in vivo model to study the infection and pathogenesis of these two viruses (9, 10).ACE2 is expressed in a diverse range of species throughout the subphylum Vertebrata. Several recent studies demonstrated that ferrets, cats, dogs, and some nonhuman primates are susceptible to SARS-CoV-2 (11–15). However, the exact host tropism of SARS-CoV-2 remains unknown and it is urgent to identify the putative zoonotic reservoirs to prevent future outbreaks. Numerous studies have predicted ACE2 orthologs/SARS-CoV-2 S binding affinity or energies but lack of support by virus infection experimentation (16–21). In this study, we experimentally assessed ACE2 orthologs from a broad range of species for their ability to support SARS-CoV-2 entry. Our data demonstrate that an evolutionarily diverse set of ACE2 species variants can mediate SARS-CoV-2 entry, suggesting that SARS-CoV-2 has a broad host range at the level of virus entry that may contribute to cross-species transmission and viral evolution.     

SARS-CoV-2 Infection Modulates ACE2 Function and Subsequent Inflammatory Responses in Swabs and Plasma of COVID-19 Patients

Angiotensin converting enzyme 2 (ACE2) is a host ectopeptidase and the receptor for the SARS-CoV-2 virus, albeit virus-ACE2 interaction goes far beyond viral entry into target cells. Controversial data exists linking viral infection to changes in ACE2 expression and function, which might influence the subsequent induction of an inflammatory response. Here, we tested the significance of soluble ACE2 enzymatic activity longitudinally in nasopharyngeal swabs and plasma samples of SARS-CoV-2 infected patients, along with the induction of inflammatory cytokines. Release of soluble functional ACE2 increases upon SARS-CoV-2 infection in swabs and plasma of infected patients, albeit rapidly decreasing during infection course in parallel with ACE2 gene expression. Similarly, SARS-CoV-2 infection also induced the expression of inflammatory cytokines. These changes positively correlated with the viral load. Overall, our results demonstrate the existence of mechanisms by which SARS-CoV-2 modulates ACE2 expression and function, intracellular viral sensing and subsequent inflammatory response, offering new insights into ACE2 dynamics in the human upper respiratory tract and pointing towards soluble ACE2 levels as a putative early biomarker of infection severity.     

Killing Two Birds with One Stone by Administration of Soluble ACE2: A Promising Strategy to Treat Both Cardiovascular Diseases and SARS-CoV-2 Infection

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells mainly by the angiotensin converting enzyme 2 (ACE2) receptor, which can recognize the spike (S) protein by its extracellular domain. Previously, recombinant soluble ACE2 (sACE2) has been clinically used as a therapeutic treatment for cardiovascular diseases. Recent data demonstrated that sACE2 can also be exploited as a decoy to effectively inhibit the cell entry of SARS-CoV-2, through blocking SARS-CoV-2 binding to membrane-anchored ACE2. In this study, we summarized the current findings on the optimized sACE2-based strategies as a therapeutic agent, including Fc fusion to prolong the half-life of sACE2, deep mutagenesis to create high-affinity decoys for SARS-CoV-2, or designing the truncated functional fragments to enhance its safety, among others. Considering that COVID-19 patients are often accompanied by manifestations of cardiovascular complications, we think that administration of sACE2 in COVID-19 patients may be a promising therapeutic strategy to simultaneously treat both cardiovascular diseases and SARS-CoV-2 infection. This review would provide insights for the development of novel therapeutic agents against the COVID-19 pandemic.     

Investigations on SARS-CoV-2 Susceptibility of Domestic and Wild Animals Using Primary Cell Culture Models Derived from the Upper and Lower Respiratory Tract

Several animal species are susceptible to SARS-CoV-2 infection, as documented by case reports and serological and in vivo infection studies. However, the susceptibility of many animal species remains unknown. Furthermore, the expression patterns of SARS-CoV-2 entry factors, such as the receptor angiotensin-converting enzyme 2 (ACE2), as well as transmembrane protease serine subtype 2 (TMPRSS2) and cathepsin L (CTSL), cellular proteases involved in SARS-CoV-2 spike protein activation, are largely unexplored in most species. Here, we generated primary cell cultures from the respiratory tract of domestic and wildlife animals to assess their susceptibility to SARS-CoV-2 infection. Additionally, the presence of ACE2, TMPRSS2 and CTSL within respiratory tract compartments was investigated in a range of animals, some with unknown susceptibility to SARS-CoV-2. Productive viral replication was observed in the nasal mucosa explants and precision-cut lung slices from dogs and hamsters, whereas culture models from ferrets and multiple ungulate species were non-permissive to infection. Overall, whereas TMPRSS2 and CTSL were equally expressed in the respiratory tract, the expression levels of ACE2 were more variable, suggesting that a restricted availability of ACE2 may contribute to reduced susceptibility. Summarized, the experimental infection of primary respiratory tract cell cultures, as well as an analysis of entry-factor distribution, enable screening for SARS-CoV-2 animal reservoirs.     

The Spike Glycoprotein of SARS-CoV-2 Binds to β1 Integrins Expressed on the Surface of Lung Epithelial Cells

The spike glycoprotein attached to the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to and exploits angiotensin-converting enzyme 2 (ACE2) as an entry receptor to infect pulmonary epithelial cells. A subset of integrins that recognize the arginyl–glycyl–aspartic acid (RGD) sequence in the cognate ligands has been predicted in silico to bind the spike glycoprotein and, thereby, to be exploited for viral infection. Here, we show experimental evidence that the β1 integrins predominantly expressed on human pulmonary epithelial cell lines and primary mouse alveolar epithelial cells bind to this spike protein. The cellular β1 integrins support adhesive interactions with the spike protein independently of ACE2, suggesting the possibility that the β1 integrins may function as an alternative receptor for SARS-CoV-2, which could be targeted for the prevention of viral infections.     

The pivotal link between ACE2 deficiency and SARS-CoV-2 infection

Angiotensin converting enzyme-2 (ACE2) receptors mediate the entry into the cell of three strains of coronavirus: SARS-CoV, NL63 and SARS-CoV-2. ACE2 receptors are ubiquitous and widely expressed in the heart, vessels, gut, lung (particularly in type 2 pneumocytes and macrophages), kidney, testis and brain. ACE2 is mostly bound to cell membranes and only scarcely present in the circulation in a soluble form. An important salutary function of membrane-bound and soluble ACE2 is the degradation of angiotensin II to angiotensin_1-7. Consequently, ACE2 receptors limit several detrimental effects resulting from binding of angiotensin II to AT1 receptors, which include vasoconstriction, enhanced inflammation and thrombosis. The increased generation of angiotensin_1-7 also triggers counter-regulatory protective effects through binding to G-protein coupled Mas receptors. Unfortunately, the entry of SARS-CoV2 into the cells through membrane fusion markedly down-regulates ACE2 receptors, with loss of the catalytic effect of these receptors at the external site of the membrane. Increased pulmonary inflammation and coagulation have been reported as unwanted effects of enhanced and unopposed angiotensin II effects via the ACE→Angiotensin II→AT1 receptor axis. Clinical reports of patients infected with SARS-CoV-2 show that several features associated with infection and severity of the disease (i.e., older age, hypertension, diabetes, cardiovascular disease) share a variable degree of ACE2 deficiency. We suggest that ACE2 down-regulation induced by viral invasion may be especially detrimental in people with baseline ACE2 deficiency associated with the above conditions. The additional ACE2 deficiency after viral invasion might amplify the dysregulation between the ‘adverse’ ACE→Angiotensin II→AT1 receptor axis and the ‘protective’ ACE2→Angiotensin_1-7→Mas receptor axis. In the lungs, such dysregulation would favor the progression of inflammatory and thrombotic processes triggered by local angiotensin II hyperactivity unopposed by angiotensin_1-7. In this setting, recombinant ACE2, angiotensin_1-7 and angiotensin II type 1 receptor blockers could be promising therapeutic approaches in patients with SARS-CoV-2 infection.     

Angiotensin converting enzyme-2 as therapeutic target in COVID-19

The pandemic of coronavirus disease 2019 (COVID-19) is a global health emergency that poses a significant threat to world people’s health. This outbreak causes major challenges to healthcare systems. Given the lack of effective treatments or vaccine for it, the identification of novel and safe drugs against COVID-19 infection is an urgent need. Angiotensin-converting enzyme 2 (ACE2) is not only an entry receptor of the SARS-CoV-2 virus, the virus that causes COVID-19, but also can protect from lung injury. In this view, we highlighted potential approaches to address ACE2-mediated SARS-CoV-2 virus, including 1) delivering an excessive soluble form of ACE2 (recombinant human ACE2: rhACE2) and 2) inhibition of the interaction between SARS-CoV-2 virus and ACE2 by some compounds with competitive effects (morphine and codeine). Further clinical trials in this regard can reveal a more definite conclusion against the COVID-19 disaster.     

Omicron mutations enhance infectivity and reduce antibody neutralization of SARS-CoV-2 virus-like particles

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant contains extensive sequence changes relative to the earlier-arising B.1, B.1.1, and Delta SARS-CoV-2 variants that have unknown effects on viral infectivity and response to existing vaccines. Using SARS-CoV-2 virus-like particles (VLPs), we examined mutations in all four structural proteins and found that Omicron and Delta showed 4.6-fold higher luciferase delivery overall relative to the ancestral B.1 lineage, a property conferred mostly by enhancements in the S and N proteins, while mutations in M and E were mostly detrimental to assembly. Thirty-eight antisera samples from individuals vaccinated with Pfizer/BioNTech, Moderna, or Johnson & Johnson vaccines and convalescent sera from unvaccinated COVID-19 survivors had 15-fold lower efficacy to prevent cell transduction by VLPs containing the Omicron mutations relative to the ancestral B.1 spike protein. A third dose of Pfizer vaccine elicited substantially higher neutralization titers against Omicron, resulting in detectable neutralizing antibodies in eight out of eight subjects compared to one out of eight preboosting. Furthermore, the monoclonal antibody therapeutics casirivimab and imdevimab had robust neutralization activity against B.1 and Delta VLPs but no detectable neutralization of Omicron VLPs, while newly authorized bebtelovimab maintained robust neutralization across variants. Our results suggest that Omicron has similar assembly efficiency and cell entry compared to Delta and that its rapid spread is due mostly to reduced neutralization in sera from previously vaccinated subjects. In addition, most currently available monoclonal antibodies will not be useful in treating Omicron-infected patients with the exception of bebtelovimab.

Understanding the molecular determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral fitness is central to effective vaccine and therapeutic development. The emergence of viral variants including Delta and Omicron underscores the need to assess both infectivity and antibody neutralization, but biosafety level 3 handling requirements slow the pace of research on intact SARS-CoV-2. Although vesicular stomatitis virus and lentivirus pseudotyped with the SARS-CoV-2 spike (S) protein enable evaluation of S-mediated cell binding and entry via the ACE2 and TMPRSS2 receptors, they cannot determine effects of mutations outside the S gene (1, 2). To address these challenges, we developed SARS-CoV-2 virus-like particles (SC2-VLPs) comprising the S, N, M, and E structural proteins and a packaging signal-containing messenger RNA (mRNA) that together form RNA-loaded capsids capable of spike-dependent cell transduction (3). This system faithfully reflects the impact of mutations in structural proteins that are observed in infections with virus isolates, enabling rapid testing of SARS-CoV-2 structural gene variants for their impact on both infection efficiency and antibody or antiserum neutralization.