ACE2 and COVID-19 and the resulting ARDS.

This article reviews the correlation between ACE2 and COVID-19 and the resulting acute respiratory distress syndrome (ARDS). ACE2 is a crucial component of the renin-angiotensin system (RAS). The classical ACE-angiotensin Ⅱ (Ang II)-angiotensin type 1 receptor (AT1R) axis and the ACE2-Ang(1-7)-Mas counter-regulatory axis play an essential role in RAS system. ACE2 antagonises the activation of the classical RAS ACE-Ang II-AT1R axis and protects against lung injury. Similar to severe acute respiratory syndrome-related coronavirus, 2019 novel coronavirus (2019-nCoV) also uses ACE2 for cell entry. ARDS is a clinical high-mortality disease which is probably due to the excessive activation of RAS caused by 2019-nCoV infection, and ACE2 has a protective effect on ARDS caused by COVID-19. Because of these protective effects of ACE2 on ARDS, the development of drugs enhancing ACE2 activity may become one of the most promising approaches for the treatment of COVID-19 in the near future. In the meantime, however, the use of RAS blockers such as ACE inhibitors and angiotensin II receptor blockers that inhibit the damaging (ACE-Ang II) arm of the RAS cascade in the lung may also be promising. Trial registration number: NCT04287686.

Highly conserved binding region of ACE2 as a receptor for SARS-CoV-2 between humans and mammals.

Several cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection transmitted from human owners to their dogs have recently been reported. The first ever case of SARS-CoV-2 transmission from a human owner to a domestic cat was confirmed on March 27, 2020. A tiger from a zoo in New York, USA, was also reportedly infected with SARS-CoV-2. It is believed that SARS-CoV-2 was transmitted to tigers from their caretakers, who were previously infected with this virus. On May 25, 2020, the Dutch Minister of Agriculture, Nature and Food Quality reported that two employees were infected with SARS-CoV-2 transmitted from minks. These reports have influenced us to perform a comparative analysis among angiotensin-converting enzyme 2 (ACE2) homologous proteins for verifying the conservation of specific protein regions. One of the most conserved peptides is represented by the peptide "353-KGDFR-357 (H. sapiens ACE2 residue numbering), which is located on the surface of the ACE2 molecule and participates in the binding of SARS-CoV-2 spike receptor binding domain (RBD). Multiple sequence alignments of the ACE2 proteins by ClustalW, whereas the three-dimensional structure of its binding region for the spike glycoprotein of SARS-CoV-2 was assessed by means of Spanner, a structural homology modeling pipeline method. In addition, evolutionary phylogenetic tree analysis by ETE3 was used. ACE2 works as a receptor for the SARS-CoV-2 spike glycoprotein between humans, dogs, cats, tigers, minks, and other animals, except for snakes. The three-dimensional structure of the KGDFR hosting protein region involved in direct interactions with SARS-CoV-2 spike RBD of the mink ACE2 appears to form a loop structurally related to the human ACE2 corresponding protein loop, despite of the reduced available protein length (401 residues of the mink ACE2 available sequence vs 805 residues of the human ACE2). The multiple sequence alignments of the ACE2 proteins shows high homology and complete conservation of the five amino acid residues: 353-KGDFR-357 with humans, dogs, cats, tigers, minks, and other animals, except for snakes. Where the information revealed from our examinations can support precision vaccine design and the discovery of antiviral therapeutics, which will accelerate the development of medical countermeasures, the World Health Organization recently reported on the possible risks of reciprocal infections regarding SARS-CoV-2 transmission from animals to humans.

hACE2-Induced Allosteric Activation in SARS-CoV versus SARS-CoV-2 Spike Assemblies Revealed by Structural Dynamics.

SARS-CoV and SARS-CoV-2 cell entry begins when spike glycoprotein (S) docks with the human ACE2 (hACE2) receptor. While the two coronaviruses share a common receptor and architecture of S, they exhibit differences in interactions with hACE2 as well as differences in proteolytic processing of S that trigger the fusion machine. Understanding how those differences impact S activation is key to understand its function and viral pathogenesis. Here, we investigate hACE2-induced activation in SARS-CoV and SARS-CoV-2 S using hydrogen/deuterium-exchange mass spectrometry (HDX-MS). HDX-MS revealed differences in dynamics in unbound S, including open/closed conformational switching and D614G-induced S stability. Upon hACE2 binding, notable differences in transduction of allosteric changes were observed extending from the receptor binding domain to regions proximal to proteolytic cleavage sites and the fusion peptide. Furthermore, we report that dimeric hACE2, the native oligomeric form of the receptor, does not lead to any more pronounced structural effect in S compared to saturated monomeric hACE2 binding. These experiments provide mechanistic insights into receptor-induced activation of Sarbecovirus spike proteins.

Novel Therapeutic Target Critical for SARS-CoV-2 Infectivity and Induction of the Cytokine Release Syndrome.

We discovered a novel therapeutic target critical for SARS-CoV-2, cellular infectivity and the induction of the cytokine release syndrome. Here, we show that the mammalian enzyme neuraminidase-1 (Neu-1) is part of a highly conserved signaling platform that regulates the dimerization and activation of the ACE2 receptors and the Toll-like receptors (TLRs) implicated in the cytokine release syndrome (CRS). Activated Neu-1 cleaves glycosylated residues that provide a steric hindrance to both ACE2 and TLR dimerization, a process critical to both viral attachment to the receptor and entry into the cell and TLR activation. Blocking Neu-1 inhibited ACE2 receptor dimerization and internalization, TLR dimerization and activation, and the expression of several key inflammatory molecules implicated in the CRS and death from ARDS. Treatments that target Neu-1 are predicted to be highly effective against infection with SARS-CoV-2, given the central role played by this enzyme in viral cellular entry and the induction of the CRS.

Discrimination of the H1N1 and H5N2 Variants of Influenza A Virus Using an Isomeric Sialic Acid-Conjugated Graphene Field-Effect Transistor.

There has been a continuous effort to fabricate a fast, sensitive, and inexpensive system for influenza virus detection to meet the demand for effective screening in point-of-care testing. Herein, we report a sialic acid (SA)-conjugated graphene field-effect transistor (SA-GFET) sensor designed using α2,3-linked sialic acid (3'-SA) and α2,6-linked sialic acid (6'-SA) for the detection and discrimination of the hemagglutinin (HA) protein of the H5N2 and H1N1 viruses. 3'-SA and 6'-SA specific for H5 and H1 influenza were used in the SA-GFET to capture the HA protein of the influenza virus. The net charge of the captured viral sample led to a change in the electrical current of the SA-GFET platform, which could be correlated to the concentration of the viral sample. This SA-GFET platform exhibited a highly sensitive response in the range of 101-106 pfu mL-1, with a limit of detection (LOD) of 101 pfu mL-1 in buffer solution and a response time of approximately 10 s. The selectivity of the SA-GFET platform for the H1N1 and H5N2 influenza viruses was verified by testing analogous respiratory viruses, i.e., influenza B and the spike protein of SARS-CoV-2 and MERS-CoV, on the SA-GFET. Overall, the results demonstrate that the developed dual-channel SA-GFET platform can potentially serve as a highly efficient and sensitive sensing platform for the rapid detection of infectious diseases.

Mechanism of an RBM-targeted rabbit monoclonal antibody 9H1 neutralizing SARS-CoV-2.

The COVID-19 pandemic, caused by SARS-CoV-2, has led to over 750 million infections and 6.8 million deaths worldwide since late 2019. Due to the continuous evolution of SARS-CoV-2, many significant variants have emerged, creating ongoing challenges to the prevention and treatment of the pandemic. Therefore, the study of antibody responses against SARS-CoV-2 is essential for the development of vaccines and therapeutics. Here we perform single particle cryo-electron microscopy (cryo-EM) structure determination of a rabbit monoclonal antibody (RmAb) 9H1 in complex with the SARS-CoV-2 wild-type (WT) spike trimer. Our structural analysis shows that 9H1 interacts with the receptor-binding motif (RBM) region of the receptor-binding domain (RBD) on the spike protein and by directly competing with angiotensin-converting enzyme 2 (ACE2), it blocks the binding of the virus to the receptor and achieves neutralization. Our findings suggest that utilizing rabbit-derived mAbs provides valuable insights into the molecular interactions between neutralizing antibodies and spike proteins and may also facilitate the development of therapeutic antibodies and expand the antibody library.

The Alpha-1 Subunit of the Na+/K+-ATPase (ATP1A1) Is a Host Factor Involved in the Attachment of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is an acute and severe atrophic enteritis caused by porcine epidemic diarrhea virus (PEDV) that infects pigs and makes huge economic losses to the global swine industry. Previously, researchers have believed that porcine aminopeptidase-N (pAPN) was the primary receptor for PEDV, but it has been found that PEDV can infect pAPN knockout pigs. Currently, the functional receptor for PEDV remains unspecified. In the present study, we performed virus overlay protein binding assay (VOPBA), found that ATP1A1 was the highest scoring protein in the mass spectrometry results, and confirmed that the CT structural domain of ATP1A1 interacts with PEDV S1. First, we investigated the effect of ATP1A1 on PEDV replication. Inhibition of hosts ATP1A1 protein expression using small interfering RNA (siRNAs) significantly reduced the cells susceptibility to PEDV. The ATP1A1-specific inhibitors Ouabain (a cardiac steroid) and PST2238 (a digitalis toxin derivative), which specifically bind ATP1A1, could block the ATP1A1 protein internalization and degradation, and consequently reduce the infection rate of host cells by PEDV significantly. Additionally, as expected, overexpression of ATP1A1 notably enhanced PEDV infection. Next, we observed that PEDV infection of target cells resulted in upregulation of ATP1A1 at the mRNA and protein levels. Furthermore, we found that the host protein ATP1A1 was involved in PEDV attachment and co-localized with PEDV S1 protein in the early stage of infection. In addition, pretreatment of IPEC-J2 and Vero-E6 cells with ATP1A1 mAb significantly reduced PEDV attachment. Our observations provided a perspective on identifying key factors in PEDV infection, and may provide valuable targets for PEDV infection, PEDV functional receptor, related pathogenesis, and the development of new antiviral drugs.

Biomimetic SARS-CoV-2 Spike Protein Nanoparticles.

COVID-19 is an infectious respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus contains a crucial coat protein that engages with target cells via a receptor binding domain (RBD) on its spike protein. To better study the RBD and its therapeutic opportunities, we genetically engineered a simple fusion with a thermo-responsive elastin-like polypeptide (ELP). These fusions express in Escherichia coli at a high yield in the soluble fraction and were easily purified using ELP-mediated phase separation (79 mg/L culture). Interestingly, they assembled peptide-based nanoparticles (Rh = 71.4 nm), which was attributed to oligomerization of RBDs (25.3 kDa) counterbalanced by steric stabilization by a soluble ELP (73.4 kDa). To investigate their biophysical properties, we explored the size, shape, and binding affinity for the human angiotensin-converting enzyme 2 (hACE2) and cellular uptake. Biomimetic nanoparticles such as these may enable future strategies to target the same cells, tissues, and cell-surface receptors as those harnessed by SARS-CoV-2.

Monocyte migration profiles define disease severity in acute COVID-19 and unique features of long COVID.

BACKGROUND: COVID-19 is associated with a dysregulated immune response but it is unclear how immune dysfunction contributes to the chronic morbidity persisting in many COVID-19 patients during convalescence (long COVID). METHODS: We assessed phenotypical and functional changes of monocytes in COVID-19 patients during hospitalisation and up to 9 months of convalescence following COVID-19, respiratory syncytial virus or influenza A. Patients with progressive fibrosing interstitial lung disease were included as a positive control for severe, ongoing lung injury. RESULTS: Monocyte alterations in acute COVID-19 patients included aberrant expression of leukocyte migration molecules, continuing into convalescence (n=142) and corresponding with specific symptoms of long COVID. Long COVID patients with unresolved lung injury, indicated by sustained shortness of breath and abnormal chest radiology, were defined by high monocyte expression of C-X-C motif chemokine receptor 6 (CXCR6) (p<0.0001) and adhesion molecule P-selectin glycoprotein ligand 1 (p<0.01), alongside preferential migration of monocytes towards the CXCR6 ligand C-X-C motif chemokine ligand 16 (CXCL16) (p<0.05), which is abundantly expressed in the lung. Monocyte CXCR6 and lung CXCL16 were heightened in patients with progressive fibrosing interstitial lung disease (p<0.001), confirming a role for the CXCR6-CXCL16 axis in ongoing lung injury. Conversely, monocytes from long COVID patients with ongoing fatigue exhibited a sustained reduction of the prostaglandin-generating enzyme cyclooxygenase 2 (p<0.01) and CXCR2 expression (p<0.05). These monocyte changes were not present in respiratory syncytial virus or influenza A convalescence. CONCLUSIONS: Our data define unique monocyte signatures that define subgroups of long COVID patients, indicating a key role for monocyte migration in COVID-19 pathophysiology. Targeting these pathways may provide novel therapeutic opportunities in COVID-19 patients with persistent morbidity.

Host range and structural analysis of bat-origin RshSTT182/200 coronavirus binding to human ACE2 and its animal orthologs.

Bat-origin RshSTT182 and RshSTT200 coronaviruses (CoV) from Rhinolophus shameli in Southeast Asia (Cambodia) share 92.6% whole-genome identity with SARS-CoV-2 and show identical receptor-binding domains (RBDs). In this study, we determined the structure of the RshSTT182/200 receptor binding domain (RBD) in complex with human angiotensin-converting enzyme 2 (hACE2) and identified the key residues that influence receptor binding. The binding of the RshSTT182/200 RBD to ACE2 orthologs from 39 animal species, including 18 bat species, was used to evaluate its host range. The RshSTT182/200 RBD broadly recognized 21 of 39 ACE2 orthologs, although its binding affinities for the orthologs were weaker than those of the RBD of SARS-CoV-2. Furthermore, RshSTT182 pseudovirus could utilize human, fox, and Rhinolophus affinis ACE2 receptors for cell entry. Moreover, we found that SARS-CoV-2 induces cross-neutralizing antibodies against RshSTT182 pseudovirus. Taken together, these findings indicate that RshSTT182/200 can potentially infect susceptible animals, but requires further evolution to obtain strong interspecies transmission abilities like SARS-CoV-2.

A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry.

It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.

The Elusive Coreceptors for the SARS-CoV-2 Spike Protein.

Evidence suggests that the N-terminal domain (NTD) of the SARS-CoV-2 spike protein interacts with host coreceptors that participate in viral entry. Resolving the identity of coreceptors has important clinical implications as it may provide the basis for the development of antiviral drugs and vaccine candidates. The majority of characteristic mutations in variants of concern (VOCs) have occurred in the NTD and receptor binding domain (RBD). Unlike the RBD, mutations in the NTD have clustered in the most flexible parts of the spike protein. Many possible coreceptors have been proposed, including various sugars such as gangliosides, sialosides, and heparan sulfate. Protein coreceptors, including neuropilin-1 and leucine-rich repeat containing 15 (LRRC15), are also proposed coreceptors that engage the NTD.

Metabolic dysfunction associated fatty liver disease and coronavirus disease 2019: clinical relationship and current management.

The coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). At present, the COVID-19 has been prevalent worldwide for more than a year and caused more than four million deaths. Liver injury was frequently observed in patients with COVID-19. Recently, a new definition of metabolic dysfunction associated fatty liver disease (MAFLD) was proposed by a panel of international experts, and the relationship between MAFLD and COVID-19 has been actively investigated. Several previous studies indicated that the patients with MAFLD had a higher prevalence of COVID-19 and a tendency to develop severe type of respiratory infection, and others indicated that liver injury would be exacerbated in the patients with MAFLD once infected with COVID-19. The mechanism underlying the relationship between MAFLD and COVID-19 infection has not been thoroughly investigated, and recent studies indicated that multifactorial mechanisms, such as altered host angiotensin converting enzyme 2 (ACE2) receptor expression, direct viral attack, disruption of cholangiocyte function, systemic inflammatory reaction, drug-induced liver injury, hepatic ischemic and hypoxic injury, and MAFLD-related glucose and lipid metabolic disorders, might jointly contribute to both of the adverse hepatic and respiratory outcomes. In this review, we discussed the relationship between MAFLD and COVID-19 based on current available literature, and summarized the recommendations for clinical management of MAFLD patients during the pandemic of COVID-19.

SARS-CoV-2 viral entry and replication is impaired in Cystic Fibrosis airways due to ACE2 downregulation.

As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis could be considered a comorbidity for coronavirus disease 2019. Instead, current clinical evidence seems to be heading in the opposite direction. To clarify whether host factors expressed by the Cystic Fibrosis epithelia may influence coronavirus disease 2019 progression, here we describe the expression of SARS-CoV-2 receptors in primary airway epithelial cells. We show that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral entry and replication in Cystic Fibrosis cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin 6 in healthy donor-derived primary airway epithelial cells, but a very weak response in primary Cystic Fibrosis cells. Collectively, these data support that Cystic Fibrosis condition may be at least partially protecting from SARS-CoV-2 infection.

Impedance-Based Neutralizing Antibody Detection Biosensor with Application in SARS-CoV-2 Infection.

Although safe and efficacious coronavirus disease-2019 (COVID-19) vaccines are available, real protective immunity is revealed by the serum COVID-19 neutralizing antibody (NAb) concentration. NAbs deactivate the virus by attaching to the viral receptor-binding domain (RBD), which interacts with angiotensin-converting enzyme 2 (ACE2) on the human cell. This paper introduces inexpensive, rapid, sensitive, and quantifiable impedance-based immunosensors to evaluate the NAb. The sensor limit of detection is experimentally determined in different buffer dilutions using bovine IgG-anti-bovine IgG interaction. The dominance of AC electrokinetic transport and molecular diffusion in the sensor is investigated using scaling analysis and numerical simulations. The results demonstrated that the sensor detection mechanism is mainly based on the diffusion of the biomolecules onto the electrode surface. After evaluating the sensor working principles, viral RBD buffers, including different NAb concentrations, are applied to the sensor, immobilized with the human ACE2 (hACE2). Results demonstrate that the sensor is capable of NAb detection in the analytical measuring interval between 45 ng/mL and 185 ng/mL. Since the present sensor provides fast test results with lower costs, it can be used to assess the NAb in people's blood serum before receiving further COVID vaccine doses.

Identification of anti-SARS-CoV-2 agents based on flavor/fragrance compositions that inhibit the interaction between the virus receptor binding domain and human angiotensin converting enzyme 2.

Coronavirus disease 2019 (COVID-19) pandemic poses a threat to human beings and numerous cases of infection as well as millions of victims have been reported. The binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor binding domain (RBD) to human angiotensin converting enzyme 2 (hACE2) is known to promote the engulfment of the virus by host cells. Employment of flavor/fragrance compositions to prevent SARS-CoV-2 infection by inhibiting the binding of viral RBD (vRBD) to hACE2 might serve as a favorable, simple, and easy method for inexpensively preventing COVID-19, as flavor/fragrance compositions are known to directly interact with the mucosa in the respiratory and digestive systems and have a long history of use and safety assessment. Herein we report the results of screening of flavor/fragrance compositions that inhibit the binding of vRBD to hACE2. We found that the inhibitory effect was observed with not only the conventional vRBD, but also variant vRBDs, such as L452R, E484K, and N501Y single-residue variants, and the K417N+E484K+N501Y triple-residue variant. Most of the examined flavor/fragrance compositions are not known to have anti-viral effects. Cinnamyl alcohol and Helional inhibited the binding of vRBD to VeroE6 cells, a monkey kidney cell line expressing ACE2. We termed the composition with inhibitory effect on vRBD-hACE2 binding as "the molecularly targeted flavor/fragrance compositions". COVID-19 development could be prevented by using these compositions with reasonable administration methods such as inhalation, oral administration, and epidermal application.

High activity of an affinity-matured ACE2 decoy against Omicron SARS-CoV-2 and pre-emergent coronaviruses.

The viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly its cell-binding spike protein gene, has undergone rapid evolution during the coronavirus disease 2019 (COVID-19) pandemic. Variants including Omicron BA.1 and Omicron BA.2 now seriously threaten the efficacy of therapeutic monoclonal antibodies and vaccines that target the spike protein. Viral evolution over a much longer timescale has generated a wide range of genetically distinct sarbecoviruses in animal populations, including the pandemic viruses SARS-CoV-2 and SARS-CoV-1. The genetic diversity and widespread zoonotic potential of this group complicates current attempts to develop drugs in preparation for the next sarbecovirus pandemic. Receptor-based decoy inhibitors can target a wide range of viral strains with a common receptor and may have intrinsic resistance to escape mutant generation and antigenic drift. We previously generated an affinity-matured decoy inhibitor based on the receptor target of the SARS-CoV-2 spike protein, angiotensin-converting enzyme 2 (ACE2), and deployed it in a recombinant adeno-associated virus vector (rAAV) for intranasal delivery and passive prophylaxis against COVID-19. Here, we demonstrate the exceptional binding and neutralizing potency of this ACE2 decoy against SARS-CoV-2 variants including Omicron BA.1 and Omicron BA.2. Tight decoy binding tracks with human ACE2 binding of viral spike receptor-binding domains across diverse clades of coronaviruses. Furthermore, in a coronavirus that cannot bind human ACE2, a variant that acquired human ACE2 binding was bound by the decoy with nanomolar affinity. Considering these results, we discuss a strategy of decoy-based treatment and passive protection to mitigate the ongoing COVID-19 pandemic and future airway virus threats.

Mapping of functional SARS-CoV-2 receptors in human lungs establishes differences in variant binding and SLC1A5 as a viral entry modulator of hACE2.

BACKGROUND: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. METHODS: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. FINDINGS: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. INTERPRETATION: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. FUNDING: This work was supported by an unrestricted grant from "Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola.

COVID-19 signalome: Potential therapeutic interventions.

The COVID-19 pandemic has triggered intensive research and development of drugs and vaccines against SARS-CoV-2 during the last two years. The major success was especially observed with development of vaccines based on viral vectors, nucleic acids and whole viral particles, which have received emergent authorization leading to global mass vaccinations. Although the vaccine programs have made a big impact on COVID-19 spread and severity, emerging novel variants have raised serious concerns about vaccine efficacy. Due to the urgent demand, drug development had originally to rely on repurposing of antiviral drugs developed against other infectious diseases. For both drug and vaccine development the focus has been mainly on SARS-CoV-2 surface proteins and host cell receptors involved in viral attachment and entry. In this review, we expand the spectrum of SARS-CoV-2 targets by investigating the COVID-19 signalome. In addition to the SARS-CoV-2 Spike protein, the envelope, membrane, and nucleoprotein targets have been subjected to research. Moreover, viral proteases have presented the possibility to develop different strategies for the inhibition of SARS-CoV-2 replication and spread. Several signaling pathways involving the renin-angiotensin system, angiotensin-converting enzymes, immune pathways, hypoxia, and calcium signaling have provided attractive alternative targets for more efficient drug development.

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors.

Middle East respiratory syndrome coronavirus (MERS-CoV) and several bat coronaviruses use dipeptidyl peptidase-4 (DPP4) as an entry receptor1-4. However, the receptor for NeoCoV-the closest known MERS-CoV relative found in bats-remains unclear5. Here, using a pseudotype virus entry assay, we found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins. Cryo-electron microscopy analysis revealed an RBD-ACE2 binding interface involving protein-glycan interactions, distinct from those of other known ACE2-using coronaviruses. We identified residues 337-342 of human ACE2 as a molecular determinant restricting NeoCoV entry, whereas a NeoCoV S pseudotyped virus containing a T510F RBD mutation efficiently entered cells expressing human ACE2. Although polyclonal SARS-CoV-2 antibodies or MERS-CoV RBD-specific nanobodies did not cross-neutralize NeoCoV or PDF-2180, an ACE2-specific antibody and two broadly neutralizing betacoronavirus antibodies efficiently inhibited these two pseudotyped viruses. We describe MERS-CoV-related viruses that use ACE2 as an entry receptor, underscoring a promiscuity of receptor use and a potential zoonotic threat.