Integrin mediates cell entry of the SARS-CoV-2 virus independent of cellular receptor ACE2

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is broadly accepted that SARS-CoV-2 utilizes its spike protein to recognize the extracellular domain of angiotensin-converting enzyme 2 (ACE2) to enter cells for viral infection. However, other mechanisms of SARS-CoV-2 cell entry may occur. We show quantitatively that the SARS-CoV-2 spike protein also binds to the extracellular domain of broadly expressed integrin α5β1 with an affinity comparable to that of SARS-CoV-2 binding to ACE2. More importantly, we provide direct evidence that such binding promotes the internalization of SARS-CoV-2 into non-ACE2 cells in a manner critically dependent upon the activation of the integrin. Our data demonstrate an alternative pathway for the cell entry of SARS-CoV-2, suggesting that upon initial ACE2-mediated invasion of the virus in the respiratory system, which is known to trigger an immune response and secretion of cytokines to activate integrin, the integrin-mediated cell invasion of SARS-CoV-2 into the respiratory system and other organs becomes effective, thereby promoting further infection and progression of COVID-19.     

Penta-peptide ATN-161 based neutralization mechanism of SARS-CoV-2 spike protein

SARS-CoV-2 has become a big challenge for the scientific community worldwide. SARS-CoV-2 enters into the host cell by the spike protein binding with an ACE2 receptor present on the host cell. Developing safe and effective inhibitor appears an urgent need to interrupt the binding of SARS-CoV-2 spike protein with ACE2 receptor in order to reduce the SARS-CoV-2 infection. We have examined the penta-peptide ATN-161 as potential inhibitor of ACE2 and SARS-CoV-2 spike protein binding, where ATN-161 has been commercially approved for the safety and possess high affinity and specificity towards the receptor binding domain (RBD) of S1 subunit in SARS-CoV-2 spike protein. We carried out experiments and confirmed these phenomena that the virus bindings were indeed minimized. ATN-161 peptide can be used as an inhibitor of protein-protein interaction (PPI) stands as a crucial interaction in biological systems. The molecular docking finding suggests that the binding energy of the ACE2-spike protein complex is reduced in the presence of ATN-161. Protein-protein docking binding energy (-40.50 kcal/mol) of the spike glycoprotein toward the human ACE2 and binding of ATN-161 at their binding interface reduced the biding energy (-26.25 kcal/mol). The finding of this study suggests that ATN-161 peptide can mask the RBD of the spike protein and be considered as a neutralizing candidate by binding with the ACE2 receptor. Peptide-based masking of spike S1 protein (RBD) and its neutralization is a highly promising strategy to prevent virus penetration into the host cell. Thus masking of the RBD leads to the loss of receptor recognition property which can reduce the chance of infection host cells.     

In silico mutagenesis of human ACE2 with S protein and translational efficiency explain SARS-CoV-2 infectivity in different species

The coronavirus disease COVID-19 constitutes the most severe pandemic of the last decades having caused more than 1 million deaths worldwide. The SARS-CoV-2 virus recognizes the angiotensin converting enzyme 2 (ACE2) on the surface of human cells through its spike protein. It has been reported that the coronavirus can mildly infect cats, and ferrets, and perhaps dogs while not pigs, mice, chicken and ducks. Differences in viral infectivity among different species or individuals could be due to amino acid differences at key positions of the host proteins that interact with the virus, the immune response, expression levels of host proteins and translation efficiency of the viral proteins among other factors. Here, first we have addressed the importance that sequence variants of different animal species, human individuals and virus isolates have on the interaction between the RBD domain of the SARS-CoV-2 spike S protein and human angiotensin converting enzyme 2 (ACE2). Second, we have looked at viral translation efficiency by using the tRNA adaptation index. We find that integration of both interaction energy with ACE2 and translational efficiency explains animal infectivity. Humans are the top species in which SARS-CoV-2 is both efficiently translated as well as optimally interacting with ACE2. We have found some viral mutations that increase affinity for hACE and some hACE2 variants affecting ACE2 stability and virus binding. These variants suggest that different sensitivities to coronavirus infection in humans could arise in some cases from allelic variability affecting ACE2 stability and virus binding.     

Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.     

Repurposing of approved drugs with potential to interact with SARS-CoV-2 receptor

Respiratory transmission is the primary route of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Angiotensin I converting enzyme 2 (ACE2) is the known receptor of SARS-CoV-2 surface spike glycoprotein for entry into human cells. A recent study reported absent to low expression of ACE2 in a variety of human lung epithelial cell samples. Three bioprojects (PRJEB4337, PRJNA270632 and PRJNA280600) invariably found abundant expression of ACE1 (a homolog of ACE2 and also known as ACE) in human lungs compared to very low expression of ACE2. In fact, ACE1 has a wider and more abundant tissue distribution compared to ACE2. Although it is not obvious from the primary sequence alignment of ACE1 and ACE2, comparison of X-ray crystallographic structures show striking similarities in the regions of the peptidase domains (PD) of these proteins, which is known (for ACE2) to interact with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Critical amino acids in ACE2 that mediate interaction with the viral spike protein are present and organized in the same order in the PD of ACE1. In silico analysis predicts comparable interaction of SARS-CoV-2 spike protein with ACE1 and ACE2. In addition, this study predicts from a list of 1263 already approved drugs that may interact with ACE2 and/or ACE1 and potentially interfere with the entry of SARS-CoV-2 inside the host cells.     

Screening of inhibitors against SARS-CoV-2 spike protein and their capability to block the viral entry mechanism: A viroinformatics study

SARS-CoV-2, previously named 2019 novel coronavirus (2019-nCoV), has been associated with the global pandemic of acute respiratory distress syndrome. First reported in December 2019 in the Wuhan province of China, this new RNA virus has several folds higher transmission among humans than its other family member (SARS-CoV and MERS-CoV). The SARS-CoV-2 spike receptor-binding domain (RBD) is the region mediating the binding of the virus to host cells via Angiotensin-converting enzyme 2 (ACE2), a critical step of viral. Here in this study, we have utilized in silico approach for the virtual screening of antiviral library extracted from the Asinex database against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein. Further, the molecules were ranked based on their binding affinity against RBD, and the top 15 molecules were selected. The affinity of these selected molecules to interrupt the ACE2-Spike interaction was also studied. It was found that the chosen molecules were demonstrating excellent binding affinity against spike protein, and these molecules were also very effectively interrupting the ACE2-RBD interaction.Furthermore, molecular dynamics (MD) simulation studies were utilized to investigate the top 3 selected molecules' stability in the ACE2-RBD complexes. To the best of our knowledge, this is the first study where molecules' inhibitory potential against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein and their inhibitory potential against the ACE2-Spike has been studied. We believe that these compounds can be further tested as a potential therapeutic option against COVID-19.     

A Comparative Study between Spanish and British SARS-CoV-2 Variants

The study of the interaction between the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor is key to understanding binding affinity and stability. In the present report, we sought to investigate the differences between two already sequenced genome variants (Spanish and British) of SARS-CoV-2. Methods: In silico model evaluating the homology, identity and similarity in the genome sequence and the structure and alignment of the predictive spike by computational docking methods. Results: The identity results between the Spanish and British variants of the Spike protein were 28.67%. This close correspondence in the results between the Spanish and British SARS-CoV-2 variants shows that they are very similar (99.99%). The alignment obtained results in four deletions. There were 23 nucleotide substitutions also predicted which could affect the functionality of the proteins produced from this sequence. The interaction between the binding receptor domain from the spike protein and the ACE2 receptor produces some of the mutations found and, therefore, the energy of this ligand varies. However, the estimated antigenicity of the British variant is higher than its Spanish counterpart. Conclusions: Our results indicate that minimal mutations could interfere in the infectivity of the virus due to changes in the fitness between host cell recognition and interaction proteins. In particular, the N501Y substitution, situated in the RBD of the spike of the British variant, might be the reason for its extraordinary infective potential.     

Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.     

Identification of Conserved Epitopes in SARS-CoV-2 Spike and Nucleocapsid Protein

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel virus that first occurred in Wuhan in December 2019. The spike glycoproteins and nucleocapsid proteins are the most common targets for the development of vaccines and antiviral drugs.ObjectiveWe herein analyze the rate of evolution along with the sequences of spike and nucleocapsid proteins in relation to the spatial locations of their epitopes, previously suggested to contribute to the immune response caused by SARS-CoV-2 infections.MethodsWe compare homologous proteins of seven human coronaviruses: HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, and SARS-CoV-2. We then focus on the local, structural order-disorder propensity of the protein regions where the SARS-CoV-2 epitopes are located. ResultsWe show that most of nucleocapsid protein epitopes overlap the RNA-binding and dimerization domains, and some of them are characterized by a low rate of evolutions. Similarly, spike protein epitopes are preferentially located in regions that are predicted to be ordered and well- conserved, in correspondence of the heptad repeats 1 and 2. Interestingly, both the receptor-binding motif to ACE2 and the fusion peptide of spike protein are characterized by a high rate of evolution.ConclusionOur results provide evidence for conserved epitopes that might help develop broad-spectrum SARS-CoV-2 vaccines.     

Receptor-binding domain of SARS-CoV-2 spike protein efficiently inhibits SARS-CoV-2 infection and attachment to mouse lung

COVID-19, caused by a novel coronavirus, SARS-CoV-2, poses a serious global threat. It was first reported in 2019 in China and has now dramatically spread across the world. It is crucial to develop therapeutics to mitigate severe disease and viral spread. The receptor-binding domains (RBDs) in the spike protein of SARS-CoV and MERS-CoV have shown anti-viral activity in previous reports suggesting that this domain has high potential for development as therapeutics. To evaluate the potential antiviral activity of recombinant SARS-CoV-2 RBD proteins, we determined the RBD residues of SARS-CoV-2 using a homology search with RBD of SARS-CoV. For efficient expression and purification, the signal peptide of spike protein was identified and used to generate constructs expressing recombinant RBD proteins. Highly purified RBD protein fused with the Fc domain of human IgG showed potent anti-viral efficacy, which was better than that of a protein fused with a histidine tag. Intranasally pre-administrated RBD protein also inhibited the attachment of SARS-COV-2 to mouse lungs. These findings indicate that RBD protein could be used for the prevention and treatment of SARS-CoV-2 infection.     

基于新型冠状病毒S蛋白结构及入胞机制的抗体与药物研发

新型冠状病毒肺炎,世界卫生组织命名为"2019冠状病毒病"(corona virus disease 2019,COVID-19),是一种由2019新型冠状病毒(2019-nCov)感染导致的肺炎.目前新冠肺炎在全球广泛流行,且疫情尚未得到全部控制.由于新型冠状病毒表面的刺突蛋白(spike protein,S)介导病...     

新冠病毒中和单克隆抗体及纳米抗体研究进展北大核心CSCD

由严重急性呼吸系统综合征冠状病毒2型(severe acute respiratory syndrome coronavirus-2,SARS-CoV-2)引起的疾病被命名为新型冠状病毒肺炎(coronavirus disease 2019,COVID-19),是一种具有强传染性、高易感性、长潜伏期的传染病。病毒刺突蛋白受体结合结构域(receptor binding domain,RBD)和细胞血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)之间的相互作用使得SARS-CoV-2顺利进入细胞。本文对SARS-CoV-2与ACE2的相关作用机制进行了简单概述,对目前针对SARS-CoV-2中和单克隆抗体、纳米抗体的最新研究进展进行了总结,探讨了新冠肺炎的发展过程和抗体药物的研究方向,以期为包括新冠肺炎在内的新发、突发传染病中和抗体药物的研发提供参考。     

Biomechanical characterization of SARS-CoV-2 spike RBD and human ACE2 protein-protein interaction

The current COVID-19 pandemic has led to a devastating impact across the world. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus causing COVID-19) is known to use the receptor-binding domain (RBD) at viral surface spike (S) protein to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD-ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has a higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002–2004 SARS outbreak. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approaches to quantify the specific interactions between SARS-CoV-2 or SARS-CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between SARS-CoV-2 RBD and ACE2 range from 70 to 105 pN and are 30–40% higher than those of SARS-CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that SARS-CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the SARS-CoV-1 RBD-ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After removing N-linked glycans on ACE2, its mechanical binding strength with SARS-CoV-2 RBD decreases to a similar level of the SARS-CoV-1 RBD-ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1 and could help develop new strategies to block SARS-CoV-2 entry.     

Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct “S-R/x3” to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.     

The role of S477N mutation in the molecular behavior of SARS-CoV-2 spike protein: An in-silico perspective

The attachment of SARA-CoV-2 happens between ACE2 and the receptor binding domain (RBD) on the spike protein. Mutations in this domain can affect the binding affinity of the spike protein for ACE2. S477N, one of the most common mutations reported in the recent variants, is located in the RBD. Today's computational approaches in biology, especially during the SARS-CoV-2 pandemic, assist researchers in predicting a protein's behavior in contact with other proteins in more detail. In this study, we investigated the interactions of the S477N-hACE2 in silico to find the impact of this mutation on its binding affinity for ACE2 and immunity responses using dynamics simulation, protein–protein docking, and immunoinformatics methods. Our computational analysis revealed an increased binding affinity of N477 for ACE2. Four new hydrogen and hydrophobic bonds in the mutant RBD-ACE2 were formed (with S19 and Q24 of ACE2), which do not exist in the wild type. Also, the protein spike structure in this mutation was associated with an increase in stabilization and a decrease in its fluctuations at the atomic level. N477 mutation can be considered as the cause of increased escape from the immune system through MHC-II.     

The binding of heparin to spike glycoprotein inhibits SARS-CoV-2 infection by three mechanisms

Heparin, a naturally occurring glycosaminoglycan, has been found to have antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. To elucidate the mechanistic basis for the antiviral activity of heparin, we investigated the binding of heparin to the SARS-CoV-2 spike glycoprotein by means of sliding window docking, molecular dynamics simulations, and biochemical assays. Our simulations show that heparin binds at long, positively charged patches on the spike glycoprotein, thereby masking basic residues of both the receptor-binding domain (RBD) and the multifunctional S1/S2 site. Biochemical experiments corroborated the simulation results, showing that heparin inhibits the furin-mediated cleavage of spike by binding to the S1/S2 site. Our simulations showed that heparin can act on the hinge region responsible for motion of the RBD between the inactive closed and active open conformations of the spike glycoprotein. In simulations of the closed spike homotrimer, heparin binds the RBD and the N-terminal domain of two adjacent spike subunits and hinders opening. In simulations of open spike conformations, heparin induces stabilization of the hinge region and a change in RBD motion. Our results indicate that heparin can inhibit SARS-CoV-2 infection by three mechanisms: by allosterically hindering binding to the host cell receptor, by directly competing with binding to host heparan sulfate proteoglycan coreceptors, and by preventing spike cleavage by furin. Furthermore, these simulations provide insights into how host heparan sulfate proteoglycans can facilitate viral infection. Our results will aid the rational optimization of heparin derivatives for SARS-CoV-2 antiviral therapy.     

Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.

This study characterizes novel neutralizing antibodies against the SARS-CoV-2 spike protein. Co-crystal structures of the spike protein receptor-binding domain and humanised mouse antibodies identify novel epitopes on the spike protein; binding to these epitopes competes with the ACE2 receptor, and one of the antibodies provides protection against SARS-CoV-2 infection in a mouse model of COVID-19.     

Virus-receptor interactions of SARS-CoV-2 spikereceptor-binding domain and human neuropilin-1 b1 domain

In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wahan, China and it causes disease which is known as COVID-19. This infection spreads everywhere in the world, and it leads to an enormous number of death among individuals. The mystery issue about SARS-CoV-2 that appears not to have functions of a hemagglutinin and neuraminidase like other coronaviruses. Angiotensin-converting enzyme 2 (ACE2) is the main surface receptor for entering SARS-CoV-2 into the host cell. This entry process is mediated by binding the SARS-CoV-2 spike receptor-binding domain (RBD) to ACE2. Recently, researchers discover a new receptor responsible for the SARS-CoV-2 entry which is neuropilin-1 (NRP1). So, this work provides afford a knowledge of how the initial interaction between SARS-CoV-2 spike RBD and NRP1 b1 domain may occur. Understanding this interaction would be very necessary for drug design against SARS-CoV-2.     

Regulation of epithelial sodium channel activity by SARS-CoV-1 and SARS-CoV-2 proteins

Severe acute respiratory syndrome (SARS) coronavirus (CoV) 2 (SARS-CoV-2), which causes the coronavirus disease 2019, encodes several proteins whose roles are poorly understood. We tested their ability either to directly form plasma membrane ion channels or to change functions of two mammalian plasma membrane ion channels, the epithelial sodium channel (ENaC) and the α3β4 nicotinic acetylcholine receptor. In mRNA-injected Xenopus oocytes, none of nine SARS-CoV-2 proteins or two SARS-CoV-1 proteins produced conductances, nor did co-injection of several combinations. Immunoblots for ORF8, spike (S), and envelope (E) proteins revealed that the proteins are expressed at appropriate molecular weights. In experiments on coexpression with ENaC, three tested SARS proteins (SARS-CoV-1 E, SARS-CoV-2 E, and SARS-CoV-2 S) markedly decrease ENaC currents. SARS-CoV-1 S protein decreases ENaC currents modestly. Coexpressing the E proteins but not the S proteins with α3β4 nicotinic acetylcholine receptors significantly reduces acetylcholine-induced currents. ENaC inhibition does not occur if the SARS-CoV protein mRNAs are injected 24 h after the ENaC mRNAs, suggesting that SARS-CoV proteins affect early step(s) in functional expression of channel proteins. Consistent with the hypothesis that the SARS-CoV-2 S protein-induced ENaC inhibition involves competition for available protease, mutating the furin cleavage site in SARS-CoV-2 S protein partially relieves inhibition of ENaC currents. Extending previous suggestions that SARS proteins affect ENaC currents via protein kinase C (PKC) activation, PKC activation via phorbol 12-myristate 13-acetate decreases ENaC and α3β4 activity. Phorbol 12-myristate 13-acetate application reduced membrane capacitance ～5%, presumably via increased endocytosis, but this decrease is much smaller than the SARS proteins’ effects on conductances. Also, incubating oocytes in Gö-6976, a PKCα and PKCβ inhibitor, did not alter E or S protein-induced channel inhibition. We conclude that SARS-CoV-1 and SARS-CoV-2 proteins alter the function of human plasma membrane channels, via incompletely understood mechanisms. These interactions may play a role in the coronavirus 2019 pathophysiology.     

Role of host factors in SARS-CoV-2 entry

The zoonotic transmission of highly pathogenic coronaviruses into the human population is a pressing concern highlighted by the ongoing SARS-CoV-2 pandemic. Recent work has helped to illuminate much about the mechanisms of SARS-CoV-2 entry into the cell, which determines host- and tissue-specific tropism, pathogenicity, and zoonotic transmission. Here we discuss current findings on the factors governing SARS-CoV-2 entry. We first reviewed key features of the viral spike protein (S) mediating fusion of the viral envelope and host cell membrane through binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2. We then examined the roles of host proteases including transmembrane protease serine 2 and cathepsins in processing S for virus entry and the impact of this processing on endosomal and plasma membrane virus entry routes. We further discussed recent work on several host cofactors that enhance SARS-CoV-2 entry including Neuropilin-1, CD147, phosphatidylserine receptors, heparan sulfate proteoglycans, sialic acids, and C-type lectins. Finally, we discussed two key host restriction factors, i.e., interferon-induced transmembrane proteins and lymphocyte antigen 6 complex locus E, which can disrupt SARS-CoV-2 entry. The features of SARS-CoV-2 are presented in the context of other human coronaviruses, highlighting unique aspects. In addition, we identify the gaps in understanding of SARS-CoV-2 entry that will need to be addressed by future studies.