Spike Protein Cleavage-Activation in the Context of the SARS-CoV-2 P681R Mutation: an Analysis from Its First Appearance in Lineage A.23.1 Identified in Uganda.

Based on its predicted ability to affect transmissibility and pathogenesis, surveillance studies have highlighted the role of a specific mutation (P681R) in the S1/S2 furin cleavage site of the SARS-CoV-2 spike protein. Here we analyzed A.23.1, first identified in Uganda, as a P681R-containing virus several months prior to the emergence of B.1.617.2 (Delta variant). We performed assays using peptides mimicking the S1/S2 from A.23.1 and B.1.617 and observed significantly increased cleavability with furin compared to both an original B lineage (Wuhan-Hu1) and B.1.1.7 (Alpha variant). We also performed cell-cell fusion and functional infectivity assays using pseudotyped particles and observed an increase in activity for A.23.1 compared to an original B lineage spike. However, these changes in activity were not reproduced in the B lineage spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution, this substitution needs to occur on the background of other spike protein changes to enable its functional consequences. IMPORTANCE During the course of the SARS-CoV-2 pandemic, viral variants have emerged that often contain notable mutations in the spike gene. Mutations that encode changes in the spike S1/S2 (furin) activation site have been considered especially impactful. The S1/S2 change from proline to arginine at position 681 (P681R) first emerged in the A.23.1 variant in Uganda, and subsequently occurred in the more widely transmitted Delta variant. We show that the A.23.1 spike is more readily activated by the host cell protease furin, but that this is not reproduced in an original SARS-CoV-2 spike containing the P681R mutation. Changes to the S1/S2 (furin) activation site play a role in SARS-CoV-2 infection and spread, but successful viruses combine these mutations with other less well identified changes, occurring as part of natural selection.

Functional evaluation of the P681H mutation on the proteolytic activation of the SARS-CoV-2 variant B.1.1.7 (Alpha) spike.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the COVID-19 pandemic. SARS-CoV-2 B.1.1.7 (Alpha), a WHO variant of concern first identified in the United Kingdom in late 2020, contains several mutations including P681H in the spike S1/S2 cleavage site, which is predicted to increase cleavage by furin, potentially impacting the viral cell entry. Here, we studied the role of the P681H mutation in B.1.1.7 cell entry. We performed assays using fluorogenic peptides mimicking the Wuhan-Hu-1 and B.1.1.7 S1/S2 sequence and observed no significant difference in furin cleavage. Functional assays using pseudoparticles harboring SARS-CoV-2 spikes and cell-to-cell fusion assays demonstrated no differences between Wuhan-Hu-1, B.1.1.7, or a P681H point mutant. Likewise, we observed no differences in viral growth between USA-WA1/2020 and a B.1.1.7 isolate in cell culture. Our findings suggest that, although the B.1.1.7 P681H mutation may slightly increase S1/S2 cleavage, this does not significantly impact viral entry or cell-cell spread.

SARS-CoV-2 electrochemical immunosensor based on the spike-ACE2 complex.

Rapid, straightforward, and massive diagnosis of coronavirus disease 2019 (COVID-19) is one of the more important measures to mitigate the current pandemics. This work reports on an immunosensor to rapidly detect the spike protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The immunosensing device entraps the spike protein linked to angiotensin-converting enzyme host receptor (ACE2) protein in a sandwich between carboxylated magnetic beads functionalized with an anti-spike antibody and an anti-ACE2 antibody, further labeled with streptavidin (poly)horseradish peroxidase (HRP) reporter enzyme. The particles were confined at the surface of screen-printed gold electrodes, whose signal resulting from the interaction of the enzyme with a mediator was recorded in a portable potentiostat. The immunosensor showed a sensitivity of 0.83 µA∗mL/µg and a limit of detection of 22.5 ng/mL of spike protein, with high reproducibility. As a proof-of-concept, it detected commercial spike protein-supplemented buffer solutions, pseudovirions, isolated viral particles and ten nasopharyngeal swab samples from infected patients compared to samples from three healthy individuals paving the way to detect the virus closer to the patient.

Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry.

Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing infection at the stage of viral entry. Coagulation factors increased SARS-CoV-2 infection in human lung organoids. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases and coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.

SARS-CoV-2 electrochemical immunosensor based on the spike-ACE2 complex

Rapid, straightforward, and massive diagnosis of coronavirus disease 2019 (COVID-19) is one of the more important measures to mitigate the current pandemics. This work reports on an immunosensor to rapidly detect the spike protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The immunosensing device entraps the spike protein linked to angiotensin-converting enzyme host receptor (ACE2) protein in a sandwich between carboxylated magnetic beads functionalized with an anti-spike antibody and an anti-ACE2 antibody, further labeled with streptavidin (poly)horseradish peroxidase (HRP) reporter enzyme. The particles were confined at the surface of screen-printed gold electrodes, whose signal resulting from the interaction of the enzyme with a mediator was recorded in a portable potentiostat. The immunosensor showed a sensitivity of 0.83 μA*mL/μg and a limit of detection of 22.5 ng/mL of spike protein, with high reproducibility. As a proof-of-concept, it detected commercial spike protein-supplemented buffer solutions, pseudovirions, isolated viral particles and ten nasopharyngeal swab samples from infected patients compared to samples from three healthy individuals paving the way to detect the virus closer to the patient. Graphical Image 1

Inhibitors of L-Type Calcium Channels Show Therapeutic Potential for Treating SARS-CoV-2 Infections by Preventing Virus Entry and Spread.

COVID-19 is caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus (CoV)-2 (SARS-CoV-2). The virus is responsible for an ongoing pandemic and concomitant public health crisis around the world. While vaccine development is proving to be highly successful, parallel drug development approaches are also critical in the response to SARS-CoV-2 and other emerging viruses. Coronaviruses require Ca2+ ions for host cell entry, and we have previously shown that Ca2+ modulates the interaction of the viral fusion peptide with host cell membranes. In an attempt to accelerate drug repurposing, we tested a panel of L-type calcium channel blocker (CCB) drugs currently developed for other conditions to determine whether they would inhibit SARS-CoV-2 infection in cell culture. All the CCBs tested showed varying degrees of inhibition, with felodipine and nifedipine strongly limiting SARS-CoV-2 entry and infection in epithelial lung cells at concentrations where cell toxicity was minimal. Further studies with pseudotyped particles displaying the SARS-CoV-2 spike protein suggested that inhibition occurs at the level of virus entry. Overall, our data suggest that certain CCBs have the potential to treat SARS-CoV-2 infections and are worthy of further examination for possible treatment of COVID-19.

SARS-CoV-2 Clinical Outcome in Domestic and Wild Cats: A Systematic Review.

Recently, it has been proved that SARS-CoV-2 has the ability to infect multiple species. This work was aimed at identifying the clinical signs of SARS-CoV-2 infection in domestic and wild felids. A PRISMA-based systematic review was performed on case reports on domestic and wild cats, reports on experimental infections, case reports in databases, preprints and published press releases. Descriptive statistical analysis of the data was performed. A total of 256 articles, 63 detailed official reports and 2 press articles on SARS-CoV-2 infection in domestic and wild cats were analyzed, of which 19 articles and 65 reports were finally included. In domestic cats, most cats' infections are likely to be asymptomatic, and 46% of the reported infected animals were symptomatic and predominantly presented respiratory signs such as sneezing and coughing. In wild felines, respiratory clinical signs were most frequent, and up to 96.5% of the reported affected animals presented coughing. It is noteworthy that, to date, symptomatic animals with SARS-CoV-2 infection have been reported to belong to two different subfamilies (Phanterinae and Felinae), with up to five different felid species affected within the Felidae family. Reported results evince that the signs developed in felids show similar progression to those occurring in humans, suggesting a relationship between the viral cycle and target tissues of the virus in different species. While viral transmission to humans in contact with animal populations has not been reported, spill-back could result in the emergence of immune-escape mutants that might pose a risk to public health. Despite the clear results in the identification of the typical clinical picture of SARS-CoV-2 infection in felines, the number of detailed academic reports and papers on the subject is scarce. Therefore, further description of these cases will allow for more accurate and statistically robust clinical approaches in the future.

SARS CoV-2 Spike Protein in silico Interaction With ACE2 Receptors From Wild and Domestic Species.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared a pandemic by the World Health Organization (WHO), and since its first report, it has become a major public health concern. SARS-CoV-2 is closely related to SARS-CoV and SARS-related bat coronaviruses, and it has been described to use angiotensin-converting enzyme 2 (ACE2) as a receptor. Natural SARS-CoV-2 infection in domestic and wildlife animals, measured by RT-qPCR, has been confirmed in different countries, especially from the Felidae family. In silico analysis of the interaction between the SARS-CoV-2 spike protein and the cellular receptor ACE2 in various animal species has suggested that wild felids and domestic cats could be susceptible to SARS-CoV-2 based on this interaction. Here, we performed a protein-protein molecular docking analysis of SARS-CoV-2 spike protein with the ACE2 receptor from different animals to elucidate the potential of those species as intermediate hosts or susceptible animals for SARS-CoV-2 infection. Compared to human ACE2, we found that ACE2 receptors from domestic cats and tigers could efficiently interact with RBD of SARS CoV-2 Spike protein. However, dog, ferret, and hamster ACE2 receptor interaction with SARS-CoV-2 S protein RBD was not predicted as favorable, demonstrating a potential differentiated susceptibility in the evaluated species.

Proteolytic Activation of SARS-CoV-2 Spike at the S1/S2 Boundary: Potential Role of Proteases beyond Furin.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2' site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.

Molecular diversity of coronavirus host cell entry receptors.

Coronaviruses are a group of viruses causing disease in a wide range of animals, and humans. Since 2002, the successive emergence of bat-borne severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), swine acute diarrhea syndrome coronavirus (SADS-CoV) and SARS-CoV-2 has reinforced efforts in uncovering the molecular and evolutionary mechanisms governing coronavirus cell tropism and interspecies transmission. Decades of studies have led to the discovery of a broad set of carbohydrate and protein receptors for many animal and human coronaviruses. As the main determinant of coronavirus entry, the spike protein binds to these receptors and mediates membrane fusion. Prone to mutations and recombination, spike evolution has been studied extensively. The interactions between spike proteins and their receptors are often complex and despite many advances in the field, there remains many unresolved questions concerning coronavirus tropism modification and cross-species transmission, potentially leading to delays in outbreak responses. The emergence of SARS-CoV-2 underscores the need to address these outstanding issues in order to better anticipate new outbreaks. In this review, we discuss the latest advances in the field of coronavirus receptors emphasizing on the molecular and evolutionary processes that underlie coronavirus receptor usage and host range expansion.

A Tale of Two Viruses: The Distinct Spike Glycoproteins of Feline Coronaviruses.

Feline coronavirus (FCoV) is a complex viral agent that causes a variety of clinical manifestations in cats, commonly known as feline infectious peritonitis (FIP). It is recognized that FCoV can occur in two different serotypes. However, differences in the S protein are much more than serological or antigenic variants, resulting in the effective presence of two distinct viruses. Here, we review the distinct differences in the S proteins of these viruses, which are likely to translate into distinct biological outcomes. We introduce a new concept related to the non-taxonomical classification and differentiation among FCoVs by analyzing and comparing the genetic, structural, and functional characteristics of FCoV and the FCoV S protein among the two serotypes and FCoV biotypes. Based on our analysis, we suggest that our understanding of FIP needs to consider whether the presence of these two distinct viruses has implications in clinical settings.

Coronaviruses in cats and other companion animals: Where does SARS-CoV-2/COVID-19 fit?

Coronaviruses (CoVs) cause disease in a range of agricultural and companion animal species, and can be important causes of zoonotic infections. In humans, several coronaviruses circulate seasonally. Recently, a novel zoonotic CoV named SARS-CoV-2 emerged from a bat reservoir, resulting in the COVID-19 pandemic. With a focus on felines, we review here the evidence for SARS-CoV-2 infection in cats, ferrets and dogs, describe the relationship between SARS-CoV-2 and the natural coronaviruses known to infect these species, and provide a rationale for the relative susceptibility of these species to SARS-CoV-2 through comparative analysis of the ACE-2 receptor.

Concerns on the Emerging Research of SARS-CoV-2 on Felines: Could They be Significant Hosts/Reservoirs?

With the growing evidence of cases and studies showing natural and experimental infection due to SARS-CoV-2 in felines, including cats, lion, and tigers, there is also an increasing concern about its susceptibility and their role in urban cycles of SARS-CoV-2 transmission, initially from humans-to-animals, but with uncertainty about reverse transmission. In this review, we addressed the evidence around this situation.

Proteolytic Cleavage of the SARS-CoV-2 Spike Protein and the Role of the Novel S1/S2 Site.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread to the entire world within a few months. The origin of SARS-CoV-2 has been related to the lineage B Betacoronavirus SARS-CoV and SARS-related coronaviruses found in bats. Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct four amino acid insert within the spike (S) protein (underlined, SPRRAR↓S), at the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this insert appears to be a distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochemical evidence for proteolytic processing by a variety of proteases. We discuss these findings in the context of the origin of SARS-CoV-2, viral stability, and transmission.

Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop.

The 2019 novel coronavirus (2019-nCoV/SARS-CoV-2) originally arose as part of a major outbreak of respiratory disease centered on Hubei province, China. It is now a global pandemic and is a major public health concern. Taxonomically, SARS-CoV-2 was shown to be a Betacoronavirus (lineage B) closely related to SARS-CoV and SARS-related bat coronaviruses, and it has been reported to share a common receptor with SARS-CoV (ACE-2). Subsequently, betacoronaviruses from pangolins were identified as close relatives to SARS-CoV-2. Here, we perform structural modeling of the SARS-CoV-2 spike glycoprotein. Our data provide support for the similar receptor utilization between SARS-CoV-2 and SARS-CoV, despite a relatively low amino acid similarity in the receptor binding module. Compared to SARS-CoV and all other coronaviruses in Betacoronavirus lineage B, we identify an extended structural loop containing basic amino acids at the interface of the receptor binding (S1) and fusion (S2) domains. We suggest this loop confers fusion activation and entry properties more in line with betacoronaviruses in lineages A and C, and be a key component in the evolution of SARS-CoV-2 with this structural loop affecting virus stability and transmission.

Coronavirus membrane fusion mechanism offers a potential target for antiviral development.

The coronavirus disease 2019 (COVID-19) pandemic has focused attention on the need to develop effective therapies against the causative agent, SARS-CoV-2, and also against other pathogenic coronaviruses (CoV) that have emerged in the past or might appear in future. Researchers are therefore focusing on steps in the CoV replication cycle that may be vulnerable to inhibition by broad-spectrum or specific antiviral agents. The conserved nature of the fusion domain and mechanism across the CoV family make it a valuable target to elucidate and develop pan-CoV therapeutics. In this article, we review the role of the CoV spike protein in mediating fusion of the viral and host cell membranes, summarizing the results of research on SARS-CoV, MERS-CoV, and recent peer-reviewed studies of SARS-CoV-2, and suggest that the fusion mechanism be investigated as a potential antiviral target. We also provide a supplemental file containing background information on the biology, epidemiology, and clinical features of all human-infecting coronaviruses, along with a phylogenetic tree of these coronaviruses.