Natural selection differences detected in key protein domains between non-pathogenic and pathogenic feline coronavirus phenotypes.

Feline coronaviruses (FCoVs) commonly cause mild enteric infections in felines worldwide (termed feline enteric coronavirus [FECV]), with around 12 per cent developing into deadly feline infectious peritonitis (FIP; feline infectious peritonitis virus [FIPV]). Genomic differences between FECV and FIPV have been reported, yet the putative genotypic basis of the highly pathogenic phenotype remains unclear. Here, we used state-of-the-art molecular evolutionary genetic statistical techniques to identify and compare differences in natural selection pressure between FECV and FIPV sequences, as well as to identify FIPV- and FECV-specific signals of positive selection. We analyzed full-length FCoV protein coding genes thought to contain mutations associated with FIPV (Spike, ORF3abc, and ORF7ab). We identified two sites exhibiting differences in natural selection pressure between FECV and FIPV: one within the S1/S2 furin cleavage site (FCS) and the other within the fusion domain of Spike. We also found fifteen sites subject to positive selection associated with FIPV within Spike, eleven of which have not previously been suggested as possibly relevant to FIP development. These sites fall within Spike protein subdomains that participate in host cell receptor interaction, immune evasion, tropism shifts, host cellular entry, and viral escape. There were fourteen sites (twelve novel sites) within Spike under positive selection associated with the FECV phenotype, almost exclusively within the S1/S2 FCS and adjacent to C domain, along with a signal of relaxed selection in FIPV relative to FECV, suggesting that furin cleavage functionality may not be needed for FIPV. Positive selection inferred in ORF7b was associated with the FECV phenotype and included twenty-four positively selected sites, while ORF7b had signals of relaxed selection in FIPV. We found evidence of positive selection in ORF3c in FCoV-wide analyses, but no specific association with the FIPV or FECV phenotype. We hypothesize that some combination of mutations in FECV may contribute to FIP development, and that it is unlikely to be one singular 'switch' mutational event. This work expands our understanding of the complexities of FIP development and provides insights into how evolutionary forces may alter pathogenesis in coronavirus genomes.

Interactions of SARS-CoV-2 and MERS-CoV fusion peptides measured using single-molecule force methods.

We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide (FP) sequences within coronavirus (CoV) spike proteins. Within the FPs of severe acute respiratory syndrome CoV 2 and Middle East respiratory syndrome CoV (MERS-CoV), a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. Although a non-polar triad (Leu-Leu-Phe (LLF)) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS-2 and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore whether single-molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask whether sequence variations between FP1 from SARS-2 and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single-molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 ± 0.03 nN versus 0.68 ± 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single-residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single-residue diversity in viral FPs, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts.

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.

Recent Zoonotic Spillover and Tropism Shift of a Canine Coronavirus Is Associated with Relaxed Selection and Putative Loss of Function in NTD Subdomain of Spike Protein.

A canine coronavirus (CCoV) has now been reported from two independent human samples from Malaysia (respiratory, collected in 2017-2018; CCoV-HuPn-2018) and Haiti (urine, collected in 2017); these two viruses were nearly genetically identical. In an effort to identify any novel adaptations associated with this apparent shift in tropism we carried out detailed evolutionary analyses of the spike gene of this virus in the context of related Alphacoronavirus 1 species. The spike 0-domain retains homology to CCoV2b (enteric infections) and Transmissible Gastroenteritis Virus (TGEV; enteric and respiratory). This domain is subject to relaxed selection pressure and an increased rate of molecular evolution. It contains unique amino acid substitutions, including within a region important for sialic acid binding and pathogenesis in TGEV. Overall, the spike gene is extensively recombinant, with a feline coronavirus type II strain serving a prominent role in the recombinant history of the virus. Molecular divergence time for a segment of the gene where temporal signal could be determined, was estimated at around 60 years ago. We hypothesize that the virus had an enteric origin, but that it may be losing that particular tropism, possibly because of mutations in the sialic acid binding region of the spike 0-domain.

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.

A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

Clinical and Molecular Relationships between COVID-19 and Feline Infectious Peritonitis (FIP).

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) has led the medical and scientific community to address questions surrounding the pathogenesis and clinical presentation of COVID-19; however, relevant clinical models outside of humans are still lacking. In felines, a ubiquitous coronavirus, described as feline coronavirus (FCoV), can present as feline infectious peritonitis (FIP)-a leading cause of mortality in young cats that is characterized as a severe, systemic inflammation. The diverse extrapulmonary signs of FIP and rapidly progressive disease course, coupled with a closely related etiologic agent, present a degree of overlap with COVID-19. This paper will explore the molecular and clinical relationships between FIP and COVID-19. While key differences between the two syndromes exist, these similarities support further examination of feline coronaviruses as a naturally occurring clinical model for coronavirus disease in humans.

Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity.

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry.

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

A 3D structural SARS-CoV-2-human interactome to explore genetic and drug perturbations.

Emergence of new viral agents is driven by evolution of interactions between viral proteins and host targets. For instance, increased infectivity of SARS-CoV-2 compared to SARS-CoV-1 arose in part through rapid evolution along the interface between the spike protein and its human receptor ACE2, leading to increased binding affinity. To facilitate broader exploration of how pathogen-host interactions might impact transmission and virulence in the ongoing COVID-19 pandemic, we performed state-of-the-art interface prediction followed by molecular docking to construct a three-dimensional structural interactome between SARS-CoV-2 and human. We additionally carried out downstream meta-analyses to investigate enrichment of sequence divergence between SARS-CoV-1 and SARS-CoV-2 or human population variants along viral-human protein-interaction interfaces, predict changes in binding affinity by these mutations/variants and further prioritize drug repurposing candidates predicted to competitively bind human targets. We believe this resource ( http://3D-SARS2.yulab.org ) will aid in development and testing of informed hypotheses for SARS-CoV-2 etiology and treatments.

Viral and Host Attributes Underlying the Origins of Zoonotic Coronaviruses in Bats.

With a presumed origin in bats, the COVID-19 pandemic has been a major source of morbidity and mortality in the hu- man population, and the causative agent, SARS-CoV-2, aligns most closely at the genome level with the bat coronaviruses RaBtCoV4991/RaTG13 and RmYN02. The ability of bats to provide reservoirs of numerous viruses in addition to coronaviruses remains an active area of research. Unique aspects of the physiology of the chiropteran immune system may contribute to the ability of bats to serve as viral reservoirs. The coronavirus spike protein plays important roles in viral pathogenesis and the immune response. Although much attention has focused on the spike receptor-binding domain, a unique aspect of SARS-CoV-2 as compared with its closest relatives is the presence of a furin cleavage site in the S1-S2 region of the spike protein. Proteolytic activation is likely an important feature that allows SARS-CoV-2-and other coronaviruses-to overcome the species barriers and thus cause human disease. The diversity of bat species limits the ability to draw broad conclusions about viral pathogenesis, but comparisons across species and with reference to humans and other susceptible mammals may guide future research in this regard.

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.

Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Spike Protein Proteolytic Processing.

The coronavirus spike envelope glycoprotein is an essential viral component that mediates virus entry events. Biochemical assessment of the spike protein is critical for understanding structure-function relationships and the roles of the protein in the viral life cycle. Coronavirus spike proteins are typically proteolytically processed and activated by host cell enzymes such as trypsin-like proteases, cathepsins, or proprotein-convertases. Analysis of coronavirus spike proteins by western blot allows the visualization and assessment of proteolytic processing by endogenous or exogenous proteases. Here, we present a method based on western blot analysis to investigate spike protein proteolytic cleavage by transient transfection of HEK-293 T cells allowing expression of the spike protein of the highly pathogenic Middle East respiratory syndrome coronavirus in the presence or absence of a cellular trypsin-like transmembrane serine protease, matriptase. Such analysis enables the characterization of cleavage patterns produced by a host protease on a coronavirus spike glycoprotein.

Furin cleavage sites in the spike proteins of bat and rodent coronaviruses: Implications for virus evolution and zoonotic transfer from rodent species.

Bats and rodents comprise two of the world's largest orders of mammals and the order Chiroptera (bats) has been implicated as a major reservoir of coronaviruses in nature and a source of zoonotic transfer to humans. However, the order Rodentia (rodents) also harbors coronaviruses, with two human coronaviruses (HCoV-OC43 and HCoV-HKU1) considered to have rodent origins. The coronavirus spike protein mediates viral entry and is a major determinant of viral tropism; importantly, the spike protein is activated by host cell proteases at two distinct sites, designated as S1/S2 and S2'. SARS-CoV-2, which is considered to be of bat origin, contains a cleavage site for the protease furin at S1/S2, absent from the rest of the currently known betacoronavirus lineage 2b coronaviruses (Sarbecoviruses). This cleavage site is thought to be critical to its replication and pathogenesis, with a notable link to virus transmission. Here, we examine the spike protein across coronaviruses identified in both bat and rodent species and address the role of furin as an activating protease. Utilizing two publicly available furin prediction algorithms (ProP and PiTou) and based on spike sequences reported in GenBank, we show that the S1/S2 furin cleavage site is typically not present in bat virus spike proteins but is common in rodent-associated sequences, and suggest this may have implications for zoonotic transfer. We provide a phylogenetic history of the Embecoviruses (betacoronavirus lineage 2a), including context for the use of furin as an activating protease for the viral spike protein. From a One Health perspective, continued rodent surveillance should be an important consideration in uncovering novel circulating coronaviruses.

Coronavirus entry: how we arrived at SARS-CoV-2.

Because of the COVID-19 pandemic, the novel coronavirus SARS-CoV-2 has risen to shape scientific research during 2020, with its spike (S) protein being a predominant focus. The S protein is likely the most complicated of all viral glycoproteins and is a key factor in immunological responses and virus pathogenesis. It is also the driving force dictating virus entry mechanisms, which are highly 'plastic' for coronaviruses, allowing a plethora of options for different virus variants and strains in different cell types. Here we review coronavirus entry as a foundation for current work on SARS-CoV-2. We focus on the post-receptor binding events and cellular pathways that direct the membrane fusion events necessary for genome delivery, including S proteolytic priming and activation. We also address aspects of the entry process important for virus evolution and therapeutic development.

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.