A linear B-cell epitope close to the furin cleavage site within the S1 domain of SARS-CoV-2 Spike protein discriminates the humoral immune response of nucleic acid- and protein-based vaccine cohorts.

Background: Understanding the humoral immune response towards viral infection and vaccination is instrumental in developing therapeutic tools to fight and restrict the viral spread of global pandemics. Of particular interest are the specificity and breadth of antibody reactivity in order to pinpoint immune dominant epitopes that remain immutable in viral variants. Methods: We used profiling with peptides derived from the Spike surface glycoprotein of SARS-CoV-2 to compare the antibody reactivity landscapes between patients and different vaccine cohorts. Initial screening was done with peptide microarrays while detailed results and validation data were obtained using peptide ELISA. Results: Overall, antibody patterns turned out to be individually distinct. However, plasma samples of patients conspicuously recognized epitopes covering the fusion peptide region and the connector domain of Spike S2. Both regions are evolutionarily conserved and are targets of antibodies that were shown to inhibit viral infection. Among vaccinees, we discovered an invariant Spike region (amino acids 657-671) N-terminal to the furin cleavage site that elicited a significantly stronger antibody response in AZD1222- and BNT162b2- compared to NVX-CoV2373-vaccinees. Conclusions: Understanding the exact function of antibodies recognizing amino acid region 657-671 of SARS-CoV-2 Spike glycoprotein and why nucleic acid-based vaccines elicit different responses from protein-based ones will be helpful for future vaccine design.

Host antiviral factors hijack furin to block SARS-CoV-2, ebola virus, and HIV-1†glycoproteins cleavage.

Viral envelope glycoproteins are crucial for viral infections. In the process of enveloped viruses budding and release from the producer cells, viral envelope glycoproteins are presented on the viral membrane surface as spikes, promoting the virus's next-round infection of target cells. However, the host cells evolve counteracting mechanisms in the long-term virus-host co-evolutionary processes. For instance, the host cell antiviral factors could potently suppress viral replication by targeting their envelope glycoproteins through multiple channels, including their intracellular synthesis, glycosylation modification, assembly into virions, and binding to target cell receptors. Recently, a group of studies discovered that some host antiviral proteins specifically recognized host proprotein convertase (PC) furin and blocked its cleavage of viral envelope glycoproteins, thus impairing viral infectivity. Here, in this review, we briefly summarize several such host antiviral factors and analyze their roles in reducing furin cleavage of viral envelope glycoproteins, aiming at providing insights for future antiviral studies.

SARS-CoV-2 spike protein-mediated cardiomyocyte fusion may contribute to increased arrhythmic risk in COVID-19.

BACKGROUND: SARS-CoV-2-mediated COVID-19 may cause sudden cardiac death (SCD). Factors contributing to this increased risk of potentially fatal arrhythmias include thrombosis, exaggerated immune response, and treatment with QT-prolonging drugs. However, the intrinsic arrhythmic potential of direct SARS-CoV-2 infection of the heart remains unknown. OBJECTIVE: To assess the cellular and electrophysiological effects of direct SARS-CoV-2 infection of the heart using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS: hiPSC-CMs were transfected with recombinant SARS-CoV-2 spike protein (CoV-2 S) or CoV-2 S fused to a modified Emerald fluorescence protein (CoV-2 S-mEm). Cell morphology was visualized using immunofluorescence microscopy. Action potential duration (APD) and cellular arrhythmias were measured by whole cell patch-clamp. Calcium handling was assessed using the Fluo-4 Ca2+ indicator. RESULTS: Transfection of hiPSC-CMs with CoV-2 S-mEm produced multinucleated giant cells (syncytia) displaying increased cellular capacitance (75±7 pF, n = 10 vs. 26±3 pF, n = 10; P<0.0001) consistent with increased cell size. The APD90 was prolonged significantly from 419±26 ms (n = 10) in untransfected hiPSC-CMs to 590±67 ms (n = 10; P<0.05) in CoV-2 S-mEm-transfected hiPSC-CMs. CoV-2 S-induced syncytia displayed delayed afterdepolarizations, erratic beating frequency, and calcium handling abnormalities including calcium sparks, large "tsunami"-like waves, and increased calcium transient amplitude. After furin protease inhibitor treatment or mutating the CoV-2 S furin cleavage site, cell-cell fusion was no longer evident and Ca2+ handling returned to normal. CONCLUSION: The SARS-CoV-2 spike protein can directly perturb both the cardiomyocyte's repolarization reserve and intracellular calcium handling that may confer the intrinsic, mechanistic substrate for the increased risk of SCD observed during this COVID-19 pandemic.

SARS-CoV-2-related bat virus behavior in human-relevant models sheds light on the origin of COVID-19.

Bat sarbecovirus BANAL-236 is highly related to SARS-CoV-2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. BANAL-236 replicates efficiently and pauci-symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS-CoV-2. BANAL-236 infection leads to protection against superinfection by a virulent strain. We find no evidence of antibodies recognizing bat sarbecoviruses in populations in close contact with bats in which the virus was identified, indicating that such spillover infections, if they occur, are rare. Six passages in humanized mice or in human intestinal cells, mimicking putative early spillover events, select adaptive mutations without appearance of a furin cleavage site and no change in virulence. Therefore, acquisition of a furin site in the spike protein is likely a pre-spillover event that did not occur upon replication of a SARS-CoV-2-like bat virus in humans or other animals. Other hypotheses regarding the origin of the SARS-CoV-2 should therefore be evaluated, including the presence of sarbecoviruses carrying a spike with a furin cleavage site in bats.

Why All the Fury over Furin?

Analysis of the SARS-CoV-2 sequence revealed a multibasic furin cleavage site at the S1/S2 boundary of the spike protein distinguishing this virus from SARS-CoV. Furin, the best-characterized member of the mammalian proprotein convertases, is an ubiquitously expressed single pass type 1 transmembrane protein. Cleavage of SARS-CoV-2 spike protein by furin promotes viral entry into lung cells. While furin knockout is embryonically lethal, its knockout in differentiated somatic cells is not, thus furin provides an exciting therapeutic target for viral pathogens including SARS-CoV-2 and bacterial infections. Several peptide-based and small-molecule inhibitors of furin have been recently reported, and select cocrystal structures have been solved, paving the way for further optimization and selection of clinical candidates. This perspective highlights furin structure, substrates, recent inhibitors, and crystal structures with emphasis on furin's role in SARS-CoV-2 infection, where the current data strongly suggest its inhibition as a promising therapeutic intervention for SARS-CoV-2.

SARS-CoV-2 S Mutations: A Lesson from the Viral World to Understand How Human Furin Works.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the etiological agent responsible for the worldwide pandemic and has now claimed millions of lives. The virus combines several unusual characteristics and an extraordinary ability to spread among humans. In particular, the dependence of the maturation of the envelope glycoprotein S from Furin enables the invasion and replication of the virus virtually within the entire body, since this cellular protease is ubiquitously expressed. Here, we analyzed the naturally occurring variation of the amino acids sequence around the cleavage site of S. We found that the virus grossly mutates preferentially at P positions, resulting in single residue replacements that associate with gain-of-function phenotypes in specific conditions. Interestingly, some combinations of amino acids are absent, despite the evidence supporting some cleavability of the respective synthetic surrogates. In any case, the polybasic signature is maintained and, as a consequence, Furin dependence is preserved. Thus, no escape variants to Furin are observed in the population. Overall, the SARS-CoV-2 system per se represents an outstanding example of the evolution of substrate-enzyme interaction, demonstrating a fast-tracked optimization of a protein stretch towards the Furin catalytic pocket. Ultimately, these data disclose important information for the development of drugs targeting Furin and Furin-dependent pathogens.

The Path to Therapeutic Furin Inhibitors: From Yeast Pheromones to SARS-CoV-2.

The spurious acquisition and optimization of a furin cleavage site in the SARS-CoV-2 spike protein is associated with increased viral transmission and disease, and has generated intense interest in the development and application of therapeutic furin inhibitors to thwart the COVID-19 pandemic. This review summarizes the seminal studies that informed current efforts to inhibit furin. These include the convergent efforts of endocrinologists, virologists, and yeast geneticists that, together, culminated in the discovery of furin. We describe the pioneering biochemical studies which led to the first furin inhibitors that were able to block the disease pathways which are broadly critical for pathogen virulence, tumor invasiveness, and atherosclerosis. We then summarize how these studies subsequently informed current strategies leading to the development of small-molecule furin inhibitors as potential therapies to combat SARS-CoV-2 and other diseases that rely on furin for their pathogenicity and progression.

State-of-the-art analytical methods of viral infections in human lung organoids.

Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors.

Loss of furin site enhances SARS-CoV-2 spike protein pseudovirus infection.

BACKGROUND: SARS-CoV-2 has a significant impact on healthcare systems all around the world. Due to its high pathogenicity, live SARS-CoV-2 must be handled under biosafety level 3 conditions. Pseudoviruses are useful virological tools because of their safety and versatility, but the low titer of these viruses remains a limitation for their more comprehensive applications. METHOD: Here, we constructed a Luc/eGFP based on a pseudotyped lentiviral HIV-1 system to transduce SARS-CoV-2 S glycoprotein to detect cell entry properties and cellular tropism. RESULTS: The furin cleavage site deletion of the S protein removed (SFko) can help SARS-CoV-2 S to be cleaved during viral packaging to improve infection efficiency. The furin cleavage site in SARS-CoV-2-S mediates membrane fusion and SFko leads to an increased level of S protein and limits S1/S2 cleavage to enhance pseudovirus infection in cells. Full-length S (SFL) pseudotyped with N, M, and E helper packaging can effectively help SFL infect cells. Finally, pseudotyped SFko particles were successfully used to detect neutralizing antibodies in RBD protein-immunized mouse serum. CONCLUSION: Overall, our study indicates a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudo-particles that may be suitable for the detection of neutralizing antibodies from COVID-19 patients.

Identification of an immunogenic epitope and protective antibody against the furin cleavage site of SARS-CoV-2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the global coronavirus disease 2019 (COVID-19) pandemic, contains a unique, four amino acid (aa) "PRRA" insertion in the spike (S) protein that creates a transmembrane protease serine 2 (TMPRSS2)/furin cleavage site and enhances viral infectivity. More research into immunogenic epitopes and protective antibodies against this SARS-CoV-2 furin cleavage site is needed. METHODS: Combining computational and experimental methods, we identified and characterized an immunogenic epitope overlapping the furin cleavage site that detects antibodies in COVID-19 patients and elicits strong antibody responses in immunized mice. We also identified a high-affinity monoclonal antibody from COVID-19 patient peripheral blood mononuclear cells; the antibody directly binds the furin cleavage site and protects against SARS-CoV-2 infection in a mouse model. FINDINGS: The presence of "PRRA" amino acids in the S protein of SARS-CoV-2 not only creates a furin cleavage site but also generates an immunogenic epitope that elicits an antibody response in COVID-19 patients. An antibody against this epitope protected against SARS-CoV-2 infection in mice. INTERPRETATION: The immunogenic epitope and protective antibody we have identified may augment our strategy in handling COVID-19 epidemic. FUNDING: The National Natural Science Foundation of China (82102371, 91542201, 81925025, 82073181, and 81802870), the Chinese Academy of Medical Sciences Initiative for Innovative Medicine (2021-I2M-1-047 and 2022-I2M-2-004), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2020-PT310-006, 2019XK310002, and 2018TX31001), the National Key Research and Development Project of China (2020YFC0841700), US National Institute of Health (NIH) funds grant AI158154, University of California Los Angeles (UCLA) AI and Charity Treks, and UCLA DGSOM BSCRC COVID-19 Award Program. H.Y. is supported by Natural Science Foundation of Jiangsu Province (BK20211554 andBE2022728).

Proprotein convertases regulate trafficking and maturation of key proteins within the secretory pathway.

Proprotein Convertases (PCs) are serine endoproteases that regulate the homeostasis of protein substrates in the cell. The PCs family counts 9 members-PC1/3, PC2, PC4, PACE4, PC5/6, PC7, Furin, SKI-1/S1P, and PCSK9. The first seven PCs are known as Basic Proprotein Convertases due to their propensity to cleave after polybasic clusters. SKI-1/S1P requires the additional presence of hydrophobic residues for processing, whereas PCSK9 is catalytically dead after autoactivation and exerts its functions using mechanisms alternative to direct cleavage. All PCs traffic through the canonical secretory pathway, reaching different compartments where the various substrates reside. Despite PCs members do not share the same subcellular localization, most of the cellular organelles count one or more Proprotein Convertases, including ER, Golgi stack, endosomes, secretory granules, and plasma membranes. The widespread expression of these enzymes at the systemic level speaks for their importance in the homeostasis of a large number of biological functions. Among others, PCs cleave precursors of hormones and growth factors and activate receptors and transcription factors. Notably, dysregulation of the enzymatic activity of Proprotein Convertases is associated to major human pathologies, such as cardiovascular diseases, cancer, diabetes, infections, inflammation, autoimmunity diseases, and Parkinson. In the current COVID-19 pandemic, Furin has further attracted the attention as a key player for conferring high pathogenicity to SARS-CoV-2. Here, we review the Proprotein Convertases family and their most important substrates along the secretory pathway. Knowledge about the complex functions of PCs is important to identify potential drug strategies targeting this class of enzymes.

Competitive cleavage of SARS-CoV-2 spike protein and epithelial sodium channel by plasmin as a potential mechanism for COVID-19 infection.

Cleavage of the furin site in SARS-CoV-2 spike (S) protein accounts for increased transmissibility of COVID-19 by promoting the entry of virus into host cells through specific angiotensin-converting enzyme 2 (ACE2) receptors. Plasmin, a key serine protease of fibrinolysis system, cleaves the furin site of γ subunit of human epithelial sodium channels (ENaCs). Sharing the plasmin cleavage by viral S and host ENaC proteins may competitively inter-regulate SARS-CoV-2 transmissibility and edema resolution via the ENaC pathway. To address this possibility, we analyzed single-cell RNA sequence (scRNA-seq) data sets and found that PLAU (encoding urokinase plasminogen activator), SCNN1G (γENaC), and ACE2 (SARS-CoV-2 receptor) were co-expressed in airway/alveolar epithelial cells. The expression levels of PLAU and FURIN were significantly higher compared with TMPRSS2 in healthy group. This difference was further amplified in both epithelial and immune cells in patients with moderate/severe COVID-19 and SARS-CoV-2 infected airway/alveolar epithelial cell lines. Of note, plasmin cleaved the S protein and facilitated the entry of pseudovirus in HEK293 cells. Conclusively, SARS-CoV-2 may expedite infusion by competing the fibrinolytic protease network with ENaC.

Reactive Centre Loop Mutagenesis of SerpinB3 to Target TMPRSS2 and Furin: Inhibition of SARS-CoV-2 Cell Entry and Replication.

The SARS-CoV-2 virus can utilize host cell proteases to facilitate cell entry, whereby the Spike (S) protein is cleaved at two specific sites to enable membrane fusion. Furin, transmembrane protease serine 2 (TMPRSS2), and cathepsin L (CatL) are the major proteases implicated, and are thus targets for anti-viral therapy. The human serpin (serine protease inhibitor) alpha-1 antitrypsin (A1AT) shows inhibitory activity for TMPRSS2, and has previously been found to suppress cell infection with SARS-CoV-2. Here, we have generated modified serpin inhibitors with increased specificity for these cellular proteases. Using SerpinB3 (SCCA-1), a cross-class inhibitor of CatL, as a scaffold, we have designed and produced reactive centre loop (RCL) variants to more specifically target both furin and TMPRSS2. Two further variants were generated by substituting the RCL P7-P1 with the spike protein S1/S2 cleavage site from either SARS-CoV-2 alpha or delta (P681R) sequences. Altered inhibitory specificity of purified recombinant proteins was verified in protease assays, with attenuated CatL inhibition and gain of furin or TMPRSS2 inhibition, as predicted, and modified serpins were shown to block S protein cleavage in vitro. Furthermore, the serpin variants were able to inhibit S-pseudoparticle entry into A549-ACE2-TMPRSS2 cells and suppress SARS-CoV-2 replication in Vero E6 cells expressing TMPRSS2. The construct designed to inhibit TMPRSS2 (B3-TMP) was most potent. It was more effective than A1AT for TMPRSS2 enzyme inhibition (with an eighteen-fold improvement in the second order inhibition rate constant) and for blocking SARS-CoV-2 viral replication. These findings advance the potential for serpin RCL mutagenesis to generate new inhibitors, and may lead to novel anti-viral biological molecules.

Essential role of TMPRSS2 in SARS-CoV-2 infection in murine airways.

In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn't an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant.

Neuropilin-1 Facilitates Pseudorabies Virus Replication and Viral Glycoprotein B Promotes Its Degradation in a Furin-Dependent Manner.

Pseudorabies virus (PRV), which is extremely infectious and can infect numerous mammals, has a risk of spillover into humans. Virus-host interactions determine viral entry and spreading. Here, we showed that neuropilin-1 (NRP1) significantly potentiates PRV infection. Mechanistically, NRP1 promoted PRV attachment and entry, and enhanced cell-to-cell fusion mediated by viral glycoprotein B (gB), gD, gH, and gL. Furthermore, through in vitro coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays, NRP1 was found to physically interact with gB, gD, and gH, and these interactions were C-end Rule (CendR) motif independent, in contrast to currently known viruses. Remarkably, we illustrated that the viral protein gB promotes NRP1 degradation via a lysosome-dependent pathway. We further demonstrate that gB promotes NRP1 degradation in a furin-cleavage-dependent manner. Interestingly, in this study, we generated gB furin cleavage site (FCS)-knockout PRV (Δfurin PRV) and evaluated its pathogenesis; in vivo, we found that Δfurin PRV virulence was significantly attenuated in mice. Together, our findings demonstrated that NRP1 is an important host factor for PRV and that NRP1 may be a potential target for antiviral intervention. IMPORTANCE Recent studies have shown accelerated PRV cross-species spillover and that PRV poses a potential threat to humans. PRV infection in humans always manifests as a high fever, tonic-clonic seizures, and encephalitis. Therefore, understanding the interaction between PRV and host factors may contribute to the development of new antiviral strategies against PRV. NRP1 has been demonstrated to be a receptor for several viruses that harbor CendR, including SARS-CoV-2. However, the relationships between NRP1 and PRV are poorly understood. Here, we found that NRP1 significantly potentiated PRV infection by promoting PRV attachment and enhanced cell-to-cell fusion. For the first time, we demonstrated that gB promotes NRP1 degradation via a lysosome-dependent pathway. Last, in vivo, Δfurin PRV virulence was significantly attenuated in mice. Therefore, NRP1 is an important host factor for PRV, and NRP1 may be a potential target for antiviral drug development.

SARS-CoV-2 requires acidic pH to infect cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein-catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.

Estimation of virus-mediated cell fusion rate of SARS-CoV-2.

Several viruses have the ability to form large multinucleated cells known as syncytia. Many properties of syncytia and the role they play in the evolution of a viral infection are not well understood. One basic question that has not yet been answered is how quickly syncytia form. We use a novel mathematical model of cell-cell fusion assays and apply it to experimental data from SARS-CoV-2 fusion assays to provide the first estimates of virus-mediated cell fusion rate. We find that for SARS-CoV2, the fusion rate is in the range of 6 × 10-4-12×10-4/h. We also use our model to compare fusion rates when the protease TMPRSS2 is overexpressed (2-4 times larger fusion rate), when the protease furin is removed (one third the original fusion rate), and when the spike protein is altered (1/10th the original fusion rate). The use of mathematical models allows us to provide additional quantitative information about syncytia formation.

Furin cleavage is required for swine acute diarrhea syndrome coronavirus spike protein-mediated cell - cell fusion.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) was reported in China in 2017 and is a causative agent of porcine enteric disease. Recent studies indicate that cells from various hosts are susceptible to SADS-CoV, suggesting the zoonotic potential of this virus. However, little is known about the mechanisms through which this virus enters cells. In this study, we investigated the role of furin in SADS-CoV spike (S)-mediated cell - cell fusion and entry. We found that the SADS-CoV S protein induced the fusion of various cells. Cell - cell fusion was inhibited by the proprotein convertase inhibitor dec-RVKR-cmk, and between cells transfected with mutant S proteins resistant to furin cleavage. These findings revealed that furin-induced cleavage of the SADS-CoV S protein is required for cell - cell fusion. Using mutagenesis analysis, we demonstrated that furin cleaves the SADS-CoV S protein near the S1/S2 cleavage site, 446RYVR449 and 543AVRR546. We used pseudotyped viruses to determine whether furin-induced S cleavage is also required for viral entry. Pseudotyped viruses expressing S proteins with a mutated furin cleavage site could be transduced into target cells, indicating that furin-induced cleavage is not required for pseudotyped virus entry. Our data indicate that S cleavage is critical for SADS-CoV S-mediated cell - cell fusion and suggest that furin might be a host target for SADS-CoV antivirals.

Intranasal administration of cold-adapted live-attenuated SARS-CoV-2 candidate vaccine confers protection against SARS-CoV-2.

With the COVID-19 pandemic globally, the ongoing threat of new challenges of mucosal infections was once again reminded human beings. Hence, access to the next-generation vaccine to elicit mucosal immunity is required to reduce virus shedding. SARS-CoV-2 retains a unique polybasic cleavage motif in its spike protein, recognized by the host furin protease. The proteolytic furin cleavage site at the junction of S1/S2 glycoprotein plays a key role in the pathogenesis of SARS-CoV-2. Here, we examined the protective immunity of a double-deleted PRRA/GTNGTKR motifs cold-adapted live-attenuated candidate vaccines as a called "KaraVac." using a hamster animal model of infected attenuated SARS-CoV-2. The KaraVac vaccinated hamsters were challenged against the wild-type (WT) SARS-CoV-2. No apparent bodyweight loss and histopathological lesions were observed in the hamsters. The establishment of sterilizing immunity was induced via stimulating a robust neutralizing antibody (NAb) response in a hamster model. Consequently, deletions in the spike sequence and inoculation into hamsters provide resistance to the subsequent challenge with WT SARS-CoV-2. We have suggested that deletion of the furin cleavage site and GTNGTKR motifs in the spike sequence attenuates the virus from the parental strain and can be used as a potent immunogen.

Rapid quantitative monitoring of SARS-CoV-2 spike protein-mediated syncytia formation using split NanoLuc.

SARS-CoV-2 infection causes syncytial pneumocyte in patients and this has been considered as a defining feature of severe COVID-19 cases. Traditional methods of syncytia quantification require the morphology characterization of fused cells either with light microscope or fluorescent microscope, which is time-consuming and not accurate. Here we developed a rapid and sensitive coculture system measuring spike-induced syncytia by using NanoLuc complementation system. We found the formation of syncytia occurred rapidly after ACE2-expressing cells exposure to spike protein. In addition, we found furin cleavage as well as the cell surface protease TMPRSS2 enhanced syncytia formation. Finally, we showed that this coculture system can be used to test the ability of different compound to inhibit syncytia formation, thus providing a useful tool to screen anti-syncytial drugs.