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1.
Nature ; 599(7883): 114-119, 2021 11.
Article in English | MEDLINE | ID: covidwho-2114880

ABSTRACT

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.


Subject(s)
Immune Evasion , SARS-CoV-2/growth & development , SARS-CoV-2/immunology , Virus Replication/immunology , Antibodies, Neutralizing/immunology , COVID-19 Vaccines/immunology , Cell Fusion , Cell Line , Female , Health Personnel , Humans , India , Kinetics , Male , Spike Glycoprotein, Coronavirus/metabolism , Vaccination
2.
Biomed Pharmacother ; 155: 113766, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2104426

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus that has caused over 6 million fatalities. SARS-CoV-2 variants with spike mutations are frequently endowed with a strong capability to escape vaccine-elicited protection. Due to this characteristic, a broad-spectrum inhibitor against SARS-CoV-2 infection is urgently demanded. Ganoderma microsporum immunomodulatory protein (GMI) was previously reported to alleviate infection of SARS-CoV-2 through ACE2 downregulation whereas the impact of GMI on virus itself was less understood. Our study aims to determine the effects of GMI on SARS-CoV-2 pseudovirus and the more detailed mechanisms of GMI inhibition against SARS-CoV-2 pseudovirus infection. METHODS: ACE2-overexpressing HEK293T cells (HEK293T/ACE2) and SARS-CoV-2 pseudoviruses carrying spike variants were used to study the effects of GMI in vitro. Infectivity was evaluated by fluorescence microscopy and flow cytometry. Fusion rate mediated by SARS-CoV-2 spike protein was examined with split fluorescent protein /luciferase systems. The interactions of GMI with SARS-CoV-2 pseudovirus and ACE2 were investigated by immunoprecipitation and immunoblotting. RESULTS: GMI broadly blocked SARS-CoV-2 infection in various cell lines. GMI effectively inhibited the infection of pseudotyped viruses carrying different emerged spike variants, including Delta and Omicron strains, on HEK293T/hACE2 cells. In cell-free virus infection, GMI dominantly impeded the binding of spike-bearing pseudotyped viruses to ACE2-expressing cells. In cell-to-cell fusion model, GMI could efficiently inhibit spike-mediated syncytium without the requirement of ACE2 downregulation. CONCLUSIONS: GMI, an FDA-approved dietary ingredient, acts as a multifunctional broad-spectrum antiviral against SARS-CoV-2 and could become a promising candidate for preventing or treating SARS-CoV-2 associated diseases.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Angiotensin-Converting Enzyme 2 , Antiviral Agents/pharmacology , Virus Attachment , COVID-19/drug therapy , Receptors, Virus/metabolism , Cell Fusion , HEK293 Cells , Protein Binding
3.
Viruses ; 14(10)2022 09 21.
Article in English | MEDLINE | ID: covidwho-2099840

ABSTRACT

SARS-CoV-2 cell-cell fusion and syncytiation is an emerging pathomechanism in COVID-19, but the precise factors contributing to the process remain ill-defined. In this study, we show that metalloproteases promote SARS-CoV-2 spike protein-induced syncytiation in the absence of established serine proteases using in vitro cell-cell fusion assays. We also show that metalloproteases promote S2'-activation of the SARS-CoV-2 spike protein, and that metalloprotease inhibition significantly reduces the syncytiation of SARS-CoV-2 variants of concern. In the presence of serine proteases, however, metalloprotease inhibition does not reduce spike protein-induced syncytiation and a combination of metalloprotease and serine protease inhibition is necessitated. Moreover, we show that the spike protein induces metalloprotease-dependent ectodomain shedding of the ACE2 receptor and that ACE2 shedding contributes to spike protein-induced syncytiation. These observations suggest a benefit to the incorporation of pharmacological inhibitors of metalloproteases into treatment strategies for patients with COVID-19.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Cell Fusion , Serine Endopeptidases/metabolism , Metalloproteases , Serine Proteases
5.
J Biol Chem ; 298(11): 102511, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2031421

ABSTRACT

Revealing the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and cell-to-cell spread might provide insights for understanding the underlying mechanisms of viral pathogenesis, tropism, and virulence. The signaling pathways involved in SARS-CoV-2 entry and viral spike-mediated cell-to-cell fusion remain elusive. In the current study, we found that macropinocytosis inhibitors significantly suppressed SARS-CoV-2 infection at both the entry and viral spike-mediated cell-to-cell fusion steps. We demonstrated that SARS-CoV-2 entry required the small GTPase Rac1 and its effector kinase p21-activated kinase 1 by dominant-negative and RNAi assays in human embryonic kidney 293T-angiotensin-converting enzyme 2 cells and that the serine protease transmembrane serine protease 2 reversed the decrease in SARS-CoV-2 entry caused by the macropinocytosis inhibitors. Moreover, in the cell-to-cell fusion assay, we confirmed that macropinocytosis inhibitors significantly decreased viral spike-mediated cell-to-cell fusion. Overall, we provided evidence that SARS-CoV-2 utilizes a macropinocytosis pathway to enter target cells and to efficiently promote viral spike-mediated cell-to-cell fusion.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Spike Glycoprotein, Coronavirus/metabolism , Cell Fusion , Virus Internalization , Serine Proteases
6.
Virology ; 575: 91-100, 2022 10.
Article in English | MEDLINE | ID: covidwho-2008177

ABSTRACT

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.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Cell Fusion , Furin/metabolism , Humans , RNA, Viral , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
7.
Methods Enzymol ; 675: 351-381, 2022.
Article in English | MEDLINE | ID: covidwho-2007357

ABSTRACT

After more than two years, COVID-19 still represents a global health burden of unprecedented extent and assessing the degree of immunity of individuals against SARS-CoV-2 remains a challenge. Virus neutralization assays represent the gold standard for assessing antibody-mediated protection against SARS-CoV-2 in sera from recovered and/or vaccinated individuals. Neutralizing antibodies block the interaction of viral spike protein with human angiotensin-converting enzyme 2 (ACE2) receptor in vitro and prevent viral entry into host cells. Classical viral neutralization assays using full replication-competent viruses are restricted to specific biosafety level 3-certified laboratories, limiting their utility for routine and large-scale applications. We developed therefore a cell-fusion-based assay building on the interaction between viral spike and ACE2 receptor expressed on two different cell lines, substantially reducing biosafety risks associated with classical viral neutralization assays. This chapter describes this simple, sensitive, safe and cost-effective approach for rapid and high-throughput evaluation of SARS-CoV-2 neutralizing antibodies relying on high-affinity NanoLuc® luciferase complementation technology (HiBiT). When applied to a variety of standards and patient samples, this method yields highly reproducible results in 96-well, as well as in 384-well format. The use of novel NanoLuc® substrates with increased signal stability like Nano-Glo® Endurazine™ furthermore allows for high flexibility in assay set-up and full automatization of all reading processes. Lastly, the assay is suitable to evaluate the neutralizing capacity of sera against the existing spike variants, and potentially variants that will emerge in the future.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Antibodies, Neutralizing , Antibodies, Viral , Cell Fusion , Humans , Luciferases , Neutralization Tests/methods , Peptidyl-Dipeptidase A/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism
8.
Emerg Microbes Infect ; 11(1): 2176-2183, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1984971

ABSTRACT

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.


Subject(s)
Furin , Spike Glycoprotein, Coronavirus , Alphacoronavirus , Animals , Antiviral Agents , Cell Fusion , Furin/metabolism , Proprotein Convertases , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Swine , Virus Internalization
9.
Int J Mol Sci ; 23(13)2022 Jul 01.
Article in English | MEDLINE | ID: covidwho-1917524

ABSTRACT

Virus-cell fusion is the key step for viral infection in host cells. Studies on virus binding and fusion with host cells are important for understanding the virus-host interaction and viral pathogenesis for the discovery of antiviral drugs. In this review, we focus on the virus-cell fusions induced by the two major pandemic viruses, including the influenza virus and SARS-CoV-2. We further compare the cell fusions induced by the influenza virus and SARS-CoV-2, especially the pH-dependent fusion of the influenza virus and the fusion of SARS-CoV-2 in the type-II transmembrane serine protease 2 negative (TMPRSS2-) cells with syncytia formation. Finally, we present the development of drugs used against SARA-CoV-2 and the influenza virus through the discovery of anti-fusion drugs and the prevention of pandemic respiratory viruses.


Subject(s)
COVID-19 , Orthomyxoviridae , Cell Fusion , Humans , Orthomyxoviridae/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
10.
Viruses ; 14(6)2022 06 15.
Article in English | MEDLINE | ID: covidwho-1911637

ABSTRACT

Screening of a protein kinase inhibitor library identified SB431542, targeting activin receptor-like kinase 5 (ALK5), as a compound interfering with SARS-CoV-2 replication. Since ALK5 is implicated in transforming growth factor ß (TGF-ß) signaling and regulation of the cellular endoprotease furin, we pursued this research to clarify the role of this protein kinase for SARS-CoV-2 infection. We show that TGF-ß1 induces the expression of furin in a broad spectrum of cells including Huh-7 and Calu-3 that are permissive for SARS-CoV-2. The inhibition of ALK5 by incubation with SB431542 revealed a dose-dependent downregulation of both basal and TGF-ß1 induced furin expression. Furthermore, we demonstrate that the ALK5 inhibitors SB431542 and Vactosertib negatively affect the proteolytic processing of the SARS-CoV-2 Spike protein and significantly reduce spike-mediated cell-cell fusion. This correlated with an inhibitory effect of ALK5 inhibition on the production of infectious SARS-CoV-2. Altogether, our study shows that interference with ALK5 signaling attenuates SARS-CoV-2 infectivity and cell-cell spread via downregulation of furin which is most pronounced upon TGF-ß stimulation. Since a TGF-ß dominated cytokine storm is a hallmark of severe COVID-19, ALK5 inhibitors undergoing clinical trials might represent a potential therapy option for COVID-19.


Subject(s)
COVID-19 , Transforming Growth Factor beta1 , Cell Fusion , Furin , Humans , Protein Serine-Threonine Kinases , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1/metabolism
11.
EMBO Rep ; 23(6): e54305, 2022 06 07.
Article in English | MEDLINE | ID: covidwho-1836040

ABSTRACT

The severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19, but host cell factors contributing to COVID-19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS-CoV-2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID-19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS-CoV-2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease-targeted inhibitors severely impair lung cell infection by the SARS-CoV-2 variants of concern alpha, beta, delta, and omicron and also reduce SARS-CoV-2 infection of primary human lung cells in a TMPRSS2 protease-independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.


Subject(s)
COVID-19 , SARS-CoV-2 , ADAM10 Protein/genetics , ADAM17 Protein , Amyloid Precursor Protein Secretases/genetics , Angiotensin-Converting Enzyme 2 , Cell Fusion , Humans , Lung , Membrane Proteins/genetics , Membrane Proteins/metabolism , Metalloproteases , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
12.
Sci Signal ; 15(729): eabg8744, 2022 04 12.
Article in English | MEDLINE | ID: covidwho-1784765

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the unprecedented coronavirus disease 2019 (COVID-19) pandemic. Critical cases of COVID-19 are characterized by the production of excessive amounts of cytokines and extensive lung damage, which is partially caused by the fusion of SARS-CoV-2-infected pneumocytes. Here, we found that cell fusion caused by the SARS-CoV-2 spike (S) protein induced a type I interferon (IFN) response. This function of the S protein required its cleavage by proteases at the S1/S2 and the S2' sites. We further showed that cell fusion damaged nuclei and resulted in the formation of micronuclei that were sensed by the cytosolic DNA sensor cGAS and led to the activation of its downstream effector STING. Phosphorylation of the transcriptional regulator IRF3 and the expression of IFNB, which encodes a type I IFN, were abrogated in cGAS-deficient fused cells. Moreover, infection with VSV-SARS-CoV-2 also induced cell fusion, DNA damage, and cGAS-STING-dependent expression of IFNB. Together, these results uncover a pathway underlying the IFN response to SARS-CoV-2 infection. Our data suggest a mechanism by which fused pneumocytes in the lungs of patients with COVID-19 may enhance the production of IFNs and other cytokines, thus exacerbating disease severity.


Subject(s)
COVID-19 , Interferon Type I , COVID-19/genetics , Cell Fusion , Cytokines , Humans , Interferon Type I/genetics , Membrane Proteins/metabolism , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
13.
Emerg Microbes Infect ; 11(1): 1024-1036, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1740712

ABSTRACT

SARS-CoV-2 has caused the COVID-19 pandemic. B.1.617 variants (including Kappa and Delta) have been transmitted rapidly in India. The transmissibility, pathogenicity, and neutralization characteristics of these variants have received considerable interest. In this study, 22 pseudotyped viruses were constructed for B.1.617 variants and their corresponding single amino acid mutations. B.1.617 variants did not exhibit significant enhanced infectivity in human cells, but mutations T478K and E484Q in the receptor binding domain led to enhanced infectivity in mouse ACE2-overexpressing cells. Furin activities were slightly increased against B.1.617 variants and cell-cell fusion after infection of B.1.617 variants were enhanced. Furthermore, B.1.617 variants escaped neutralization by several mAbs, mainly because of mutations L452R, T478K, and E484Q in the receptor binding domain. The neutralization activities of sera from convalescent patients, inactivated vaccine-immunized volunteers, adenovirus vaccine-immunized volunteers, and SARS-CoV-2 immunized animals against pseudotyped B.1.617 variants were reduced by approximately twofold, compared with the D614G variant.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antibodies, Neutralizing , Cell Fusion , Humans , Mice , Mutation , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus , Viral Tropism
14.
J Virol ; 95(24): e0136821, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1691427

ABSTRACT

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.


Subject(s)
COVID-19/virology , Myocytes, Cardiac/virology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Animals , Cathepsin B/metabolism , Cell Fusion , Chlorocebus aethiops , Embryonic Stem Cells/metabolism , Exocytosis , Humans , Induced Pluripotent Stem Cells/metabolism , Microscopy, Confocal , Serine Endopeptidases/metabolism , Vero Cells , Viral Proteins/metabolism , Virus Internalization , Virus Replication
15.
Mil Med Res ; 8(1): 68, 2021 12 20.
Article in English | MEDLINE | ID: covidwho-1639349
16.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Article in English | MEDLINE | ID: covidwho-1599544

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.


Subject(s)
COVID-19/immunology , COVID-19/transmission , SARS-CoV-2/immunology , Virus Internalization , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral , COVID-19/therapy , Cell Fusion , Chlorocebus aethiops , HEK293 Cells , Humans , Immunization, Passive , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
17.
Viruses ; 13(12)2021 12 11.
Article in English | MEDLINE | ID: covidwho-1572661

ABSTRACT

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. Here, we compared B.1.617 variants for neutralization resistance by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. B.1.617.1, B.1.617.2, and AY.1 pseudoviruses showed a modest 1.5- to 4.4-fold reduction in neutralization by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for C.37, P.1, R.1, and B.1.526 pseudoviruses, but 7- and 16-fold reductions for vaccine-elicited and convalescent sera, respectively, were seen for B.1.351 pseudoviruses. Among twenty-three therapeutic antibodies tested, four antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses and six antibodies showed either complete or partial loss of neutralization against B.1.617.1 and AY.1 pseudoviruses. Our results indicate that the current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants. Finally, the P681R substitution confers efficient cleavage of B.1.617 variants' spike proteins and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.


Subject(s)
Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Antigenic Variation , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/immunology , Cell Fusion , Furin/metabolism , Humans , Protein Binding , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
18.
Emerg Microbes Infect ; 11(1): 182-194, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1550502

ABSTRACT

The ubiquitously-expressed proteolytic enzyme furin is closely related to the pathogenesis of SARS-CoV-2 and therefore represents a key target for antiviral therapy. Based on bioinformatic analysis and pseudovirus tests, we discovered a second functional furin site located in the spike protein. Furin still increased the infectivity of mutated SARS-CoV-2 pseudovirus in 293T-ACE2 cells when the canonical polybasic cleavage site (682-686) was deleted. However, K814A mutation eliminated the enhancing effect of furin on virus infection. Furin inhibitor prevented infection by 682-686-deleted SARS-CoV-2 in 293T-ACE2-furin cells, but not the K814A mutant. K814A mutation did not affect the activity of TMPRSS2 and cathepsin L but did impact the cleavage of S2 into S2' and cell-cell fusion. Additionally, we showed that this functional furin site exists in RaTG13 from bat and PCoV-GD/GX from pangolin. Therefore, we discovered a new functional furin site that is pivotal in promoting SARS-CoV-2 infection.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Cathepsin L/metabolism , Furin/metabolism , SARS-CoV-2/genetics , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/genetics , Animals , Cathepsin L/genetics , Cell Fusion , Chiroptera , Furin/genetics , Gene Expression , HEK293 Cells , Humans , Mice , Mice, Transgenic , Mutation , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2/growth & development , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/metabolism
19.
Nat Commun ; 12(1): 4502, 2021 07 23.
Article in English | MEDLINE | ID: covidwho-1550282

ABSTRACT

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Membrane/metabolism , Cell Nucleus/metabolism , Membrane Glycoproteins/metabolism , Transcription Factors/metabolism , Transcription, Genetic/genetics , Viral Envelope Proteins/metabolism , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Biological Transport , Cell Fusion , Cell Line , Cell Line, Tumor , Cells, Cultured , Giant Cells/metabolism , HEK293 Cells , Humans , Membrane Glycoproteins/genetics , Mice , RNA-Seq/methods , Signal Transduction/genetics , Transcription Factors/genetics , Viral Envelope Proteins/genetics
20.
Proc Natl Acad Sci U S A ; 118(47)2021 11 23.
Article in English | MEDLINE | ID: covidwho-1500833

ABSTRACT

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation in cells. Here, we show that O-glycosylation near the furin cleavage site is mediated by members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible alpha and delta variants abrogate O-glycosylation, increase furin cleavage, and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible alpha and delta variants.


Subject(s)
SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , Cell Fusion , Cell Line , Furin/metabolism , Giant Cells , Glycosylation , Humans , N-Acetylgalactosaminyltransferases/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
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