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2.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-320953

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pneumonia in humans. The virus is enzootic in dromedary camels across the Middle East and Africa. It is acquired through animal contact and undergoes limited onward transmission particularly in hospitals. Because of this initial potential for human-to-human transmission, we monitor the virus for phenotypic changes related to its pandemic potential. Potential phenotypic changes have been suspected since the year 2015, when a novel recombinant clade (MERS-CoV lineage 5) caused large nosocomial outbreaks in Saudi Arabia and South Korea that effectively swept other, hitherto co-circulating viral lineages. To this day, lineage 5 remains the only circulating MERS-CoV lineage on the Arabian Peninsula. In spite of available sequence data, no studies of viral phenotype have been carried out to date. Here we performed a comprehensive in-vitro and ex-vivo comparison of live virus isolates taken in Saudi Arabia immediately before and after the shift toward lineage 5. We characterized seven isolates representing the recombination-parental lineage 3, eight isolates representing parental lineage 4, as well as eight isolates representing lineage 5. Replication of lineage 5 viruses is significantly increased over isolates from parental lineages in cell culture and ex-vivo lung models. Transcriptional profiling by real-time RT-PCR shows that several key immune genes (IFNb1, CCL5, IFNL1) are significantly less induced in lung cells infected with lineage 5 MERS-CoV compared to parental strains. In IFN receptor knock out cells, as well as under chemical inhibition of IFN signalling, the differences in replication level between lineage 5 and parental lineages are reduced, suggesting that phenotypic differences may be determined by IFN antagonism. Concordantly, lineage 5 shows increased resilience against interferon (IFN) pre-treatment of Calu-3 cells and maintains a 10-fold higher replication level under low and high concentrations of IFN. Reduced immune activation combined with enhanced virus replication and IFN resilience may explain the dominance of lineage 5 on the Arabian Peninsula. This phenotypic difference is highly relevant with regard to pandemic potential, and has remained undiscovered in spite of viral sequence surveillance.

3.
Mol Inform ; : e2100231, 2022 Jan 23.
Article in English | MEDLINE | ID: covidwho-1648973

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) as global pandemic disease has been adversely affecting public health and social life with considerable loss of human life worldwide. Therefore, there is an urgent need for developing novel therapeutics to combat COVID-19. The causative agent of COVID-19 is SARS-CoV-2 which targets human angiotensin converting enzyme 2 (ACE2) as cellular receptor via its spike (S) protein. In this context, interfering with the binding of SARS-CoV-2 S protein to target molecules could provide a promising strategy to find novel therapeutic agents against SARS-CoV-2. The purpose of the current study was to identify potential peptidomimetics against S protein with a combination of structure-based virtual screening methods and in vitro assays. METHODS: The candidates were inspected in terms of ADME properties, drug-likeness, as well as toxicity profiles. Additionally, molecular docking and dynamics simulations were performed to predict binding of the studied ligands to spike protein. RESULTS: Biological evaluation of the compounds revealed that PM2 molecule exhibits some antiviral activity. CONCLUSION: In summary, this study highlights the importance of combining in silico and in vitro techniques in order to identify antiviral compound to tackle COVID-19 and presents a new scaffold that may be structurally optimized for improved antiviral activity.

4.
Cell Rep ; 35(3): 109017, 2021 04 20.
Article in English | MEDLINE | ID: covidwho-1163486

ABSTRACT

Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to farmed mink has been observed in Europe and the US. In the infected animals, viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink are mostly compatible with efficient entry into human cells and its inhibition by soluble angiotensin-converting enzyme 2 (ACE2). In contrast, mutation Y453F reduces neutralization by an antibody with emergency use authorization for coronavirus disease 2019 (COVID-19) therapy and sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 , Mink , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , A549 Cells , Angiotensin-Converting Enzyme 2/immunology , Animals , COVID-19/genetics , COVID-19/immunology , Chlorocebus aethiops , Cricetinae , Humans , Mink/immunology , Mink/virology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
5.
J Virol ; 95(9)2021 04 12.
Article in English | MEDLINE | ID: covidwho-1093846

ABSTRACT

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S (SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to that of SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector cell-target cell fusion when ACE2 or TMPRSS2 was limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target cell-effector cell fusion was unaltered compared to that of wild-type (wt) SARS2-S, but syncytia remained smaller. Mutation of the S2 cleavage (S2') site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor bromhexine, unlike the inhibitor camostat, was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S. Paradoxically, bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite ambroxol exhibited inhibitory activity under some conditions. On Calu-3 lung cells, ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend toward weak inhibition of authentic SARS-CoV-2.IMPORTANCE Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2' cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently being tested in clinical trials against coronavirus disease 2019. Our results indicate that bromhexine enhances fusion under some conditions. We therefore caution against the use of bromhexine in high dosages until its effects on SARS-CoV-2 spike activation are better understood. The related compound ambroxol, which similarly to bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for ambroxol.


Subject(s)
COVID-19/metabolism , SARS Virus/metabolism , SARS-CoV-2/metabolism , Severe Acute Respiratory Syndrome/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Ambroxol/pharmacology , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Bromhexine/pharmacology , COVID-19/genetics , Cell Line , Humans , Mutation, Missense , Proteolysis/drug effects , SARS Virus/genetics , SARS-CoV-2/genetics , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Severe Acute Respiratory Syndrome/genetics , Spike Glycoprotein, Coronavirus/genetics
6.
Cell Mol Immunol ; 18(4): 936-944, 2021 04.
Article in English | MEDLINE | ID: covidwho-899921

ABSTRACT

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells via surface-expressed angiotensin-converting enzyme 2 (ACE2). We used a surrogate virus neutralization test (sVNT) and SARS-CoV-2 S protein-pseudotyped vesicular stomatitis virus (VSV) vector-based neutralization assay (pVNT) to assess the degree to which serum antibodies from coronavirus disease 2019 (COVID-19) convalescent patients interfere with the binding of SARS-CoV-2 S to ACE2. Both tests revealed neutralizing anti-SARS-CoV-2 S antibodies in the sera of ~90% of mildly and 100% of severely affected COVID-19 convalescent patients. Importantly, sVNT and pVNT results correlated strongly with each other and to the levels of anti-SARS-CoV-2 S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies correlated with the duration and severity of clinical symptoms but not with patient age. Compared to pVNT, sVNT is less sophisticated and does not require any biosafety labs. Since this assay is also much faster and cheaper, sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/blood , Cell Line , Convalescence , Female , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Male , Middle Aged , Neutralization Tests/methods
7.
Nat Microbiol ; 5(11): 1330-1339, 2020 11.
Article in English | MEDLINE | ID: covidwho-676586

ABSTRACT

Zoonotic coronaviruses (CoVs) are substantial threats to global health, as exemplified by the emergence of two severe acute respiratory syndrome CoVs (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome CoV (MERS-CoV) within two decades1-3. Host immune responses to CoVs are complex and regulated in part through antiviral interferons. However, interferon-stimulated gene products that inhibit CoVs are not well characterized4. Here, we show that lymphocyte antigen 6 complex, locus E (LY6E) potently restricts infection by multiple CoVs, including SARS-CoV, SARS-CoV-2 and MERS-CoV. Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in immune cells were highly susceptible to a murine CoV-mouse hepatitis virus. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic immune cells, higher splenic viral burden and reduction in global antiviral gene pathways. Accordingly, we found that constitutive Ly6e directly protects primary B cells from murine CoV infection. Our results show that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo-knowledge that could help inform strategies to combat infection by emerging CoVs.


Subject(s)
Antigens, Surface/metabolism , Coronavirus Infections/immunology , Coronavirus Infections/virology , Coronavirus/physiology , GPI-Linked Proteins/metabolism , Angiotensin-Converting Enzyme 2 , Animals , Antigens, Surface/genetics , Antigens, Surface/immunology , Betacoronavirus/immunology , Betacoronavirus/physiology , COVID-19 , Coronavirus/immunology , Female , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/physiology , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , SARS Virus/immunology , SARS Virus/physiology , SARS-CoV-2 , Virus Internalization
8.
Nature ; 585(7826): 588-590, 2020 09.
Article in English | MEDLINE | ID: covidwho-663955

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been associated with more than 780,000 deaths worldwide (as of 20 August 2020). To develop antiviral interventions quickly, drugs used for the treatment of unrelated diseases are currently being repurposed to treat COVID-19. Chloroquine is an anti-malaria drug that is used for the treatment of COVID-19 as it inhibits the spread of SARS-CoV-2 in the African green monkey kidney-derived cell line Vero1-3. Here we show that engineered expression of TMPRSS2, a cellular protease that activates SARS-CoV-2 for entry into lung cells4, renders SARS-CoV-2 infection of Vero cells insensitive to chloroquine. Moreover, we report that chloroquine does not block infection with SARS-CoV-2 in the TMPRSS2-expressing human lung cell line Calu-3. These results indicate that chloroquine targets a pathway for viral activation that is not active in lung cells and is unlikely to protect against the spread of SARS-CoV-2 in and between patients.


Subject(s)
Chloroquine/pharmacology , Chloroquine/therapeutic use , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Lung/cytology , Lung/drug effects , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Animals , Betacoronavirus/drug effects , COVID-19 , Cell Line , Chlorocebus aethiops , Humans , In Vitro Techniques , Lung/virology , Pandemics , SARS-CoV-2 , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Treatment Failure , Vero Cells , Virus Internalization
9.
Emerg Microbes Infect ; 9(1): 155-168, 2020.
Article in English | MEDLINE | ID: covidwho-326251

ABSTRACT

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) causes a severe respiratory disease in humans. The MERS-CoV spike (S) glycoprotein mediates viral entry into target cells. For this, MERS-CoV S engages the host cell protein dipeptidyl peptidase 4 (DPP4, CD26) and the interface between MERS-CoV S and DPP4 has been resolved on the atomic level. Here, we asked whether naturally-occurring polymorphisms in DPP4, that alter amino acid residues required for MERS-CoV S binding, influence cellular entry of MERS-CoV. By screening of public databases, we identified fourteen such polymorphisms. Introduction of the respective mutations into DPP4 revealed that all except one (Δ346-348) were compatible with robust DPP4 expression. Four polymorphisms (K267E, K267N, A291P and Δ346-348) strongly reduced binding of MERS-CoV S to DPP4 and S protein-driven host cell entry, as determined using soluble S protein and S protein bearing rhabdoviral vectors, respectively. Two polymorphisms (K267E and A291P) were analyzed in the context of authentic MERS-CoV and were found to attenuate viral replication. Collectively, we identified naturally-occurring polymorphisms in DPP4 that negatively impact cellular entry of MERS-CoV and might thus modulate MERS development in infected patients.


Subject(s)
Coronavirus Infections/genetics , Dipeptidyl Peptidase 4/genetics , Middle East Respiratory Syndrome Coronavirus/physiology , Virus Internalization , Coronavirus Infections/immunology , Coronavirus Infections/virology , Dipeptidyl Peptidase 4/immunology , Host-Pathogen Interactions , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Polymorphism, Genetic
10.
Mol Cell ; 78(4): 779-784.e5, 2020 05 21.
Article in English | MEDLINE | ID: covidwho-88548

ABSTRACT

The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.


Subject(s)
Betacoronavirus/chemistry , Coronavirus Infections/virology , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus/chemistry , Animals , Betacoronavirus/physiology , COVID-19 , Cell Line , Chlorocebus aethiops , Furin/chemistry , Furin/genetics , Furin/metabolism , Humans , Lung/metabolism , Lung/virology , Pandemics , SARS-CoV-2 , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Attachment
11.
Cell ; 181(2): 271-280.e8, 2020 04 16.
Article in English | MEDLINE | ID: covidwho-4561

ABSTRACT

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/drug therapy , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/drug therapy , Protease Inhibitors/pharmacology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects , Ammonium Chloride/pharmacology , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/chemistry , Betacoronavirus/genetics , COVID-19 , Cell Line , Coronavirus/chemistry , Coronavirus/genetics , Coronavirus/physiology , Coronavirus Infections/immunology , Coronavirus Infections/therapy , Drug Development , Esters , Gabexate/analogs & derivatives , Gabexate/pharmacology , Guanidines , Humans , Immunization, Passive , Leucine/analogs & derivatives , Leucine/pharmacology , Pandemics , Peptidyl-Dipeptidase A/chemistry , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS Virus/physiology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vesiculovirus/genetics
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