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1.
J Med Virol ; 94(5): 1815-1820, 2022 05.
Article in English | MEDLINE | ID: covidwho-1777571

ABSTRACT

The polybasic furin cleavage site insertion with four amino acid motifs (PRRA) in spike protein's S1/S2 junction site is important in determining viral infectivity, transmission, and host range. However, there is no review so far explaining the effect of the furin cleavage site of the spike protein on SARS-CoV-2 replication and pathogenesis in the host and immune responses and vaccination. Therefore, here we specifically focused on genomic evolution and properties of the cleavage site of spike protein in the context of SARS-CoV-2 followed by its effect on viral entry, replication, and pathogenesis. We also explored whether the spike protein furin cleavage site affected the host immune responses and SARS-CoV-2 vaccination. This review will help to provide novel insights into the effects of polybasic furin cleavage site on the current COVID-19 pandemic.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/prevention & control , COVID-19 Vaccines , Furin/metabolism , Humans , Immunity , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vaccination
2.
J Virol ; 96(8): e0012822, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1765079

ABSTRACT

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2' as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2' cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.


Subject(s)
COVID-19 , Furin , SARS-CoV-2 , Serine Endopeptidases , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/pathology , COVID-19/virology , Furin/metabolism , HeLa Cells , Humans , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
3.
Int J Biol Sci ; 18(6): 2362-2371, 2022.
Article in English | MEDLINE | ID: covidwho-1753909

ABSTRACT

CTSL is expressed by cancerous tissues and encodes a lysosomal cysteine proteinase that regulates cancer progression and SARS-CoV-2 entry. Therefore, it is critical to predict the susceptibility of cancer patients for SARS-CoV-2 and evaluate the correlation between disease outcomes and the expression of CTSL in malignant cancer tissues. In the current study, we analyzed CTSL expression, mutation rate, survival and COVID-19 disease outcomes in cancer and normal tissues, using online databases. We also performed immunohistochemistry (IHC) to test CTSL expression and western blot to monitor its regulation by cordycepin (CD), and N6, N6-dimethyladenosine (m6 2A), respectively. We found that CTSL is conserved across different species, and highly expressed in both normal and cancer tissues from human, as compared to ACE2 or other proteinases/proteases. Additionally, the expression of CTSL protein was the highest in the lung tissue. We show that the mRNA expression of CTSL is 66.4-fold higher in normal lungs and 54.8-fold higher in cancer tissues, as compared to ACE2 mRNA expression in the respective tissues. Compared to other proteases/proteinases/convertases such as TMPRSS2 and FURIN, the expression of CTSL was higher in both normal lungs and lung cancer samples. All these data indicate that CTSL might play an important role in COVID-19 pathogenesis in normal and cancer tissues of the lungs. Additionally, the CTSL-002 isoform containing both the inhibitor_I29 and Peptidase_C1 domains was highly prevalent in all cancers, suggesting its potential role in tumor progression and SARS-CoV-2 entry in multiple types of cancers. Further analysis of the expression of CTSL mutant showed a correlation with FURIN and TMPRSS2, suggesting a potential role of CTSL mutations in modulating SARS-CoV-2 entry in cancers. Moreover, high expression of CTSL significantly correlated with a short overall survival (OS) in lung cancer and glioma. Thus, CTSL might play a major role in the susceptibility of lung cancer and glioma patients to SARS-CoV-2 uptake and COVID-19 severity. Furthermore, CD or m6 2A inhibited CTSL expression in the cancer cell lines A549, MDA-MB-231, and/or PC3 in a dose dependent manner. In conclusion, we show that CTSL is highly expressed in normal tissues and increased in most cancers, and CD or m6 2A could inhibit its expression, suggesting the therapeutic potential of targeting CTSL for cancer and COVID-19 treatment.


Subject(s)
COVID-19 , Glioma , Lung Neoplasms , Angiotensin-Converting Enzyme 2 , COVID-19/drug therapy , COVID-19/genetics , Cathepsin L , Furin/genetics , Furin/metabolism , Humans , RNA, Messenger , SARS-CoV-2
4.
J Virol ; 96(5): e0218621, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1736028

ABSTRACT

Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS2+ human cells. Our data show that the spike protein of SARS-CoV-2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.


Subject(s)
COVID-19/metabolism , Furin/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Adaptation, Physiological , Animals , COVID-19/genetics , COVID-19/virology , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Furin/genetics , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Mutation , SARS-CoV-2/genetics , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Virus Replication
5.
Int J Mol Sci ; 23(5)2022 Mar 03.
Article in English | MEDLINE | ID: covidwho-1732066

ABSTRACT

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.


Subject(s)
Amino Acid Chloromethyl Ketones/pharmacology , Enoxaparin/pharmacology , Furin/antagonists & inhibitors , Spermine/analogs & derivatives , Zeaxanthins/pharmacology , Amino Acid Chloromethyl Ketones/chemistry , Amino Acid Chloromethyl Ketones/metabolism , COVID-19/transmission , COVID-19/virology , Catalytic Domain , Cell Line, Tumor , Cell Survival/drug effects , Enoxaparin/chemistry , Enoxaparin/metabolism , Furin/chemistry , Furin/metabolism , HEK293 Cells , Humans , Molecular Docking Simulation , Molecular Structure , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Proteolysis , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spermine/chemistry , Spermine/metabolism , Spermine/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Virus Replication , Zeaxanthins/chemistry , Zeaxanthins/metabolism
6.
Microbiol Spectr ; 10(1): e0236421, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1703367

ABSTRACT

The COVID-19 causing coronavirus (SARS-CoV-2) remains a public health threat worldwide. SARS-CoV-2 enters human lung cells via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). Notably, the cleavage of the spike by the host cell protease furin in virus-producing cells is critical for subsequent spike-driven entry into lung cells. Thus, effective targeted therapies blocking the spike cleavage and activation in viral producing cells may provide an alternate strategy to break the viral transmission cycle and to overcome disease pathology. Here we engineered and described an antibody-based targeted strategy, which directly competes with the furin mediated proteolytic activation of the spike in virus-producing cells. The described approach involves engineering competitive furin substrate residues in the IgG1 Fc-extended flexible linker domain of SARS-CoV-2 spike targeting antibodies. Considering the site of spike furin cleavage and SARS-CoV-2 egress remains uncertain, the experimental strategy pursued here revealed novel mechanistic insights into proteolytic processing of the spike protein, which suggest that processing does not occur in the constitutive secretory pathway. Furthermore, our results show blockade of furin-mediated cleavage of the spike protein for membrane fusion activation and virus host-cell entry function. These findings provide an alternate insight of targeting applicability to SARS-CoV-2 and the future coronaviridae family members, exploiting the host protease system to gain cellular entry and subsequent chain of infections. IMPORTANCE Since its emergence in December 2019, COVID-19 has remained a global economic and health threat. Although RNA and DNA vector-based vaccines induced antibody response and immunological memory have proven highly effective against hospitalization and mortality, their long-term efficacy remains unknown against continuously evolving SARS-CoV-2 variants. As host cell-enriched furin-mediated cleavage of SARS-CoV-2 spike protein is critical for viral entry and chain of the infection cycle, the solution described here of an antibody Fc-conjugated furin competing peptide is significant. In a scenario where spike mutational drifts do not interfere with the Fc-conjugated antibody's epitope, the proposed furin competing strategy confers a broad-spectrum targeting design to impede the production of efficiently transmissible SARS-CoV-2 viral particles. In addition, the proposed approach is plug-and-play against other potentially deadly viruses that exploit secretory pathway independent host protease machinery to gain cellular entry and subsequent transmissions to host cells.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/enzymology , COVID-19/virology , Furin/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , COVID-19/genetics , Furin/genetics , Host-Pathogen Interactions , Humans , Proteolysis , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
7.
Microbiol Spectr ; 10(1): e0236621, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1703078

ABSTRACT

The Amazonas was one of the most heavily affected Brazilian states by the COVID-19 epidemic. Despite a large number of infected people, particularly during the second wave associated with the spread of the Variant of Concern (VOC) Gamma (lineage P.1), SARS-CoV-2 continues to circulate in the Amazonas. To understand how SARS-CoV-2 persisted in a human population with a high immunity barrier, we generated 1,188 SARS-CoV-2 whole-genome sequences from individuals diagnosed in the Amazonas state from 1st January to 6th July 2021, of which 38 were vaccine breakthrough infections. Our study reveals a sharp increase in the relative prevalence of Gamma plus (P.1+) variants, designated Pango Lineages P.1.3 to P.1.6, harboring two types of additional Spike changes: deletions in the N-terminal (NTD) domain (particularly Δ144 or Δ141-144) associated with resistance to anti-NTD neutralizing antibodies or mutations at the S1/S2 junction (N679K or P681H) that probably enhance the binding affinity to the furin cleavage site, as suggested by our molecular dynamics simulations. As lineages P.1.4 (S:N679K) and P.1.6 (S:P681H) expanded (Re > 1) from March to July 2021, the lineage P.1 declined (Re < 1) and the median Ct value of SARS-CoV-2 positive cases in Amazonas significantly decreases. Still, we did not find an increased incidence of P.1+ variants among breakthrough cases of fully vaccinated patients (71%) in comparison to unvaccinated individuals (93%). This evidence supports that the ongoing endemic transmission of SARS-CoV-2 in the Amazonas is driven by the spread of new local Gamma/P.1 sublineages that are more transmissible, although not more efficient to evade vaccine-elicited immunity than the parental VOC. Finally, as SARS-CoV-2 continues to spread in human populations with a declining density of susceptible hosts, the risk of selecting more infectious variants or antibody evasion mutations is expected to increase. IMPORTANCE The continuous evolution of SARS-CoV-2 is an expected phenomenon that will continue to happen due to the high number of cases worldwide. The present study analyzed how a Variant of Concern (VOC) could still circulate in a population hardly affected by two COVID-19 waves and with vaccination in progress. Our results showed that the answer behind that was a new generation of Gamma-like viruses, which emerged locally carrying mutations that made it more transmissible and more capable of spreading, partially evading prior immunity triggered by natural infections or vaccines. With thousands of new cases daily, the current pandemics scenario suggests that SARS-CoV-2 will continue to evolve and efforts to reduce the number of infected subjects, including global equitable access to COVID-19 vaccines, are mandatory. Thus, until the end of pandemics, the SARS-CoV-2 genomic surveillance will be an essential tool to better understand the drivers of the viral evolutionary process.


Subject(s)
COVID-19/enzymology , Furin/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Motifs , Brazil/epidemiology , COVID-19/epidemiology , COVID-19/transmission , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Furin/genetics , Genomics , Humans , Mutation , Phylogeny , SARS-CoV-2/classification , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
8.
Cells ; 11(3)2022 01 27.
Article in English | MEDLINE | ID: covidwho-1662647

ABSTRACT

In this contribution, we report on the possibility that cryptococcal protease(s) could activate the SARS-CoV-2 spike (S) protein. The S protein is documented to have a unique four-amino-acid sequence (underlined, SPRRAR↓S) at the interface between the S1 and S2 sites, that serves as a cleavage site for the human protease, furin. We compared the biochemical efficiency of cryptococcal protease(s) and furin to mediate the proteolytic cleavage of the S1/S2 site in a fluorogenic peptide. We show that cryptococcal protease(s) processes this site in a manner comparable to the efficiency of furin (p > 0.581). We conclude the paper by discussing the impact of these findings in the context of a SARS-CoV-2 disease manifesting while there is an underlying cryptococcal infection.


Subject(s)
Aspartic Acid Proteases/metabolism , Bacterial Proteins/metabolism , Cryptococcus neoformans/enzymology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Aspartic Acid Proteases/genetics , Bacterial Proteins/genetics , Binding Sites , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/virology , Cryptococcus neoformans/genetics , Fluorescent Dyes/chemistry , Furin/genetics , Furin/metabolism , Humans , Pandemics , Peptides/chemistry , Peptides/metabolism , Proteolysis , SARS-CoV-2/physiology
9.
Nat Commun ; 13(1): 222, 2022 01 11.
Article in English | MEDLINE | ID: covidwho-1621242

ABSTRACT

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.


Subject(s)
SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Animals , COVID-19/virology , Cell Line , Cryoelectron Microscopy , Evolution, Molecular , Furin/metabolism , Humans , Linoleic Acid/metabolism , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Conformation , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Tropism , Virus Internalization
10.
Immunol Lett ; 242: 1-7, 2022 02.
Article in English | MEDLINE | ID: covidwho-1611776

ABSTRACT

SARS-CoV-2 harbors a unique S1/S2 furin cleavage site within its spike protein, which can be cleaved by furin and other proprotein convertases. Proteolytic activation of SARS-CoV-2 spike protein at the S1/S2 boundary facilitates interaction with host ACE2 receptor for cell entry. To address this, high titer antibody was generated against the SARS-CoV-2-specific furin motif. Using a series of innovative ELISA-based assays, this furin site blocking antibody displayed high sensitivity and specificity for the S1/S2 furin cleavage site, including with a P681R mutation, and demonstrated effective blockage of both enzyme-mediated cleavage and spike-ACE2 interaction. The results suggest that immunological blocking of the furin cleavage site may afford a suitable approach to stem proteolytic activation of SARS-CoV-2 spike protein and curtail viral infectivity.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Furin/metabolism , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Motifs/genetics , Amino Acid Motifs/immunology , Antibodies, Viral/pharmacology , Humans , Mutation , Nose/enzymology , Proprotein Convertases/metabolism , Protein Binding/drug effects , Proteolysis/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
11.
Emerg Microbes Infect ; 11(1): 483-497, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1606402

ABSTRACT

Coronavirus disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has set off a global pandemic. There is an urgent unmet need for safe, affordable, and effective therapeutics against COVID-19. In this regard, drug repurposing is considered as a promising approach. We assessed the compounds that affect the endosomal acidic environment by applying human angiotensin-converting enzyme 2 (hACE2)- expressing cells infected with a SARS-CoV-2 spike (S) protein-pseudotyped HIV reporter virus and identified that obatoclax resulted in the strongest inhibition of S protein-mediated virus entry. The potent antiviral activity of obatoclax at nanomolar concentrations was confirmed in different human lung and intestinal cells infected with the SARS-CoV-2 pseudotype system as well as clinical virus isolates. Furthermore, we uncovered that obatoclax executes a double-strike against SARS-CoV-2. It prevented SARS-CoV-2 entry by blocking endocytosis of virions through diminished endosomal acidification and the corresponding inhibition of the enzymatic activity of the endosomal cysteine protease cathepsin L. Additionally, obatoclax impaired the SARS-CoV-2 S-mediated membrane fusion by targeting the MCL-1 protein and reducing furin protease activity. In accordance with these overarching mechanisms, obatoclax blocked the virus entry mediated by different S proteins derived from several SARS-CoV-2 variants of concern such as, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Taken together, our results identified obatoclax as a novel effective antiviral compound that keeps SARS-CoV-2 at bay by blocking both endocytosis and membrane fusion. Our data suggested that obatoclax should be further explored as a clinical drug for the treatment of COVID-19.


Subject(s)
Cathepsins/metabolism , Furin/metabolism , Indoles/pharmacology , Pyrroles/pharmacology , SARS-CoV-2 , Virus Internalization/drug effects , COVID-19 , Humans , Hydrogen-Ion Concentration , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus
12.
Viruses ; 13(12)2021 12 14.
Article in English | MEDLINE | ID: covidwho-1572669

ABSTRACT

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants' evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was predicted to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation could confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we have shown that Q675H spike mutation is documented in all the VOCs. This finding highlights that VOCs are still evolving to enhance viral fitness and to adapt to the human host. At the same time, it may suggest Q675H spike mutation involvement in SARS-CoV-2 evolution.


Subject(s)
Furin/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites , Genetic Fitness , Humans , Hydrogen Bonding , Molecular Dynamics Simulation , Mutation , Phylogeny , Protein Binding , Protein Conformation , SARS-CoV-2/classification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry
13.
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
14.
Viruses ; 13(12)2021 12 04.
Article in English | MEDLINE | ID: covidwho-1554793

ABSTRACT

SARS-CoV-2 pathogenesis, vaccine, and therapeutic studies rely on the use of animals challenged with highly pathogenic virus stocks produced in cell cultures. Ideally, these virus stocks should be genetically and functionally similar to the original clinical isolate, retaining wild-type properties to be reliably used in animal model studies. It is well-established that SARS-CoV-2 isolates serially passaged on Vero cell lines accumulate mutations and deletions in the furin cleavage site; however, these can be eliminated when passaged on Calu-3 lung epithelial cell lines, as presented in this study. As numerous stocks of SARS-CoV-2 variants of concern are being grown in cell cultures with the intent for use in animal models, it is essential that propagation methods generate virus stocks that are pathogenic in vivo. Here, we found that the propagation of a B.1.351 SARS-CoV-2 stock on Calu-3 cells eliminated viruses that previously accumulated mutations in the furin cleavage site. Notably, there were alternative variants that accumulated at the same nucleotide positions in virus populations grown on Calu-3 cells at multiple independent facilities. When a Calu-3-derived B.1.351 virus stock was used to infect hamsters, the virus remained pathogenic and the Calu-3-specific variants persisted in the population. These results suggest that Calu-3-derived virus stocks are pathogenic but care should still be taken to evaluate virus stocks for newly arising mutations during propagation.


Subject(s)
SARS-CoV-2/growth & development , Serial Passage/methods , Spike Glycoprotein, Coronavirus/genetics , Animals , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Cricetinae , Furin/metabolism , Humans , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Vero Cells
15.
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
16.
PLoS Pathog ; 17(11): e1009820, 2021 11.
Article in English | MEDLINE | ID: covidwho-1528735

ABSTRACT

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.


Subject(s)
COVID-19/virology , Furin/metabolism , Nuclear Receptor Coactivators/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Cell Line , Endosomes/metabolism , Furin/genetics , Gene Expression , Humans , Immune Evasion , Interferons/metabolism , Lysosomes/enzymology , Nuclear Receptor Coactivators/genetics , Protein Isoforms , Proteolysis , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
17.
Infect Genet Evol ; 97: 105153, 2022 01.
Article in English | MEDLINE | ID: covidwho-1521407

ABSTRACT

Amid the ongoing COVID-19 pandemic, it has become increasingly important to monitor the mutations that arise in the SARS-CoV-2 virus, to prepare public health strategies and guide the further development of vaccines and therapeutics. The spike (S) protein and the proteins comprising the RNA-Dependent RNA Polymerase (RdRP) are key vaccine and drug targets, respectively, making mutation surveillance of these proteins of great importance. Full protein sequences were downloaded from the GISAID database, aligned, and the variants identified. 437,006 unique viral genomes were analyzed. Polymorphisms in the protein sequence were investigated and examined longitudinally to identify sequence and strain variants appearing between January 5th, 2020 and January 16th, 2021. A structural analysis was also performed to investigate mutations in the receptor binding domain and the N-terminal domain of the spike protein. Within the spike protein, there were 766 unique mutations observed in the N-terminal domain and 360 in the receptor binding domain. Four residues that directly contact ACE2 were mutated in more than 100 sequences, including positions K417, Y453, S494, and N501. Within the furin cleavage site of the spike protein, a high degree of conservation was observed, but the P681H mutation was observed in 10.47% of sequences analyzed. Within the RNA dependent RNA polymerase complex proteins, 327 unique mutations were observed in Nsp8, 166 unique mutations were observed in Nsp7, and 1157 unique mutations were observed in Nsp12. Only 4 sequences analyzed contained mutations in the 9 residues that directly interact with the therapeutic Remdesivir, suggesting limited mutations in drug interacting residues. The identification of new variants emphasizes the need for further study on the effects of the mutations and the implications of increased prevalence, particularly for vaccine or therapeutic efficacy.


Subject(s)
COVID-19/epidemiology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Genome, Viral , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Viral Nonstructural Proteins/chemistry , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Africa/epidemiology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/pharmacology , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Asia/epidemiology , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Databases, Factual , Epidemiological Monitoring , Europe/epidemiology , Evolution, Molecular , Furin/genetics , Furin/metabolism , Gene Expression , Humans , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/classification , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , United States/epidemiology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
18.
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
19.
Clin Exp Dent Res ; 8(1): 117-122, 2022 02.
Article in English | MEDLINE | ID: covidwho-1490746

ABSTRACT

OBJECTIVE: Besides angiotensin converting enzyme 2 (ACE2), an active involvement of proteases (FURIN and/or TMPRSS2) is important for cellular entry of SARS-CoV-2. Therefore, a simultaneous expression profiling of entry proteins in a tissue might provide a better risk assessment of SARS-CoV-2 infection as compared to individual proteins. In an attempt to understand the relative susceptibility of oral squamous cell carcinoma (OSCC) lesions as compared to the normal oral mucosa (NOM) for SARS-CoV-2 attachment/entry, this study examined the mRNA and protein expression profiles of ACE2, FURIN, and TMPRSS2 in the corresponding tissues using public transcriptomic and proteomics datasets. METHODS AND METHODS: Public transcriptomic and proteomics datasets (the Cancer Genome Atlas (TCGA)/the Genotype-Tissue Expression (GTEx), the Human Protein Atlas (HPA), and two independent microarray datasets) were used to examine the expression profiles of ACE2, TMPRSS2 and FURIN in NOM and OSCC. RESULTS: ACE2, TMPRSS2, and FURIN mRNAs were detected in NOM, however, at lower levels as compared to other body tissues. Except for moderate up-regulation of FURIN, expression levels of ACE2 and TMPRSS2 mRNA were unchanged/down-regulated in OSCC as compared to the NOM. CONCLUSIONS: These results indicate that NOM may serve as a possible site for SARS-CoV-2 attachment, however, to a lesser extent as compared to organs with higher expression levels of the SARS-CoV-2 entry proteins. However, the evidence is lacking to suggest that expression status of entry proteins predisposes OSCC lesions to additional risk for SARS-CoV-2 attachment/entry as compared to NOM.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/pathology , Furin/genetics , Gene Expression/genetics , Mouth Neoplasms/pathology , RNA, Messenger/genetics , SARS-CoV-2/genetics , Serine Endopeptidases/genetics , COVID-19/genetics , Carcinoma, Squamous Cell/genetics , Furin/metabolism , Head and Neck Neoplasms , Humans , Mouth Mucosa , Mouth Neoplasms/genetics , Mucous Membrane/pathology , Squamous Cell Carcinoma of Head and Neck , Tongue/metabolism
20.
J Virol ; 95(21): e0135721, 2021 10 13.
Article in English | MEDLINE | ID: covidwho-1476390

ABSTRACT

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.


Subject(s)
Evolution, Molecular , Heparitin Sulfate/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Adaptation, Biological , Animals , Binding Sites , Chlorocebus aethiops , Cytopathogenic Effect, Viral , DNA, Complementary , Furin/metabolism , Heparin/metabolism , Host-Pathogen Interactions , Protein Binding , Protein Domains , Protein Processing, Post-Translational , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Serial Passage , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Viral Plaque Assay , Virus Attachment
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