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1.
Commun Biol ; 5(1): 651, 2022 07 01.
Article in English | MEDLINE | ID: covidwho-1972669

ABSTRACT

Angiotensin-converting enzyme 2 (ACE2) has been identified as a primary receptor for severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2). Here, we investigated the expression regulation of ACE2 in enterocytes under amino acid deprivation conditions. In this study, we found that ACE2 expression was upregulated upon all or single essential amino acid deprivation in human colonic epithelial CCD841 cells. Furthermore, we found that knockdown of general control nonderepressible 2 (GCN2) reduced intestinal ACE2 mRNA and protein levels in vitro and in vivo. Consistently, we revealed two GCN2 inhibitors, GCN2iB and GCN2-IN-1, downregulated ACE2 protein expression in CCD841 cells. Moreover, we found that increased ACE2 expression in response to leucine deprivation was GCN2 dependent. Through RNA-sequencing analysis, we identified two transcription factors, MAFB and MAFF, positively regulated ACE2 expression under leucine deprivation in CCD841 cells. These findings demonstrate that amino acid deficiency increases ACE2 expression and thereby likely aggravates intestinal SARS-CoV-2 infection.


Subject(s)
Amino Acids , Angiotensin-Converting Enzyme 2 , COVID-19 , Enterocytes , Protein Serine-Threonine Kinases , Amino Acids/deficiency , Amino Acids/metabolism , Angiotensin-Converting Enzyme 2/biosynthesis , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/enzymology , COVID-19/genetics , COVID-19/virology , Enterocytes/enzymology , Enterocytes/metabolism , Humans , Leucine/pharmacology , Peptidyl-Dipeptidase A/physiology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , SARS-CoV-2/metabolism
2.
Iran J Allergy Asthma Immunol ; 21(3): 254-262, 2022 Jun 18.
Article in English | MEDLINE | ID: covidwho-1924808

ABSTRACT

The role of microRNA (miR)200c-3p in regulating ACE2 gene expression in viral and bacterial respiratory diseases has been established. Since ACE2 reduces the acute inflammatory effects in lung diseases and acts as a coronavirus receptor to invade the lung cells, this study investigates the relationship between miR-200c-3p and ACE2 expression in COVID -19 patients. In this study, COVID-19 patients were divided into two groups: mild phase (PCR-positive and mild symptoms) and severe phase (PCR-positive with acute pulmonary symptoms and inflammation). Then, the subjects' demographic, clinical, and paraclinical characteristics were recorded using a prepared checklist. Total RNA was isolated from all samples according to the Trizol kit protocol to evaluate gene expression. Subsequently, the extracted product was analysed for miR-200c expression and ACE2 target gene expression by real-time PCR. The results of the checklist data showed that smoking, cough, and the factors ESR and HCT were statistically significant between the two groups of patients in the mild and acute phases. Also, the mean expression of the miR-200c gene in the mild and acute patients was 1.87±0.70 and 1.87±0.62, respectively, which was not statistically significant. Still, the mean expression of the ACE2 gene, which was 3.96±0.76 and 3.28±0.52 in the mild and acute disease groups, respectively, showed a significant difference between the two groups. This study showed that the expression levels of ACE2 were significantly reduced in people with severe inflammation compared to people with mild inflammation.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , MicroRNAs , Angiotensin-Converting Enzyme 2/biosynthesis , Angiotensin-Converting Enzyme 2/genetics , COVID-19/blood , COVID-19/enzymology , COVID-19/genetics , Gene Expression , Humans , MicroRNAs/blood , MicroRNAs/genetics
3.
Biomolecules ; 12(7)2022 06 23.
Article in English | MEDLINE | ID: covidwho-1911171

ABSTRACT

The development of inexpensive, fast, and reliable screening tests for COVID-19 is, as yet, an unmet need. The present study was aimed at evaluating the usefulness of serum arylesterase activity of paraoxonase-1 (PON1) measurement as a screening test in patients with different severity levels of COVID-19 infection. We included 615 COVID-19-positive patients who were classified as asymptomatic, mildly symptomatic, severely symptomatic, or fatally symptomatic. Results were compared with 50 healthy volunteers, 330 patients with cancer, and 343 with morbid obesity. Results showed PON1 activity greatly decreased in COVID-19 compared to healthy volunteers; a receiver operating characteristics plot showed a high diagnostic accuracy. The degree of COVID-19 severity did not influence PON1 levels. Our results indicated that PON1 determination was efficient for disease diagnosis, but not for prognosis. Furthermore, patients with obesity or cancer presented alterations similar to those of COVID-19 patients. As such, elevated levels of PON1 indicate the absence of COVID-19, but low levels may be present in various other chronic diseases. The assay is fast and inexpensive. We suggest that PON1 measurement could be used as an initial, high cut-off point screening method, while lower values should be confirmed with the more expensive nucleic acid amplification test.


Subject(s)
Aryldialkylphosphatase , COVID-19 , Aryldialkylphosphatase/blood , COVID-19/blood , COVID-19/diagnosis , COVID-19/enzymology , Carboxylic Ester Hydrolases , Humans , Serum
4.
Sci Rep ; 12(1): 1299, 2022 01 25.
Article in English | MEDLINE | ID: covidwho-1908217

ABSTRACT

Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CLpro) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CLpro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CLpro revealed that the Kcat/Km of the 3CLpro enzyme containing Ser108 was 58% lower than that of Pro108 3CLpro. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CLpro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CLpro inhibitor.


Subject(s)
COVID-19 , Coronavirus 3C Proteases , Mutation, Missense , Patient Acuity , Adult , Aged , Amino Acid Substitution , COVID-19/enzymology , COVID-19/genetics , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Female , Humans , Male , Middle Aged
5.
Front Immunol ; 13: 842740, 2022.
Article in English | MEDLINE | ID: covidwho-1903003

ABSTRACT

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a significant global health emergency with new variants in some cases evading current therapies and approved vaccines. COVID-19 presents with a broad spectrum of acute and long-term manifestations. Severe COVID-19 is characterized by dysregulated cytokine release profile, dysfunctional immune responses, and hypercoagulation with a high risk of progression to multi-organ failure and death. Unraveling the fundamental immunological processes underlying the clinical manifestations of COVID-19 is vital for the identification and design of more effective therapeutic interventions for individuals at the highest risk of severe outcomes. Caspases are expressed in both immune and non-immune cells and mediate inflammation and cell death, including apoptosis and pyroptosis. Here we review accumulating evidence defining the importance of the expression and activity of caspase family members following SARS-CoV-2 infection and disease. Research suggests SARS-CoV-2 infection is linked to the function of multiple caspases, both mechanistically in vitro as well as in observational studies of individuals with severe COVID-19, which may further the impact on disease severity. We also highlight immunological mechanisms that occur in severe COVID-19 pathology upstream and downstream of activated caspase pathways, including innate recognition receptor signaling, inflammasomes, and other multiprotein complex assembly, inflammatory mediators IL-1ß and IL-18, and apoptotic and pyroptotic cell death. Finally, we illuminate discriminate and indiscriminate caspase inhibitors that have been identified for clinical use that could emerge as potential therapeutic interventions that may benefit clinical efforts to prevent or ameliorate severe COVID-19.


Subject(s)
COVID-19/enzymology , Caspases/immunology , SARS-CoV-2 , Animals , COVID-19/drug therapy , COVID-19/immunology , Humans , Inflammation/immunology
6.
Front Endocrinol (Lausanne) ; 13: 862789, 2022.
Article in English | MEDLINE | ID: covidwho-1896674

ABSTRACT

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although males and females are at equivalent risk of infection, males are more prone to develop a higher severity disease, regardless of age. The factors that mediate susceptibility to SARS-CoV-2 and transmission are still under investigation. A potential role has been attributed to differences in the immune systems response to viral antigens between males and females as well as to different regulatory actions played by sex-related hormones on the two crucial molecular effectors for SARS-CoV-2 infection, TMPRSS2 and ACE2. While few and controversial data about TMPRSS2 transcript regulation in lung cells are emerging, no data on protein expression and activity of TMPRSS2 have been reported. Aim of the present study was to search for possible modulatory actions played by sex-related hormones on TMPRSS2 and ACE2 expression in Calu-3 cells, to test the effects of sex-steroids on the expression of the 32kDa C-term fragment derived from autocatalitic cleavage of TMPRSS2 and its impact on priming of transiently transfected spike protein. Cells were stimulated with different concentrations of methyltrienolone (R1881) or estradiol for 30 h. No difference in mRNA and protein expression levels of full length TMPRSS2 was observed. However, the 32 kDa cleaved serine protease domain was increased after 100 nM R1881 (+2.36 ± 1.13 fold-increase vs control untreated cells, p < 0.05) and 10 nM estradiol (+1.90 ± 0.64, fold-increase vs control untreated cells, p < 0.05) treatment. Both R1881 and estradiol significantly increased the activating proteolytic cleavage of SARS-CoV-2 Spike (S) transfected in Calu-3 cells (+1.76 ± 0.18 and +1.99±,0.76 increase in S cleavage products at R1881 100nM and 10 nM estradiol treatment, respectively, p < 0.001 and p < 0.05 vs control untreated cells, respectively). Finally, no significant differences in ACE2 expression were observed between hormones-stimulated cells and untreated control cells. Altogether, these data suggest that both male and female sex-related hormones are able to induce a proteolityc activation of TMPRSS2, thus promoting viral infection, in agreement with the observation that males and females are equally infected by SARS-CoV-2.


Subject(s)
COVID-19 , Serine Endopeptidases , Angiotensin-Converting Enzyme 2/genetics , COVID-19/enzymology , Cell Line , Estradiol/pharmacology , Female , Humans , Lung/metabolism , Male , Metribolone/pharmacology , Peptidyl-Dipeptidase A/genetics , SARS-CoV-2 , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism
7.
Proc Natl Acad Sci U S A ; 119(21): e2202012119, 2022 05 24.
Article in English | MEDLINE | ID: covidwho-1852638

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS­CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS­CoV-2 infections and that CASP4 expression correlates with severity of SARS­CoV-2 infection in humans. SARS­CoV-2­infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS­CoV-2­infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1ß, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS­CoV-2­induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.


Subject(s)
COVID-19 , Caspases, Initiator/metabolism , SARS-CoV-2 , Thromboinflammation , Animals , COVID-19/enzymology , COVID-19/pathology , Caspases, Initiator/genetics , Disease Progression , Humans , Lung/pathology , Mice , Mice, Knockout , Severity of Illness Index , Thromboinflammation/enzymology , Thromboinflammation/genetics
8.
J Infect ; 85(1): 86-89, 2022 07.
Article in English | MEDLINE | ID: covidwho-1814751

ABSTRACT

BACKGROUND: Variations in the ACE2 activity in saliva could explain the striking differences of susceptibility to infection and risk of severe disease. METHODS: We analyze the activity of ACE2 in saliva in different population groups across a wide age range and disease status during April to June 2020, before SARS-CoV-2 vaccine implementation, and we establish differences between infected people and participants considered resistant (highly exposed healthcare workers and children who cohabited with parents with COVID-19 without isolation and remain IgG negative). RESULTS: We included 74 adults, of which 47 (64%) were susceptible and 27 (36%) were resistant, and 79 children, of which 41 (52%) were susceptible and 38 (48%) were resistant. Resistant adults have significantly lower ACE2 activity in saliva than susceptible adults and non-significant higher values than susceptible and resistant children. ACE2 activity is similar in the susceptible and resistant pediatric population (p = 0.527). In contrast, we observe an increase in activity as the disease's severity increases among the adult population (mild disease vs. severe disease, 39 vs. 105 FU, p = 0.039; severe disease vs. resistant, 105 vs. 31 FU, p < 0.001). CONCLUSIONS: using an enzymatic test, we show that ACE2 activity in saliva correlates with the susceptibility to SARS-Cov-2 infection and disease severity. Children and adults with low-susceptibility to SARS-Cov-2 infection showed the lowest ACE2 activity. These findings could inform future strategies to identify at-risk individuals.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , SARS-CoV-2 , Adult , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/enzymology , COVID-19 Vaccines , Child , Humans , Saliva/enzymology
9.
Appl Biochem Biotechnol ; 194(8): 3507-3526, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1777819

ABSTRACT

Coronavirus disease 2019 (COVID-19) is a severe disease caused by a new variant of beta-coronavirus that first appeared in China. Human genetic factors, including polymorphisms, serve pivotal roles in the high transmission of SARS-CoV-2 and the stubbornly progressing sickness seen in a small but significant percentage of infected people; however, but these factors remain ill-defined. A total of 288 COVID-19 patients and 288 controls were genotyped for TMPRSS2 polymorphisms using both restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) and amplification refractory mutation system (ARMS)-PCR techniques. Different genotypes of TMPRSS2 polymorphisms were compared in terms of disease susceptibility and mortality. The statistical analysis showed that minor alleles of all studied variants statistically increased the risk of COVID-19, except for the rs75603675 C > A variant. The T allele of rs12329760 conferred an increased risk of COVID-19. Moreover, the AG/AC/TT/AG combination of genotypes significantly enhanced the risk of COVID-19 in our population. Different haplotypes of rs17854725/rs75603675/rs12329760/rs4303795 polymorphisms, including GACA, GACG, GATG, GATA, AATA, ACCG, ACTG, ACTA, GCCA, and GCTG, were found to be associated with increased risk of the disease (odds ratio > 1). Regarding the clinical and paraclinical characteristics, a statistically significant difference was found between non-severe and severe forms except for gender, platelet, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and underlying diseases. In addition, case genotypes of TMPRSS2 rs17854725 A > G, rs12329760 C > T, and rs4303795 A > G were significantly different regarding severe and non-severe forms of the disease (P-value < 0.001). Specifically, death was more frequent in carriers of the AG genotype of rs17854725 A > G (P-value = 0.022). Patients who carry the minor alleles of the four studied TMPRSS2 variants were rather vulnerable to COVID-19 infection. Our findings indicated that rs17854725 A > G (AA vs. AG and AA vs. GG), rs12329760 C > T (CC vs. CT and CC vs. TT), and rs4303795 A > G (AA vs. AG) genotypes of TMPRSS2 variations are associated with a more invasive disorder pattern. More studies on larger populations are needed to confirm our results.


Subject(s)
COVID-19 , Serine Endopeptidases , Alleles , COVID-19/enzymology , COVID-19/epidemiology , COVID-19/genetics , Case-Control Studies , Genetic Predisposition to Disease , Genotype , Humans , Polymorphism, Single Nucleotide , SARS-CoV-2 , Serine Endopeptidases/genetics
10.
Redox Rep ; 27(1): 85-91, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1774187

ABSTRACT

Objectives: Due to the role of oxidative stress in the pathophysiology of COVID-19, it is biologically plausible that inter-individual differences in patients' clinical manifestations might be affected by antioxidant genetic profile. The aim of our study was to assess the distribution of antioxidant genetic polymorphisms Nrf2 rs6721961, SOD2 rs4880, GPX1 rs1050450, GPX3 rs8177412, and GSTP1 (rs1695 and rs1138272) haplotype in COVID-19 patients and controls, with special emphasis on their association with laboratory biochemical parameters.Methods: The antioxidant genetic polymorphisms were assessed by appropriate PCR methods in 229 COVID-19 patients and 229 matched healthy individuals.Results: Among examined polymorphisms, only GSTP1 haplotype was associated with COVID-19 risk (p = 0.009). Polymorphisms of SOD2 and GPX1 influenced COVID-19 patients' laboratory biochemical profile: SOD2*Val allele was associated with increased levels of fibrinogen (p = 0.040) and ferritin (p = 0.033), whereas GPX1*Leu allele was associated with D-dimmer (p = 0.009).Discussion: Our findings regarding the influence of SOD2 and GPX1 polymorphisms on inflammation and coagulation parameters might be of clinical importance. If confirmed in larger cohorts, these developments could provide a more personalized approach for better recognition of patients prone to thrombosis and those for the need of targeted antiox-idant therapy.


Subject(s)
COVID-19 , Glutathione Peroxidase , Superoxide Dismutase , Blood Coagulation , COVID-19/enzymology , COVID-19/genetics , Glutathione Peroxidase/genetics , Humans , Inflammation/genetics , Polymorphism, Single Nucleotide , Serbia , Superoxide Dismutase/genetics
11.
Proc Natl Acad Sci U S A ; 119(15): e2120913119, 2022 04 12.
Article in English | MEDLINE | ID: covidwho-1758464

ABSTRACT

SignificanceThe coronavirus main protease (Mpro) is required for viral replication. Here, we obtained the extended conformation of the native monomer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro by trapping it with nanobodies and found that the catalytic domain and the helix domain dissociate, revealing allosteric targets. Another monomeric state is termed compact conformation and is similar to one protomer of the dimeric form. We designed a Nanoluc Binary Techonology (NanoBiT)-based high-throughput allosteric inhibitor assay based on structural conformational change. Our results provide insight into the maturation, dimerization, and catalysis of the coronavirus Mpro and pave a way to develop an anticoronaviral drug through targeting the maturation process to inhibit the autocleavage of Mpro.


Subject(s)
Antiviral Agents , COVID-19 , Coronavirus 3C Proteases , Protease Inhibitors , SARS-CoV-2 , Allosteric Regulation/drug effects , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/enzymology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Humans , Luciferases , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Conformation , Protein Multimerization
13.
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
14.
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
15.
Molecules ; 27(4)2022 Feb 11.
Article in English | MEDLINE | ID: covidwho-1686903

ABSTRACT

Two rare 2-phenoxychromone derivatives, 6-demethoxy-4`-O-capillarsine (1) and tenuflorin C (2), were isolated from the areal parts of Artemisia commutata and A. glauca, respectively, for the first time. Being rare in nature, the inhibition potentialities of 1 and 2 against SARS-CoV-2 was investigated using multistage in silico techniques. At first, molecular similarity and fingerprint studies were conducted for 1 and 2 against co-crystallized ligands of eight different COVID-19 enzymes. The carried-out studies indicated the similarity of 1 and 2 with TTT, the co-crystallized ligand of COVID-19 Papain-Like Protease (PLP), (PDB ID: 3E9S). Therefore, molecular docking studies of 1 and 2 against the PLP were carried out and revealed correct binding inside the active site exhibiting binding energies of -18.86 and -18.37 Kcal/mol, respectively. Further, in silico ADMET in addition to toxicity evaluation of 1 and 2 against seven models indicated the general safety and the likeness of 1 and 2 to be drugs. Lastly, to authenticate the binding and to investigate the thermodynamic characters, molecular dynamics (MD) simulation studies were conducted on 1 and PLP.


Subject(s)
Artemisia/chemistry , COVID-19/enzymology , Chromones/chemistry , Coronavirus Papain-Like Proteases , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , COVID-19/drug therapy , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/chemistry , Humans
16.
J Virol ; 96(6): e0000222, 2022 03 23.
Article in English | MEDLINE | ID: covidwho-1673349

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the largest RNA genome, approximately 30 kb, among RNA viruses. The DDX DEAD box RNA helicase is a multifunctional protein involved in all aspects of RNA metabolism. Therefore, host RNA helicases may regulate and maintain such a large viral RNA genome. In this study, I investigated the potential role of several host cellular RNA helicases in SARS-CoV-2 infection. Notably, DDX21 knockdown markedly accumulated intracellular viral RNA and viral production, as well as viral infectivity of SARS-CoV-2, indicating that DDX21 strongly restricts the SARS-CoV-2 infection. In addition, MOV10 RNA helicase also suppressed the SARS-CoV-2 infection. In contrast, DDX1, DDX5, and DDX6 RNA helicases were required for SARS-CoV-2 replication. Indeed, SARS-CoV-2 infection dispersed the P-body formation of DDX6 and MOV10 RNA helicases as well as XRN1 exonuclease, while the viral infection did not induce stress granule formation. Accordingly, the SARS-CoV-2 nucleocapsid (N) protein interacted with DDX1, DDX3, DDX5, DDX6, DDX21, and MOV10 and disrupted the P-body formation, suggesting that SARS-CoV-2 N hijacks DDX6 to carry out viral replication. Conversely, DDX21 and MOV10 restricted SARS-CoV-2 infection through an interaction of SARS-CoV-2 N with host cellular RNA helicases. Altogether, host cellular RNA helicases seem to regulate the SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has a large RNA genome, of approximately 30 kb. To regulate and maintain such a large viral RNA genome, host RNA helicases may be involved in SARS-CoV-2 replication. In this study, I have demonstrated that DDX21 and MOV10 RNA helicases limit viral infection and replication. In contrast, DDX1, DDX5, and DDX6 are required for SARS-CoV-2 infection. Interestingly, SARS-CoV-2 infection disrupted P-body formation and attenuated or suppressed stress granule formation. Thus, SARS-CoV-2 seems to hijack host cellular RNA helicases to play a proviral role by facilitating viral infection and replication and by suppressing the host innate immune system.


Subject(s)
COVID-19 , Host Microbial Interactions , RNA Helicases , RNA, Viral , COVID-19/enzymology , Host Microbial Interactions/physiology , Humans , RNA Helicases/genetics , RNA Helicases/metabolism , RNA, Viral/metabolism , SARS-CoV-2 , Virus Replication/physiology
17.
Nat Commun ; 13(1): 601, 2022 02 01.
Article in English | MEDLINE | ID: covidwho-1671558

ABSTRACT

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. We show that two consecutive mutations (R203K/G204R) in the nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found increased interaction of GSK3A kinase simultaneously with hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein. Furthermore, the host cell transcriptome analysis suggests that the mutant N protein produces dysregulated interferon response genes. Here, we provide crucial information in linking the R203K/G204R mutations in the N protein to modulations of host-virus interactions and underline the potential of the nucleocapsid protein as a drug target during infection.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Genome, Viral , Mutation, Missense , SARS-CoV-2/genetics , COVID-19/enzymology , COVID-19/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Host-Pathogen Interactions , Humans , Nucleocapsid/genetics , Nucleocapsid/metabolism , Phosphorylation , Phylogeny , Protein Binding , SARS-CoV-2/classification , SARS-CoV-2/physiology , Saudi Arabia , Viral Load , Virus Replication
18.
Oxid Med Cell Longev ; 2022: 5397733, 2022.
Article in English | MEDLINE | ID: covidwho-1635531

ABSTRACT

The infection of coronavirus disease (COVID-19) seriously threatens human life. It is urgent to generate effective and safe specific antibodies (Abs) against the pathogenic elements of COVID-19. Mice were immunized with SARS-CoV-2 spike protein antigens: S ectodomain-1 (CoV, in short) mixed in Alum adjuvant for 2 times and boosted with CoV weekly for 6 times. A portion of mice were treated with Maotai liquor (MTL, in short) or/and heat stress (HS) together with CoV boosting. We observed that the anti-CoV Ab was successfully induced in mice that received the CoV/Alum immunization for 2 times. However, upon boosting with CoV, the CoV Ab production diminished progressively; spleen CoV Ab-producing plasma cell counts reduced, in which substantial CoV-specific Ab-producing plasma cells (sPC) were apoptotic. Apparent oxidative stress signs were observed in sPCs; the results were reproduced by exposing sPCs to CoV in the culture. The presence of MTL or/and HS prevented the CoV-induced oxidative stress in sPCs and promoted and stabilized the CoV Ab production in mice in re-exposure to CoV. In summary, CoV/Alum immunization can successfully induce CoV Ab production in mice that declines upon reexposure to CoV. Concurrent administration of MTL/HS stabilizes and promotes the CoV Ab production in mice.


Subject(s)
Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Apoptosis , COVID-19/immunology , Plasma Cells/immunology , SARS-CoV-2/physiology , Superoxide Dismutase-1/physiology , Adjuvants, Immunologic , Alcoholic Beverages , Alum Compounds , Angiotensin-Converting Enzyme 2/physiology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/enzymology , COVID-19 Vaccines/immunology , Heat-Shock Response , Immunization, Secondary , Immunogenicity, Vaccine , Janus Kinase 2/physiology , Male , Mice , Mice, Inbred C57BL , Oxidative Stress , Plasma Cells/drug effects , Plasma Cells/pathology , Reactive Oxygen Species/metabolism , STAT1 Transcription Factor/physiology , Signal Transduction , Specific Pathogen-Free Organisms , Spike Glycoprotein, Coronavirus/immunology , Vaccination
19.
Nat Genet ; 54(2): 125-127, 2022 02.
Article in English | MEDLINE | ID: covidwho-1625297

ABSTRACT

The OAS1/2/3 cluster has been identified as a risk locus for severe COVID-19 among individuals of European ancestry, with a protective haplotype of approximately 75 kilobases (kb) derived from Neanderthals in the chromosomal region 12q24.13. This haplotype contains a splice variant of OAS1, which occurs in people of African ancestry independently of gene flow from Neanderthals. Using trans-ancestry fine-mapping approaches in 20,779 hospitalized cases, we demonstrate that this splice variant is likely to be the SNP responsible for the association at this locus, thus strongly implicating OAS1 as an effector gene influencing COVID-19 severity.


Subject(s)
2',5'-Oligoadenylate Synthetase/genetics , COVID-19/genetics , COVID-19/pathology , Genetic Predisposition to Disease , Physical Chromosome Mapping , RNA Splicing/genetics , Severity of Illness Index , Blacks/genetics , COVID-19/enzymology , Humans , Linkage Disequilibrium/genetics , Risk Factors , Whites/genetics
20.
PLoS Biol ; 19(12): e3001510, 2021 12.
Article in English | MEDLINE | ID: covidwho-1592147

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infects a broader range of mammalian species than previously predicted, binding a diversity of angiotensin converting enzyme 2 (ACE2) orthologs despite extensive sequence divergence. Within this sequence degeneracy, we identify a rare sequence combination capable of conferring SARS-CoV-2 resistance. We demonstrate that this sequence was likely unattainable during human evolution due to deleterious effects on ACE2 carboxypeptidase activity, which has vasodilatory and cardioprotective functions in vivo. Across the 25 ACE2 sites implicated in viral binding, we identify 6 amino acid substitutions unique to mouse-one of the only known mammalian species resistant to SARS-CoV-2. Substituting human variants at these positions is sufficient to confer binding of the SARS-CoV-2 S protein to mouse ACE2, facilitating cellular infection. Conversely, substituting mouse variants into either human or dog ACE2 abolishes viral binding, diminishing cellular infection. However, these same substitutions decrease human ACE2 activity by 50% and are predicted as pathogenic, consistent with the extreme rarity of human polymorphisms at these sites. This trade-off can be avoided, however, depending on genetic background; if substituted simultaneously, these same mutations have no deleterious effect on dog ACE2 nor that of the rodent ancestor estimated to exist 70 million years ago. This genetic contingency (epistasis) may have therefore opened the road to resistance for some species, while making humans susceptible to viruses that use these ACE2 surfaces for binding, as does SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Disease Resistance/genetics , Epistasis, Genetic , SARS-CoV-2/physiology , Amino Acids , Angiotensin II/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites , COVID-19/enzymology , COVID-19/genetics , Dogs , Evolution, Molecular , Gene Frequency , Humans , Hydrolysis , Mice , Mutation , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment
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