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1.
Front Immunol ; 13: 854327, 2022.
Article in English | MEDLINE | ID: covidwho-1887100

ABSTRACT

Tuberculosis (TB) remains a significant global health crisis and the number one cause of death for an infectious disease. The health consequences in high-burden countries are significant. Barriers to TB control and eradication are in part caused by difficulties in diagnosis. Improvements in diagnosis are required for organisations like the World Health Organisation (WHO) to meet their ambitious target of reducing the incidence of TB by 50% by the year 2025, which has become hard to reach due to the COVID-19 pandemic. Development of new tests for TB are key priorities of the WHO, as defined in their 2014 report for target product profiles (TPPs). Rapid triage and biomarker-based confirmatory tests would greatly enhance the diagnostic capability for identifying and diagnosing TB-infected individuals. Protein-based test methods e.g. lateral flow devices (LFDs) have a significant advantage over other technologies with regard to assay turnaround time (minutes as opposed to hours) field-ability, ease of use by relatively untrained staff and without the need for supporting laboratory infrastructure. Here we evaluate the diagnostic performance of nine biomarkers from our previously published biomarker qPCR validation study; CALCOCO2, CD274, CD52, GBP1, IFIT3, IFITM3, SAMD9L, SNX10 and TMEM49, as protein targets assayed by ELISA. This preliminary evaluation study was conducted to quantify the level of biomarker protein expression across latent, extra-pulmonary or pulmonary TB groups and negative controls, collected across the UK and India, in whole lysed blood samples (WLB). We also investigated associative correlations between the biomarkers and assessed their suitability for ongoing diagnostic test development, using receiver operating characteristic/area under the curve (ROC) analyses, singly and in panel combinations. The top performing single biomarkers for pulmonary TB versus controls were CALCOCO2, SAMD9L, GBP1, IFITM3, IFIT3 and SNX10. TMEM49 was also significantly differentially expressed but downregulated in TB groups. CD52 expression was not highly differentially expressed across most of the groups but may provide additional patient stratification information and some limited use for incipient latent TB infection. These show therefore great potential for diagnostic test development either in minimal configuration panels for rapid triage or more complex formulations to capture the diversity of disease presentations.


Subject(s)
COVID-19 , Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Tuberculosis , Biomarkers , COVID-19/diagnosis , Diagnostic Tests, Routine , Enzyme-Linked Immunosorbent Assay , Humans , Membrane Proteins/metabolism , Mycobacterium tuberculosis/metabolism , Pandemics , RNA-Binding Proteins , Sorting Nexins/metabolism , Tuberculosis/diagnosis , Tuberculosis, Pulmonary/diagnosis
2.
J Virol ; 96(12): e0041222, 2022 Jun 22.
Article in English | MEDLINE | ID: covidwho-1874504

ABSTRACT

SARS-CoV-2 is the causative agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19) and poses a significant threat to global health. N protein (NP), which is a major pathogenic protein among betacoronaviruses, binds to the viral RNA genome to allow viral genome packaging and viral particle release. Recent studies showed that NP antagonizes interferon (IFN) induction and mediates phase separation. Using live SARS-CoV-2 viruses, this study provides solid evidence showing that SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming G3BP1-mediated antiviral innate immunity. G3BP1 conditional knockout mice (g3bp1fl/fL, Sftpc-Cre) exhibit significantly higher lung viral loads after SARS-CoV-2 infection than wild-type mice. Our findings contribute to the growing body of knowledge regarding the pathogenicity of NPSARS-CoV-2 and provide insight into new therapeutics targeting NPSARS-CoV-2. IMPORTANCE In this study, by in vitro assay and live SARS-CoV-2 virus infection, we provide solid evidence that the SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming antiviral innate immunity mediated by G3BP1 in A549 cell lines and G3BP1 conditional knockout mice (g3bp1-cKO) mice, which provide in-depth evidence showing the mechanism underlying NP-related SARS-CoV-2 pathogenesis through G3BPs.


Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins , Poly-ADP-Ribose Binding Proteins , SARS-CoV-2 , Virus Replication , Adaptor Proteins, Signal Transducing/metabolism , Animals , COVID-19/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , DNA Helicases/metabolism , Host Microbial Interactions/immunology , Mice , Phosphoproteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , RNA-Binding Proteins/metabolism , Virus Replication/genetics
3.
mBio ; 13(3): e0040122, 2022 Jun 28.
Article in English | MEDLINE | ID: covidwho-1854237

ABSTRACT

Influenza viruses cause respiratory tract infections, which lead to human disease outbreaks and pandemics. Influenza A virus (IAV) circulates in diverse animal species, predominantly aquatic birds. This often results in the emergence of novel viral strains causing severe human disease upon zoonotic transmission. Innate immune sensing of the IAV infection promotes host cell death and inflammatory responses to confer antiviral host defense. Dysregulated respiratory epithelial cell death and excessive proinflammatory responses drive immunopathology in highly pathogenic influenza infections. Here, we discuss the critical mechanisms regulating IAV-induced cell death and proinflammatory responses. We further describe the essential role of the Z-form nucleic acid sensor ZBP1/DAI and RIPK3 in triggering apoptosis, necroptosis, and pyroptosis during IAV infection and their impact on host defense and pathogenicity in vivo. We also discuss the functional importance of ZBP1-RIPK3 signaling in recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viral infections. Understanding these mechanisms of RNA virus-induced cytopathic and pathogenic inflammatory responses is crucial for targeting pathogenic lung infections and human respiratory illness.


Subject(s)
COVID-19 , Influenza A virus , Influenza, Human , Pneumonia , Animals , Cell Death , Humans , Influenza A virus/physiology , RNA , RNA-Binding Proteins/genetics , SARS-CoV-2
4.
J Mol Biol ; 434(6): 167277, 2022 03 30.
Article in English | MEDLINE | ID: covidwho-1851576

ABSTRACT

Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions induced by IFN. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent manner, opening new perspectives to target weakness points in the life cycle of these viruses.


Subject(s)
COVID-19 , Flavivirus , Zika Virus Infection , Zika Virus , Antiviral Agents/pharmacology , Flavivirus/genetics , Histone Deacetylases , Humans , Interferons/genetics , Membrane Proteins , RNA-Binding Proteins , Repressor Proteins , SARS-CoV-2 , Virus Replication/genetics , Zika Virus/genetics , Zika Virus Infection/genetics
5.
BMC Res Notes ; 14(1): 401, 2021 Oct 29.
Article in English | MEDLINE | ID: covidwho-1841025

ABSTRACT

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus causing severe respiratory illness (COVID-19). This virus was initially identified in Wuhan city, a populated area of the Hubei province in China, and still remains one of the major global health challenges. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing that plays a crucial role in innate viral defense mechanisms by inhibiting the virus replication as well as expression of various viral proteins. Dicer, Drosha, Ago2, and DGCR8 are essential components of the RNAi system, which is supposed to be dysregulated in COVID-19 patients. This study aimed to assess the expression level of the mentioned mRNAs in COVID-19patients compared to healthy individuals. RESULTS: Our findings demonstrated that the expression of Dicer, Drosha, and Ago2 was statistically altered in COVID-19 patients compared to healthy subjects. Ultimately, the RNA interference mechanism as a crucial antiviral defense system was suggested to be dysregulated in COVID-19 patients.


Subject(s)
COVID-19 , MicroRNAs , Humans , RNA Interference , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , SARS-CoV-2
6.
J Virol ; 96(11): e0059422, 2022 Jun 08.
Article in English | MEDLINE | ID: covidwho-1840553

ABSTRACT

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human lung cells, cardiomyocytes, and gut organoids. To date, several "variants of concern" (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the five current SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, and Omicron) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had modest effects, while knockdown of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than 4 orders of magnitude. In addition, an antibody directed against the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells, thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominant Omicron variant. IMPORTANCE Recent data indicate that SARS-CoV-2 requires endogenously expressed IFITM proteins for efficient infection. However, the results were obtained with an early SARS-CoV-2 isolate. Thus, it remained to be determined whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that outcompeted the original strains in the meantime. This includes the Omicron VOC, which currently dominates the pandemic. Here, we show that depletion of endogenous IFITM2 expression almost entirely prevents productive infection of Alpha, Beta, Gamma, Delta, and Omicron SARS-CoV-2 VOCs in human lung cells. In addition, an antibody targeting the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs, including the currently dominant Omicron variant, are strongly dependent on IFITM2 for efficient replication, suggesting a key proviral role of IFITMs in viral transmission and pathogenicity.


Subject(s)
Lung , Membrane Proteins , SARS-CoV-2 , Virus Replication , COVID-19/virology , Cell Line, Tumor , Humans , Lung/virology , Membrane Proteins/genetics , Membrane Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Virus Internalization
7.
J Infect ; 84(6): 825-833, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1799830

ABSTRACT

BACKGROUND: Recent evidence has linked the interferon-induced transmembrane protein 3 gene (IFITM3) to coronavirus disease 2019 (COVID-19) outcomes, but the results are inconsistent. The purpose of this meta-analysis was to evaluate the association of IFITM3 gene polymorphisms with COVID-19 susceptibility and severity. METHOD: A systematic search was performed with PubMed, Web of Science, Cochrane Library, and Embase from the date of inception to 20 December 2021. The results were analyzed with pooled odds ratios (ORs) and 95% confidence intervals (95% CIs). The robustness was performed using the method of sequential removal for each trial. RESULTS: Four studies involving 1989 subjects were included, from which 1114 patients were positive for COVID-19. For IFITM3 rs12252, the pooled OR showed that there was a significant association between the genotype frequencies and infection with COVID-19 in any of the gene models, i.e., the allelic model (OR = 1.91, 95% CI, 1.36-2.68), the dominant model (OR = 1.80, 95% CI, 1.27-2.56), the recessive model (OR = 5.67, 95% CI, 1.01-31.77), the heterozygous model (OR = 1.65, 95% CI, 1.16-2.36) and the homozygous model (OR = 5.88, 95% CI, 1.05-32.98). The results stratified by severity showed that there was a significant correlation only between the allelic (OR = 0.69, 95% CI, 0.49-0.97) and recessive (OR = 0.43, 95% CI, 0.20-0.93) models. Our results did not support the associations between the IFITM3 rs34481144 gene polymorphism and COVID-19 susceptibility or severity in any of the gene models. CONCLUSIONS: The findings indicated that IFITM3 rs12252 gene polymorphisms were associated with COVID-19 susceptibility and that the rs12252-C variant was particularly critical for severity. Genetic factors should be considered in future vaccine development.


Subject(s)
COVID-19 , Influenza, Human , COVID-19/genetics , Genetic Predisposition to Disease , Humans , Interferons/genetics , Membrane Proteins/genetics , Polymorphism, Single Nucleotide , RNA-Binding Proteins/genetics
8.
Cell Death Dis ; 13(3): 269, 2022 03 25.
Article in English | MEDLINE | ID: covidwho-1764162

ABSTRACT

Stimulator of interferon genes (STING) contributes to immune responses against tumors and may control viral infection including SARS-CoV-2 infection. However, activation of the STING pathway by airway silica or smoke exposure leads to cell death, self-dsDNA release, and STING/type I IFN dependent acute lung inflammation/ARDS. The inflammatory response induced by a synthetic non-nucleotide-based diABZI STING agonist, in comparison to the natural cyclic dinucleotide cGAMP, is unknown. A low dose of diABZI (1 µg by endotracheal route for 3 consecutive days) triggered an acute neutrophilic inflammation, disruption of the respiratory barrier, DNA release with NET formation, PANoptosis cell death, and inflammatory cytokines with type I IFN dependent acute lung inflammation. Downstream upregulation of DNA sensors including cGAS, DDX41, IFI204, as well as NLRP3 and AIM2 inflammasomes, suggested a secondary inflammatory response to dsDNA as a danger signal. DNase I treatment, inhibition of NET formation together with an investigation in gene-deficient mice highlighted extracellular DNA and TLR9, but not cGAS, as central to diABZI-induced neutrophilic response. Therefore, activation of acute cell death with DNA release may lead to ARDS which may be modeled by diABZI. These results show that airway targeting by STING activator as a therapeutic strategy for infection may enhance lung inflammation with severe ARDS. STING agonist diABZI induces neutrophilic lung inflammation and PANoptosis A, Airway STING priming induce a neutrophilic lung inflammation with epithelial barrier damage, double-stranded DNA release in the bronchoalvelolar space, cell death, NETosis and type I interferon release. B, 1. The diamidobenzimidazole (diABZI), a STING agonist is internalized into the cytoplasm through unknown receptor and induce the activation and dimerization of STING followed by TBK1/IRF3 phosporylation leading to type I IFN response. STING activation also leads to NF-kB activation and the production of pro-inflammatory cytokines TNFα and IL-6. 2. The activation of TNFR1 and IFNAR1 signaling pathway results in ZBP1 and RIPK3/ASC/CASP8 activation leading to MLKL phosphorylation and necroptosis induction. 3. This can also leads to Caspase-3 cleavage and apoptosis induction. 4. Self-dsDNA or mtDNA sensing by NLRP3 or AIM2 induces inflammsome formation leading to Gasdermin D cleavage enabling Gasdermin D pore formation and the release mature IL-1ß and pyroptosis. NLRP3 inflammasome formation can be enhanced by the ZBP1/RIPK3/CASP8 complex. 5. A second signal of STING activation with diABZI induces cell death and the release of self-DNA which is sensed by cGAS and form 2'3'-cGAMP leading to STING hyper activation, the amplification of TBK1/IRF3 and NF-kB pathway and the subsequent production of IFN-I and inflammatory TNFα and IL-6. This also leads to IFI204 and DDX41 upregulation thus, amplifying the inflammatory loop. The upregulation of apoptosis, pyroptosis and necroptosis is indicative of STING-dependent PANoptosis.


Subject(s)
COVID-19 , Respiratory Distress Syndrome , Animals , Cytokines/metabolism , DNA , Inflammasomes/metabolism , Interleukin-6/metabolism , Mice , NF-kappa B/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , RNA-Binding Proteins , Respiratory Distress Syndrome/genetics , SARS-CoV-2 , Tumor Necrosis Factor-alpha/metabolism
9.
J Biol Chem ; 297(6): 101362, 2021 12.
Article in English | MEDLINE | ID: covidwho-1751075

ABSTRACT

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Diterpenes, Kaurane/pharmacology , RNA-Binding Proteins/metabolism , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Binding Sites/drug effects , Biological Products/chemistry , Biological Products/pharmacology , COVID-19/metabolism , COVID-19/virology , Chlorocebus aethiops , Diterpenes, Kaurane/chemistry , Humans , Molecular Docking Simulation , RNA-Binding Proteins/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , Vero Cells , Viral Nonstructural Proteins/chemistry
10.
Nucleic Acids Res ; 50(5): 2509-2521, 2022 03 21.
Article in English | MEDLINE | ID: covidwho-1722548

ABSTRACT

Upon SARS-CoV-2 infection, viral intermediates specifically activate the IFN response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to viral A-to-I RNA editing. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, varied between tissue and cell types, and was correlated with the intensity of innate immune response. On average, 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans during the epidemic. Our study highlights the ability of SARS-CoV-2 to hijack components of the host antiviral machinery to edit its genome and fuel its evolution, and also provides a framework and resource for studying viral RNA editing.


Subject(s)
COVID-19/immunology , Immunity, Innate/immunology , RNA Editing/immunology , SARS-CoV-2/immunology , Adenosine Deaminase/genetics , Adenosine Deaminase/immunology , Adenosine Deaminase/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Base Sequence , Binding Sites/genetics , COVID-19/genetics , COVID-19/virology , Evolution, Molecular , Gene Expression/immunology , Humans , Immunity, Innate/genetics , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferon-Induced Helicase, IFIH1/metabolism , Mutation , Protein Binding , RNA Editing/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/immunology , RNA-Binding Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Homology, Nucleic Acid , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
11.
Brief Bioinform ; 23(3)2022 05 13.
Article in English | MEDLINE | ID: covidwho-1713563

ABSTRACT

SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein-coding loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modeling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/genetics , Genome, Viral , Humans , Pandemics , RNA, Viral/chemistry , RNA, Viral/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics
12.
Sci Rep ; 12(1): 2420, 2022 02 14.
Article in English | MEDLINE | ID: covidwho-1684102

ABSTRACT

The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA degradation. This enzyme has recently been shown to be capable of restricting SARS-CoV-2. These data have led to the hypothesis that the low abundance of CpG in the SARS-CoV-2 genome is due to an evolutionary pressure exerted by the host ZAP. To investigate this hypothesis, we performed a detailed analysis of many coronavirus sequences and ZAP RNA binding preference data. Our analyses showed neither evidence for an evolutionary pressure acting specifically on CpG dinucleotides, nor a link between the activity of ZAP and the low CpG abundance of the SARS-CoV-2 genome.


Subject(s)
COVID-19/genetics , Dinucleoside Phosphates/genetics , Genome, Viral/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Animals , Base Sequence , Binding Sites/genetics , COVID-19/virology , Dinucleoside Phosphates/metabolism , Evolution, Molecular , Host-Pathogen Interactions/genetics , Humans , Nucleotide Motifs/genetics , Protein Binding , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/physiology , Virus Replication/genetics
13.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Article in English | MEDLINE | ID: covidwho-1642083

ABSTRACT

Adenosine deaminases acting on RNA (ADAR) are RNA-editing enzymes that may restrict viral infection. We have utilized deep sequencing to determine adenosine to guanine (A→G) mutations, signifying ADAR activity, in clinical samples retrieved from 93 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients in the early phase of the COVID-19 pandemic. A→G mutations were detected in 0.035% (median) of RNA residues and were predominantly nonsynonymous. These mutations were rarely detected in the major viral population but were abundant in minor viral populations in which A→G was more prevalent than any other mutation (P < 0.001). The A→G substitutions accumulated in the spike protein gene at positions corresponding to amino acids 505 to 510 in the receptor binding motif and at amino acids 650 to 655. The frequency of A→G mutations in minor viral populations was significantly associated with low viral load (P < 0.001). We additionally analyzed A→G mutations in 288,247 SARS-CoV-2 major (consensus) sequences representing the dominant viral population. The A→G mutations observed in minor viral populations in the initial patient cohort were increasingly detected in European consensus sequences between March and June 2020 (P < 0.001) followed by a decline of these mutations in autumn and early winter (P < 0.001). We propose that ADAR-induced deamination of RNA is a significant source of mutated SARS-CoV-2 and hypothesize that the degree of RNA deamination may determine or reflect viral fitness and infectivity.


Subject(s)
Adenosine Deaminase/genetics , COVID-19/epidemiology , Point Mutation , RNA Editing , RNA, Viral/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Adenosine/metabolism , Adenosine Deaminase/metabolism , Adult , Aged , Aged, 80 and over , Amino Acid Substitution , COVID-19/genetics , COVID-19/transmission , COVID-19/virology , Deamination , Female , Genetic Fitness , Genome, Viral , Guanine/metabolism , Host-Pathogen Interactions/genetics , Humans , Male , Middle Aged , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , Signal Transduction , Spike Glycoprotein, Coronavirus/metabolism , Sweden/epidemiology , Viral Load , Virulence
14.
Nucleic Acids Res ; 50(3): 1551-1561, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1636373

ABSTRACT

During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying variants of concern (VOC). Viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host single nucleotide variations (iSNVs). Analysing 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.


Subject(s)
Evolution, Molecular , Genetic Variation/genetics , Genome, Viral/genetics , Host-Pathogen Interactions/genetics , SARS-CoV-2/genetics , APOBEC-1 Deaminase/genetics , Adenosine Deaminase/genetics , Animals , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/virology , Chlorocebus aethiops , Coronavirus RNA-Dependent RNA Polymerase/genetics , Databases, Genetic , Immune Evasion/genetics , India/epidemiology , Phylogeny , RNA-Binding Proteins/genetics , SARS-CoV-2/classification , SARS-CoV-2/growth & development , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
15.
BMC Infect Dis ; 22(1): 47, 2022 Jan 12.
Article in English | MEDLINE | ID: covidwho-1622215

ABSTRACT

BACKGROUND: COVID-19, caused by SARS-CoV-2 has become the most threatening issue to all populations around the world. It is, directly and indirectly, affecting all of us and thus, is an emerging topic dealt in global health. To avoid the infection, various studies have been done and are still ongoing. COVID-19 cases are reported all over the globe, and among the millions of cases, genetic similarity may be seen. The genetical common features seen within confirmed cases may help outline the tendency of infection and degree severity of the disease. Here, we reviewed multiple papers on SNPs related to SARS-CoV-2 infection and analyzed their results. METHODS: The PubMed databases were searched for papers discussing SNPs associated with SARS-CoV-2 infection and severity. Clinical studies with human patients and statistically showing the relevance of the SNP with virus infection were included. Quality Assessment of all papers was done with Newcastle Ottawa Scale. RESULTS: In the analysis, 21 full-text literature out of 2956 screened titles and abstracts, including 63,496 cases, were included. All were human-based clinical studies, some based on certain regions gathered patient data and some based on big databases obtained online. ACE2, TMPRSS2, and IFITM3 are the genes mentioned most frequently that are related to SARS-CoV-2 infection. 20 out of 21 studies mentioned one or more of those genes. The relevant genes according to SNPs were also analyzed. rs12252-C, rs143936283, rs2285666, rs41303171, and rs35803318 are the SNPs that were mentioned at least twice in two different studies. CONCLUSIONS: We found that ACE2, TMPRSS2, and IFITM3 are the major genes that are involved in SARS-CoV-2 infection. The mentioned SNPs were all related to one or more of the above-mentioned genes. There were discussions on certain SNPs that increased the infection and severity to certain groups more than the others. However, as there is limited follow-up and data due to a shortage of time history of the disease, studies may be limited.


Subject(s)
COVID-19 , Population Health , Angiotensin-Converting Enzyme 2/genetics , Humans , Membrane Proteins/genetics , Polymorphism, Single Nucleotide , RNA-Binding Proteins , SARS-CoV-2 , Serine Endopeptidases/genetics
16.
Sci Rep ; 12(1): 364, 2022 01 10.
Article in English | MEDLINE | ID: covidwho-1617003

ABSTRACT

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNAs directing numerous essential roles in cellular physiology. Nuclear Factor 90 (NF90) is an RBP encoded by the interleukin enhancer-binding factor 3 (ILF3) gene that has been found to influence RNA metabolism at several levels, including pre-RNA splicing, mRNA turnover, and translation. To systematically identify the RNAs that interact with NF90, we carried out iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) analysis in the human embryonic fibroblast cell line HEK-293. Interestingly, many of the identified RNAs encoded proteins involved in the response to viral infection and RNA metabolism. We validated a subset of targets and investigated the impact of NF90 on their expression levels. Two of the top targets, IRF3 and IRF9 mRNAs, encode the proteins IRF3 and IRF9, crucial regulators of the interferon pathway involved in the SARS-CoV-2 immune response. Our results support a role for NF90 in modulating key genes implicated in the immune response and offer insight into the immunological response to the SARS-CoV-2 infection.


Subject(s)
COVID-19/metabolism , Immunoprecipitation/methods , Nuclear Factor 90 Proteins/metabolism , RNA-Binding Proteins/metabolism , RNA/metabolism , SARS-CoV-2/metabolism , COVID-19/virology , Cells, Cultured , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics , Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism , Nuclear Factor 90 Proteins/genetics , Protein Binding , RNA/genetics , RNA Interference , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Seq/methods , SARS-CoV-2/genetics , SARS-CoV-2/physiology
17.
Genes (Basel) ; 13(1)2021 12 23.
Article in English | MEDLINE | ID: covidwho-1580896

ABSTRACT

ADAR1-mediated deamination of adenosines in long double-stranded RNAs plays an important role in modulating the innate immune response. However, recent investigations based on metatranscriptomic samples of COVID-19 patients and SARS-COV-2-infected Vero cells have recovered contrasting findings. Using RNAseq data from time course experiments of infected human cell lines and transcriptome data from Vero cells and clinical samples, we prove that A-to-G changes observed in SARS-COV-2 genomes represent genuine RNA editing events, likely mediated by ADAR1. While the A-to-I editing rate is generally low, changes are distributed along the entire viral genome, are overrepresented in exonic regions, and are (in the majority of cases) nonsynonymous. The impact of RNA editing on virus-host interactions could be relevant to identify potential targets for therapeutic interventions.


Subject(s)
Adenosine Deaminase/genetics , COVID-19/genetics , Genome, Viral , Host-Pathogen Interactions/genetics , RNA Editing , RNA, Viral/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Adenosine/metabolism , Adenosine Deaminase/immunology , Animals , COVID-19/metabolism , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , DEAD Box Protein 58/genetics , DEAD Box Protein 58/immunology , Deamination , Epithelial Cells/immunology , Epithelial Cells/virology , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Inosine/metabolism , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferon-beta/genetics , Interferon-beta/immunology , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA, Viral/immunology , RNA-Binding Proteins/immunology , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Transcriptome , Vero Cells
18.
Infect Genet Evol ; 97: 105188, 2022 01.
Article in English | MEDLINE | ID: covidwho-1568934

ABSTRACT

The best and most effective way to combat pandemics is to use effective vaccines and live attenuated vaccines are among the most effective vaccines. However, one of the major problems is the length of time it takes to get the attenuated vaccines. Today, the CRISPR toolkit (Clustered Regularly Inerspaced Short Palindromic Repeats) has made it possible to make changes with high efficiency and speed. Using this toolkit to make point mutations on the RNA virus's genome in a coculture of permissive and nonpermissive cells and under controlled conditions can accelerate changes in the genome and accelerate natural selection to obtain live attenuated vaccines.


Subject(s)
COVID-19 Vaccines/genetics , COVID-19/prevention & control , CRISPR-Cas Systems , Gene Editing/methods , Mutation Rate , SARS-CoV-2/genetics , Viral Proteins/genetics , APOBEC Deaminases/genetics , APOBEC Deaminases/immunology , Adenosine Deaminase/genetics , Adenosine Deaminase/immunology , Bacterial Proteins/genetics , Bacterial Proteins/immunology , COVID-19/immunology , COVID-19 Vaccines/biosynthesis , Endonucleases/genetics , Endonucleases/immunology , Gene Expression , Genome, Viral , Humans , RNA-Binding Proteins/genetics , RNA-Binding Proteins/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/immunology , Selection, Genetic , Vaccines, Attenuated , Viral Proteins/immunology
19.
Nat Commun ; 12(1): 7193, 2021 12 10.
Article in English | MEDLINE | ID: covidwho-1565717

ABSTRACT

Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.


Subject(s)
Frameshifting, Ribosomal , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , SARS-CoV-2/genetics , COVID-19 , HEK293 Cells , Host-Pathogen Interactions , Humans , Nucleic Acid Conformation , Protein Isoforms , Proteome , RNA, Viral/genetics , SARS-CoV-2/physiology , Virus Replication
20.
J Biol Chem ; 297(6): 101362, 2021 12.
Article in English | MEDLINE | ID: covidwho-1545135

ABSTRACT

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Diterpenes, Kaurane/pharmacology , RNA-Binding Proteins/metabolism , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Binding Sites/drug effects , Biological Products/chemistry , Biological Products/pharmacology , COVID-19/metabolism , COVID-19/virology , Chlorocebus aethiops , Diterpenes, Kaurane/chemistry , Humans , Molecular Docking Simulation , RNA-Binding Proteins/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , Vero Cells , Viral Nonstructural Proteins/chemistry
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