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1.
Viruses ; 14(9)2022 08 24.
Article in English | MEDLINE | ID: covidwho-1997812

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) have significantly impacted the global epidemiology of the pandemic. From December 2020 to April 2022, we conducted genomic surveillance of SARS-CoV-2 in the Southern Province of Zambia, a region that shares international borders with Botswana, Namibia, and Zimbabwe and is a major tourist destination. Genetic analysis of 40 SARS-CoV-2 whole genomes revealed the circulation of Alpha (B.1.1.7), Beta (B.1.351), Delta (AY.116), and multiple Omicron subvariants with the BA.1 subvariant being predominant. Whereas Beta, Delta, and Omicron variants were associated with the second, third, and fourth pandemic waves, respectively, the Alpha variant was not associated with any wave in the country. Phylogenetic analysis showed evidence of local transmission and possible multiple introductions of SARS-CoV-2 VOCs in Zambia from different European and African countries. Across the 40 genomes analysed, a total of 292 mutations were observed, including 182 missense mutations, 66 synonymous mutations, 23 deletions, 9 insertions, 1 stop codon, and 11 mutations in the non-coding region. This study stresses the need for the continued monitoring of SARS-CoV-2 circulation in Zambia, particularly in strategically positioned regions such as the Southern Province which could be at increased risk of introduction of novel VOCs.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiology , Codon, Terminator , Genomics , Humans , Mutation , Phylogeny , SARS-CoV-2/genetics , Zambia/epidemiology
2.
PLoS One ; 17(5): e0267295, 2022.
Article in English | MEDLINE | ID: covidwho-1865340

ABSTRACT

Since the first reported case of coronavirus disease 2019 (COVID-19) in China, SARS-CoV-2 has been spreading worldwide. Genomic surveillance of SARS-CoV-2 has had a critical role in tracking the emergence, introduction, and spread of new variants, which may affect transmissibility, pathogenicity, and escape from infection or vaccine-induced immunity. As anticipated, the rapid increase in COVID-19 infections in Iraq in February 2021 is due to the introduction of variants of concern during the second wave of the COVID-19 pandemic. To understand the molecular epidemiology of SARS-CoV-2 during the second wave in Iraq (2021), we sequenced 76 complete SARS-CoV-2 genomes using NGS technology and identified genomic mutations and proportions of circulating variants among these. Also, we performed an in silico study to predict the effect of the truncation of NS7a protein (ORF7a) on its function. We detected nine different lineages of SARS-CoV-2. The B.1.1.7 lineage was predominant (80.20%) from February to May 2021, while only one B.1.351 strain was detected. Interestingly, the phylogenetic analysis showed that multiple strains of the B.1.1.7 lineage clustered closely with those from European countries. A notable frequency (43.33%) of stop codon mutation (NS7a Q62stop) was detected among the B.1.1.7 lineage sequences. In silico analysis of NS7a with Q62stop found that this stop codon had no considerable effect on the function of NS7a. This work provides molecular epidemiological insights into the spread variants of SARS-CoV-2 in Iraq, which are most likely imported from Europe.


Subject(s)
COVID-19 , SARS-CoV-2 , Viral Proteins/genetics , COVID-19/epidemiology , Codon, Nonsense , Codon, Terminator , Humans , Iraq/epidemiology , Mutation , Pandemics , Phylogeny , Prevalence , SARS-CoV-2/genetics
3.
RNA ; 28(2): 239-249, 2022 02.
Article in English | MEDLINE | ID: covidwho-1542151

ABSTRACT

SARS-CoV-2 produces two long viral protein precursors from one open reading frame using a highly conserved RNA pseudoknot that enhances programmed -1 ribosomal frameshifting. The 1.3 Å-resolution X-ray structure of the pseudoknot reveals three coaxially stacked helices buttressed by idiosyncratic base triples from loop residues. This structure represents a frameshift-stimulating state that must be deformed by the ribosome and exhibits base-triple-adjacent pockets that could be targeted by future small-molecule therapeutics.


Subject(s)
Frameshifting, Ribosomal , Nucleic Acid Conformation , RNA, Viral/chemistry , SARS-CoV-2/genetics , Codon, Terminator , Crystallography, X-Ray , Models, Molecular , Mutation , RNA, Viral/genetics
4.
RNA Biol ; 18(sup2): 804-817, 2021 11 12.
Article in English | MEDLINE | ID: covidwho-1522048

ABSTRACT

Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.


Subject(s)
Protein Biosynthesis , SARS-CoV-2 , Viral Nonstructural Proteins/physiology , Animals , Cell-Free System , Codon, Terminator/metabolism , GTP Phosphohydrolases/metabolism , HeLa Cells , Humans , Mutation , Peptide Chain Termination, Translational , Peptide Termination Factors/chemistry , Peptide Termination Factors/metabolism , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Domains , RNA, Messenger/metabolism , Rabbits , Ribosomes/metabolism
5.
Viruses ; 13(7)2021 06 25.
Article in English | MEDLINE | ID: covidwho-1389549

ABSTRACT

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.


Subject(s)
Codon, Terminator/genetics , Coronavirus/genetics , Frameshifting, Ribosomal/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Viral Proteins/metabolism , Base Sequence , Escherichia coli/genetics , Gene Expression Regulation, Viral , Humans , Leukemia Virus, Murine/genetics , RNA Recognition Motif Proteins , RNA, Viral/genetics , Virus Replication
6.
Nucleic Acids Res ; 50(D1): D883-D887, 2022 01 07.
Article in English | MEDLINE | ID: covidwho-1358469

ABSTRACT

Rapidly appearing SARS-CoV-2 mutations can affect T cell epitopes, which can help the virus to evade either CD8 or CD4 T-cell responses. We developed T-cell COVID-19 Atlas (T-CoV, https://t-cov.hse.ru) - the comprehensive web portal, which allows one to analyze how SARS-CoV-2 mutations alter the presentation of viral peptides by HLA molecules. The data are presented for common virus variants and the most frequent HLA class I and class II alleles. Binding affinities of HLA molecules and viral peptides were assessed with accurate in silico methods. The obtained results highlight the importance of taking HLA alleles diversity into account: mutation-mediated alterations in HLA-peptide interactions were highly dependent on HLA alleles. For example, we found that the essential number of peptides tightly bound to HLA-B*07:02 in the reference Wuhan variant ceased to be tight binders for the Indian (Delta) and the UK (Alpha) variants. In summary, we believe that T-CoV will help researchers and clinicians to predict the susceptibility of individuals with different HLA genotypes to infection with variants of SARS-CoV-2 and/or forecast its severity.


Subject(s)
COVID-19/immunology , Databases, Factual , HLA Antigens/metabolism , SARS-CoV-2/genetics , Alleles , COVID-19/virology , Codon, Terminator , Epitopes, T-Lymphocyte/immunology , HLA Antigens/genetics , HLA Antigens/immunology , HLA-B7 Antigen/immunology , Host-Pathogen Interactions , Humans , India , Mutation , SARS-CoV-2/pathogenicity , United Kingdom , Viral Proteins/genetics , Viral Proteins/immunology
7.
Viruses ; 13(7)2021 06 27.
Article in English | MEDLINE | ID: covidwho-1289026

ABSTRACT

Many viruses, especially RNA viruses, utilize programmed ribosomal frameshifting and/or stop codon readthrough in their expression, and in the decoding of a few a UGA is dynamically redefined to specify selenocysteine. This recoding can effectively increase viral coding capacity and generate a set ratio of products with the same N-terminal domain(s) but different C-terminal domains. Recoding can also be regulatory or generate a product with the non-universal 21st directly encoded amino acid. Selection for translation speed in the expression of many viruses at the expense of fidelity creates host immune defensive opportunities. In contrast to host opportunism, certain viruses, including some persistent viruses, utilize recoding or adventitious frameshifting as part of their strategy to evade an immune response or specific drugs. Several instances of recoding in small intensively studied viruses escaped detection for many years and their identification resolved dilemmas. The fundamental importance of ribosome ratcheting is consistent with the initial strong view of invariant triplet decoding which however did not foresee the possibility of transitory anticodon:codon dissociation. Deep level dynamics and structural understanding of recoding is underway, and a high level structure relevant to the frameshifting required for expression of the SARS CoV-2 genome has just been determined.


Subject(s)
DNA Viruses/genetics , DNA Viruses/immunology , Histocompatibility Antigens Class I/immunology , Immune Evasion , RNA Viruses/genetics , Antiviral Agents/pharmacology , Codon, Terminator , DNA Viruses/drug effects , Frameshifting, Ribosomal , Histocompatibility Antigens Class I/genetics , Nucleic Acid Conformation , Peptides/immunology , Protein Biosynthesis , RNA Viruses/drug effects , RNA Viruses/immunology
8.
Science ; 372(6548): 1306-1313, 2021 06 18.
Article in English | MEDLINE | ID: covidwho-1228853

ABSTRACT

Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.


Subject(s)
Frameshifting, Ribosomal , RNA, Viral/genetics , Ribosomes/ultrastructure , SARS-CoV-2/genetics , Viral Proteins/biosynthesis , Animals , Antiviral Agents/pharmacology , Codon, Terminator , Coronavirus RNA-Dependent RNA Polymerase/biosynthesis , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Cryoelectron Microscopy , Fluoroquinolones/pharmacology , Frameshifting, Ribosomal/drug effects , Genome, Viral , Humans , Image Processing, Computer-Assisted , Models, Molecular , Nucleic Acid Conformation , Open Reading Frames , Protein Folding , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Viral Proteins/chemistry , Viral Proteins/genetics , Virus Replication/drug effects
9.
Biochem Biophys Res Commun ; 550: 8-14, 2021 04 23.
Article in English | MEDLINE | ID: covidwho-1101113

ABSTRACT

The SARS-CoV-2 Variant of Concern 202012/01 (VOC-202012/01) emerged in southeast England and rapidly spread worldwide. This variant is believed to be more transmissible, with all attention being given to its spike mutations. However, VOC-202012/01 has also a mutation (Q27stop) that truncates the ORF8, a likely immune evasion protein. Removal of ORF8 changes the clinical outset of the disease, which may affect the virus transmissibility. Here I provide a detailed analysis of all reported ORF8-deficient lineages found in the background of relevant spike mutations, identified among 231,433 SARS-CoV-2 genomes. I found 19 ORF8 nonsense mutations, most of them occurring in the 5' half of the gene. The ORF8-deficient lineages were rare, representing 0.67% of sequenced genomes. Nevertheless, I identified two clusters of related sequences that emerged recently and spread in different countries. The widespread D614G spike mutation was found in most ORF-deficient lineages. Although less frequent, HV69-70del and L5F spike mutations occurred in the background of six different ORF8 nonsense mutations. I also confirmed that VOC-202012/01 is the ORF8-deficient variant with more spike mutations reported to date, although other variants could have up to six spike mutations, some of putative biological relevance. Overall, these results suggest that monitoring ORF8-deficient lineages is important for the progression of the COVID-19 pandemic, particularly when associated with relevant spike mutations.


Subject(s)
COVID-19/transmission , COVID-19/virology , Epidemiological Monitoring , Gene Deletion , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics , COVID-19/epidemiology , Codon, Nonsense , Codon, Terminator/genetics , Evolution, Molecular , Genes, Viral/genetics , Humans , Phylogeny , SARS-CoV-2/pathogenicity , Selection, Genetic , Time Factors , United Kingdom/epidemiology
10.
Emerg Microbes Infect ; 10(1): 252-255, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1059032

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was isolated from the oro/pharyngeal swabs of two Italian COVID-19 patients, physicians in a COVID-19 division hospital, with different courses of the disease. The complete genome sequences show that the two isolates belong to the B1.1 lineage, but contain a nucleotide mutation in the ORF6, leading to a stop codon and to the deletion of 6 amino acids in the C terminus. This deletion was unique, compared to the currently available sequences deposited in the GISAID and GenBank database. It did not affect the in vitro viral replication, neither the neutralizing activities of the patients' antibodies. Based on homology analysis with other Coronaviruses, the two isolated lacked the ORF6 aminoacidic portion responsible for the inhibition of the antiviral Interferon (IFN)-based host response. IFN seems to have a dual role of in SARS-CoV-2 infected patients: not only antiviral activity, but also a detrimental role in case of excessive production. A deletion in the SARS-CoV-2 ORF6 protein might have a specific, still unknown role in the viral pathogenesis.


Subject(s)
COVID-19/virology , Codon, Terminator/genetics , Point Mutation , SARS-CoV-2/genetics , Viral Proteins/genetics , COVID-19/diagnosis , Female , Humans , Male , Middle Aged , SARS-CoV-2/growth & development , SARS-CoV-2/isolation & purification
11.
J Biomol Struct Dyn ; 40(6): 2475-2488, 2022 04.
Article in English | MEDLINE | ID: covidwho-900176

ABSTRACT

The global spread of the coronavirus infections disease - 2019 (COVID-19) and the search for new drugs from natural products particularly from plants are receiving much attention recently. In this study, the therapeutic potential of a new iridoid glycoside isolated from the leaves of Clerodendrum volubile against COVID-19 was investigated. Harpagide 5-O-ß-D-glucopyranoside (HG) was isolated, characterised and investigated for its druglikeness, optimized geometry, and pharmacokinetics properties. Its immunomodulatory was determined by chemiluminescence assay using polymorphonuclear neutrophils (PMNs) in addition to T-cell proliferation assay. In silico analysis was used in determining its molecular interaction with severe acute respiratory syndrome coronavirus-2 (SARS-COV-2). HG displayed potent druglikeness properties, with no inhibitory effect on cytochrome P450 (1A2, 2C19, 2C9, 2D6 and 3A4) and a predicted LD50 of 2000 mg/kg. Its 1H-NMR chemical shifts showed a little deviation of 0.01 and 0.11 ppm for H-4 and H-9, respectively. HG significantly suppressed oxidative bursts in PMNs, while concomitantly inhibiting T-cell proliferation. It also displayed a very strong binding affinity with the translation initiation and termination sequence sites of spike (S) protein mRNA of SARS-COV-2, its gene product, and host ACE2 receptor. These results suggest the immunomodulatory properties and anti-SARS-COV-2 potentials of HG which can be explored in the treatment and management of COVID-19.Communicated by Ramaswamy H. Sarma.


Subject(s)
Clerodendrum , Glucosides/pharmacology , Iridoid Glycosides/pharmacology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus , COVID-19/drug therapy , Clerodendrum/chemistry , Codon, Terminator , Humans , Pyrans , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
12.
Genomics ; 113(1 Pt 2): 892-895, 2021 01.
Article in English | MEDLINE | ID: covidwho-880627

ABSTRACT

Recently the first genome sequences for 11 SARS-CoV-2 isolates from Lebanon became available. Here, we report the detection of variants within the genome of these strains. Pairwise alignment analysis using blastx was performed between these sequences and the UniProtKB data for the SARS-CoV-2 coronavirus to identify amino acid variations. Variants analysis was performed using multiple Bioinformatics tools. We noticed for the first time 18 mutations that have never been reported before. Among those, a frame shift (8651A>) in NSP4, a stop codon 6887A > T in NSP3 and two missense mutations in spike S2 were found. In addition, we found 28 variants in ORF1ab alone. A previously reported variant, 23403A > G, in the spike protein S2 was mostly seen. Two other known mutations 25563G > T in ORF3a and 14408C > T in ORF1ab were detected respectively in 6 and 8 out of the 11 isolates. Our results may help to prognose forthcoming infections in this region.


Subject(s)
COVID-19/virology , Genetic Variation , Genome, Viral , Mutation , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing , Codon, Terminator , Evolution, Molecular , Frameshift Mutation , Humans , Lebanon/epidemiology , Mutation, Missense , Pandemics , SARS-CoV-2/isolation & purification , Sequence Alignment , Spike Glycoprotein, Coronavirus/genetics
13.
RNA Biol ; 18(4): 447-456, 2021 04.
Article in English | MEDLINE | ID: covidwho-786941

ABSTRACT

The SARS-CoV-2, a positive-sense single-stranded RNA Coronavirus, is a global threat to human health. Thus, understanding its life cycle mechanistically would be important to facilitate the design of antiviral drugs. A key aspect of viral progression is the synthesis of viral proteins by the ribosome of the human host. In Coronaviruses, this process is regulated by the viral 5' and 3' untranslated regions (UTRs), but the precise regulatory mechanism has not yet been well understood. In particular, the 5'-UTR of the viral genome is most likely involved in translation initiation of viral proteins. Here, we performed inline probing and RNase V1 probing to establish a model of the secondary structure of SARS-CoV-2 5'-UTR. We found that the 5'-UTR contains stable structures including a very stable four-way junction close to the AUG start codon. Sequence alignment analysis of SARS-CoV-2 variants 5'-UTRs revealed a highly conserved structure with few co-variations that confirmed our secondary structure model based on probing experiments.


Subject(s)
5' Untranslated Regions , RNA, Viral/chemistry , SARS-CoV-2/genetics , Codon, Terminator , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Nucleic Acid Conformation , RNA, Viral/metabolism , Viral Nonstructural Proteins/genetics
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