Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 186
Filter
1.
Viruses ; 12(6)2020 06 25.
Article in English | MEDLINE | ID: covidwho-1726024

ABSTRACT

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide has highlighted the importance of reliable and rapid diagnostic testing to prevent and control virus circulation. Dozens of monoplex in-house RT-qPCR assays are already available; however, the development of dual-target assays is suited to avoid false-negative results caused by polymorphisms or point mutations, that can compromise the accuracy of diagnostic and screening tests. In this study, two mono-target assays recommended by WHO (E-Sarbeco (enveloppe gene, Charite University, Berlin, Germany) and RdRp-IP4 (RdRp, Institut Pasteur, Paris, France)) were selected and combined in a unique robust test; the resulting duo SARS-CoV-2 RT-qPCR assay was compared to the two parental monoplex tests. The duo SARS-CoV-2 assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. We demonstrated that combining two single systems into a dual-target assay (with or without an MS2-based internal control) did not impair performances, providing a potent tool adapted for routine molecular diagnosis in clinical microbiology laboratories.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA-Dependent RNA Polymerase/genetics , Real-Time Polymerase Chain Reaction/methods , Viral Envelope Proteins/genetics , Viral Nonstructural Proteins/genetics , Betacoronavirus/genetics , COVID-19 , Coronavirus Envelope Proteins , Coronavirus Infections/virology , Coronavirus RNA-Dependent RNA Polymerase , Humans , Pandemics , Pneumonia, Viral/virology , RNA, Viral/analysis , SARS-CoV-2 , Sensitivity and Specificity , World Health Organization
2.
J Med Virol ; 94(4): 1494-1501, 2022 04.
Article in English | MEDLINE | ID: covidwho-1718394

ABSTRACT

Coronavirus disease 2019 (COVID-19) is a severe respiratory disease caused by the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As the COVID-19 pandemic continues, mutations of SARS-CoV-2 accumulate. These mutations may not only make the virus spread faster, but also render current vaccines less effective. In this study, we established a reference sequence for each clade defined using the GISAID typing method. Homology analysis of each reference sequence confirmed a low mutation rate for SARS-CoV-2, with the latest clade GRY having the lowest homology with other clades (99.89%-99.93%), and the homology between other clade being greater than or equal to 99.95%. Variation analyses showed that the earliest genotypes S, V, and G had 2, 3, and 3 characterizing mutations in the genome respectively. The G-derived clades GR, GH, and GV had 5, 6, and 13 characterizing mutations in the genome respectively. A total of 28 characterizing mutations existed in the genome of the latest clades GRY. In addition, we found differences in the geographic distribution of different clades. G, GH, and GR are popular in the USA, while GV and GRY are common in the UK. Our work may facilitate the custom design of antiviral strategies depending on the molecular characteristics of SARS-CoV-2.


Subject(s)
COVID-19/pathology , SARS-CoV-2/genetics , Amino Acid Sequence , COVID-19/virology , Humans , Mutation , Phylogeny , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics
3.
Protein J ; 39(3): 198-216, 2020 06.
Article in English | MEDLINE | ID: covidwho-1718840

ABSTRACT

The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Betacoronavirus/genetics , COVID-19 , Coronavirus Envelope Proteins , Coronavirus Infections/drug therapy , Coronavirus Infections/metabolism , Coronavirus Nucleocapsid Proteins , Drug Discovery/methods , Genome, Viral , Host-Pathogen Interactions/drug effects , Humans , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/drug therapy , Pneumonia, Viral/metabolism , Polyproteins , Protein Interaction Maps/drug effects , SARS-CoV-2 , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Proteins/genetics , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Viroporin Proteins
4.
Commun Biol ; 5(1): 154, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1699831

ABSTRACT

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Exonucleases/antagonists & inhibitors , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Amino Acid Sequence , Anilides/pharmacology , Animals , Base Sequence , Benzimidazoles/pharmacology , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Drug Synergism , Exonucleases/genetics , Exonucleases/metabolism , Humans , Proline/pharmacology , Pyrrolidines/pharmacology , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Valine/pharmacology , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Virus Replication/genetics
5.
Biochem Biophys Res Commun ; 601: 129-136, 2022 04 23.
Article in English | MEDLINE | ID: covidwho-1699331

ABSTRACT

COVID-19, caused by SARS-CoV-2, has been spreading worldwide for more than two years and has led to immense challenges to human health. Despite the great efforts that have been made, our understanding of SARS-CoV-2 is still limited. The viral helicase, NSP13 is an important enzyme involved in SARS-CoV-2 replication and transcription. Here we highlight the important role of the stalk domain in the enzymatic activity of NSP13. Without the stalk domain, NSP13 loses its dsRNA unwinding ability due to the lack of ATPase activity. The stalk domain of NSP13 also provides a rigid connection between the ZBD and helicase domain. We found that the tight connection between the stalk and helicase is necessary for NSP13-mediated dsRNA unwinding. When a short flexible linker was inserted between the stalk and helicase domains, the helicase activity of NSP13 was impaired, although its ATPase activity remained intact. Further study demonstrated that linker insertion between the stalk and helicase domains attenuated the RNA binding ability and affected the thermal stability of NSP13. In summary, our results suggest the crucial role of the stalk domain in NSP13 enzymatic activity and provide mechanistic insight into dsRNA unwinding by SARS-CoV-2 NSP13.


Subject(s)
COVID-19/prevention & control , Methyltransferases/metabolism , RNA Helicases/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Binding Sites/genetics , COVID-19/virology , Enzyme Stability , Humans , Methyltransferases/chemistry , Methyltransferases/genetics , Models, Molecular , Mutation , Protein Conformation , RNA/chemistry , RNA/genetics , RNA/metabolism , RNA Helicases/chemistry , RNA Helicases/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Temperature , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
6.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Article in English | MEDLINE | ID: covidwho-1684241

ABSTRACT

SARS-CoV-2 is a highly pathogenic virus that evades antiviral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the messenger RNA (mRNA) entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here, we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the stem-loop 1 (SL1) region of the SARS-CoV-2 5' untranslated region (5' UTR) is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides inhibits viral translation and makes SARS-CoV-2 5' UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration, as well as providing protection against SARS-CoV-2-induced lethality in transgenic mice expressing human ACE2. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus' own virulence mechanism against itself could force a critical trade-off between drug resistance and pathogenicity.


Subject(s)
5' Untranslated Regions/genetics , Immune Evasion/genetics , Protein Biosynthesis , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Animals , Base Sequence , Chlorocebus aethiops , HEK293 Cells , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Humans , Immune Evasion/drug effects , Mice, Transgenic , Models, Biological , Oligonucleotides, Antisense/pharmacology , Protein Biosynthesis/drug effects , SARS-CoV-2/drug effects , Vero Cells , Virus Replication/drug effects
7.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Article in English | MEDLINE | ID: covidwho-1684239

ABSTRACT

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.


Subject(s)
Exoribonucleases/chemistry , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein Domains , RNA, Viral/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/chemistry , Binding Sites/genetics , COVID-19/virology , Catalytic Domain , Crystallography, X-Ray , Exoribonucleases/genetics , Exoribonucleases/metabolism , Humans , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Mutation, Missense , Protein Binding , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/physiology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
8.
PLoS Comput Biol ; 18(1): e1009804, 2022 01.
Article in English | MEDLINE | ID: covidwho-1637205

ABSTRACT

Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 180-residue protein that blocks translation of host mRNAs in SARS-CoV-2-infected cells. Although it is known that SARS-CoV-2's own RNA evades nsp1's host translation shutoff, the molecular mechanism underlying the evasion was poorly understood. We performed an extended ensemble molecular dynamics simulation to investigate the mechanism of the viral RNA evasion. Simulation results suggested that the stem loop structure of the SARS-CoV-2 RNA 5'-untranslated region (SL1) binds to both nsp1's N-terminal globular region and intrinsically disordered region. The consistency of the results was assessed by modeling nsp1-40S ribosome structure based on reported nsp1 experiments, including the X-ray crystallographic structure analysis, the cryo-EM electron density map, and cross-linking experiments. The SL1 binding region predicted from the simulation was open to the solvent, yet the ribosome could interact with SL1. Cluster analysis of the binding mode and detailed analysis of the binding poses suggest residues Arg124, Lys47, Arg43, and Asn126 may be involved in the SL1 recognition mechanism, consistent with the existing mutational analysis.


Subject(s)
COVID-19/virology , Host-Pathogen Interactions/genetics , SARS-CoV-2 , Untranslated Regions/genetics , Viral Nonstructural Proteins , Computational Biology , Humans , Models, Genetic , Molecular Dynamics Simulation , Protein Binding , Protein Biosynthesis , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
9.
Protein Sci ; 31(2): 333-344, 2022 02.
Article in English | MEDLINE | ID: covidwho-1568299

ABSTRACT

The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-are under intense current scrutiny to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent proteomic screens of the interactions between viral and host proteins have identified the human proteins targeted by SARS-CoV-2. The DNA polymerase α (Pol α)-primase complex or primosome-responsible for initiating DNA synthesis during genomic duplication-was identified as a target of nonstructural protein 1 (nsp1), a major virulence factor in the SARS-CoV-2 infection. Here, we validate the published reports of the interaction of nsp1 with the primosome by demonstrating direct binding with purified recombinant components and providing a biochemical characterization of their interaction. Furthermore, we provide a structural basis for the interaction by elucidating the cryo-electron microscopy structure of nsp1 bound to the primosome. Our findings provide biochemical evidence for the reported targeting of Pol α by the virulence factor nsp1 and suggest that SARS-CoV-2 interferes with Pol α's putative role in the immune response during the viral infection.


Subject(s)
COVID-19 , SARS-CoV-2 , Viral Nonstructural Proteins , Cryoelectron Microscopy , DNA Polymerase I , DNA Primase , Humans , Proteomics , Viral Nonstructural Proteins/genetics , Virulence Factors
10.
PLoS One ; 16(12): e0251834, 2021.
Article in English | MEDLINE | ID: covidwho-1556859

ABSTRACT

Structural characterization of the SARS-CoV-2 full length nsp1 protein will be an essential tool for developing new target-directed antiviral drugs against SARS-CoV-2 and for further understanding of intra- and intermolecular interactions of this protein. As a first step in the NMR studies of the protein, we report the 1H, 13C and 15N resonance backbone assignment as well as the Cß of the apo form of the full-lengthSARS-CoV-2 nsp1 including the folded domain together with the flaking N- and C- terminal intrinsically disordered fragments. The 19.8 kD protein was characterized by high-resolution NMR. Validation of assignment have been done by using two different mutants, H81P and K129E/D48E as well as by amino acid specific experiments. According to the obtained assignment, the secondary structure of the folded domain in solution was almost identical to its previously published X-ray structure as well as another published secondary structure obtained by NMR, but some discrepancies have been detected. In the solution SARS-CoV-2 nsp1 exhibited disordered, flexible N- and C-termini with different dynamic characteristics. The short peptide in the beginning of the disordered C-terminal domain adopted two different conformations distinguishable on the NMR time scale. We propose that the disordered and folded nsp1 domains are not fully independent units but are rather involved in intramolecular interactions. Studies of the structure and dynamics of the SARS-CoV-2 mutant in solution are on-going and will provide important insights into the molecular mechanisms underlying these interactions.


Subject(s)
Magnetic Resonance Spectroscopy/methods , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/chemistry , Amino Acid Sequence , COVID-19/pathology , COVID-19/virology , Carbon-13 Magnetic Resonance Spectroscopy , Humans , Mutation , Nitrogen Isotopes/chemistry , Protein Structure, Secondary , Proton Magnetic Resonance Spectroscopy , SARS-CoV-2/isolation & purification , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
11.
Proc Natl Acad Sci U S A ; 118(49)2021 12 07.
Article in English | MEDLINE | ID: covidwho-1541316

ABSTRACT

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.


Subject(s)
Exoribonucleases/chemistry , Models, Molecular , Protein Conformation , Viral Nonstructural Proteins/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Catalytic Domain , Conserved Sequence , Exoribonucleases/genetics , Exoribonucleases/metabolism , Microbial Viability , Nucleotide Motifs , RNA, Viral/chemistry , RNA, Viral/genetics , RNA-Binding Proteins , Structure-Activity Relationship , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/genetics
12.
Infect Genet Evol ; 97: 105154, 2022 01.
Article in English | MEDLINE | ID: covidwho-1521408

ABSTRACT

The pandemic of COVID-19 has been haunting us for almost the past two years. Although, the vaccination drive is in full swing throughout the world, different mutations of the SARS-CoV-2 virus are making it very difficult to put an end to the pandemic. The second wave in India, one of the worst sufferers of this pandemic, can be mainly attributed to the Delta variant i.e. B.1.617.2. Thus, it is very important to analyse and understand the mutational trajectory of SARS-CoV-2 through the study of the 26 virus proteins. In this regard, more than 17,000 protein sequences of Indian SARS-CoV-2 genomes are analysed using entropy-based approach in order to find the monthly mutational trajectory. Furthermore, Hellinger distance is also used to show the difference of the mutation events between the consecutive months for each of the 26 SARS-CoV-2 protein. The results show that the mutation rates and the mutation events of the viral proteins though changing in the initial months, start stabilizing later on for mainly the four structural proteins while the non-structural proteins mostly exhibit a more constant trend. As a consequence, it can be inferred that the evolution of the new mutative configurations will eventually reduce.


Subject(s)
COVID-19/epidemiology , Genome, Viral , Mutation Rate , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics , Viral Structural Proteins/genetics , COVID-19/virology , Entropy , Epidemiological Monitoring , Evolution, Molecular , Gene Expression , Humans , India/epidemiology , Phylogeny , SARS-CoV-2/classification , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Viral Nonstructural Proteins/classification , Viral Nonstructural Proteins/metabolism , Viral Structural Proteins/classification , Viral Structural Proteins/metabolism
13.
Infect Genet Evol ; 97: 105153, 2022 01.
Article in English | MEDLINE | ID: covidwho-1521407

ABSTRACT

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


Subject(s)
COVID-19/epidemiology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Genome, Viral , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Viral Nonstructural Proteins/chemistry , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Africa/epidemiology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/pharmacology , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Asia/epidemiology , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Databases, Factual , Epidemiological Monitoring , Europe/epidemiology , Evolution, Molecular , Furin/genetics , Furin/metabolism , Gene Expression , Humans , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/classification , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , United States/epidemiology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
14.
Biochem Biophys Res Commun ; 586: 137-142, 2022 01 01.
Article in English | MEDLINE | ID: covidwho-1520712

ABSTRACT

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Host Microbial Interactions/physiology , Membrane Glycoproteins/metabolism , Nuclear Pore Complex Proteins/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Active Transport, Cell Nucleus , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Gene Knockdown Techniques , HeLa Cells , Humans , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/genetics , Models, Biological , Nuclear Envelope/metabolism , Nuclear Envelope/virology , Nuclear Pore Complex Proteins/antagonists & inhibitors , Nuclear Pore Complex Proteins/genetics , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factor RelA/metabolism , Viral Nonstructural Proteins/genetics
15.
BMC Genom Data ; 22(1): 48, 2021 11 13.
Article in English | MEDLINE | ID: covidwho-1515435

ABSTRACT

BACKGROUND: This study aims to characterize SARS-CoV-2 mutations which are primarily prevalent in the Cypriot population. Moreover, using computational approaches, we assess whether these mutations are associated with changes in viral virulence. METHODS: We utilize genetic data from 144 sequences of SARS-CoV-2 strains from the Cypriot population obtained between March 2020 and January 2021, as well as all data available from GISAID. We combine this with countries' regional information, such as deaths and cases per million, as well as COVID-19-related public health austerity measure response times. Initial indications of selective advantage of Cyprus-specific mutations are obtained by mutation tracking analysis. This entails calculating specific mutation frequencies within the Cypriot population and comparing these with their prevalence world-wide throughout the course of the pandemic. We further make use of linear regression models to extrapolate additional information that may be missed through standard statistical analysis. RESULTS: We report a single mutation found in the ORF1ab gene (nucleotide position 18,440) that appears to be significantly enriched within the Cypriot population. The amino acid change is denoted as S6059F, which maps to the SARS-CoV-2 NSP14 protein. We further analyse this mutation using regression models to investigate possible associations with increased deaths and cases per million. Moreover, protein structure prediction tools show that the mutation infers a conformational change to the protein that significantly alters its structure when compared to the reference protein. CONCLUSIONS: Investigating Cyprus-specific mutations for SARS-CoV-2 can lead to a better understanding of viral pathogenicity. Researching these mutations can generate potential links between viral-specific mutations and the unique genomics of the Cypriot population. This can not only lead to important findings from which to battle the pandemic on a national level, but also provide insights into viral virulence worldwide.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/virology , Cyprus , Exoribonucleases/genetics , Humans , Mutation , Phylogeny , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics
16.
Sci Rep ; 11(1): 22164, 2021 11 12.
Article in English | MEDLINE | ID: covidwho-1514425

ABSTRACT

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (ΔNS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of ΔNS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a ΔNS1 virus results in an antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that ΔNS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on ΔNS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.


Subject(s)
Influenza A virus/immunology , Influenza Vaccines/therapeutic use , Interferon Type I/immunology , Orthomyxoviridae Infections/prevention & control , Vaccines, Attenuated/therapeutic use , Viral Nonstructural Proteins/immunology , Adaptive Immunity , Animals , COVID-19/immunology , COVID-19/prevention & control , Chickens , Gene Deletion , Humans , Influenza A virus/genetics , Influenza Vaccines/genetics , Influenza Vaccines/immunology , Influenza, Human/immunology , Influenza, Human/prevention & control , Mice , Mice, Inbred C57BL , Orthomyxoviridae Infections/immunology , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Viral Nonstructural Proteins/genetics
17.
Exp Biol Med (Maywood) ; 246(21): 2332-2337, 2021 11.
Article in English | MEDLINE | ID: covidwho-1507096

ABSTRACT

The coronavirus disease COVID-19 has been the cause of millions of deaths worldwide. Among the SARS-CoV-2 proteins, the non-structural protein 1 (NSP1) has great importance during the virus infection process and is present in both alpha and beta-CoVs. Therefore, monitoring of NSP1 polymorphisms is crucial in order to understand their role during infection and virus-induced pathogenicity. Herein, we analyzed how mutations detected in the circulating SARS-CoV-2 in the population of the city of Manaus, Amazonas state, Brazil could modify the tertiary structure of the NSP1 protein. Three mutations were detected in the SARS-CoV-2 NSP1 gene: deletion of the amino acids KSF from positions 141 to 143 (delKSF), SARS-CoV-2, lineage B.1.195; and two substitutions, R29H and R43C, SARS-CoV-2 lineage B.1.1.28 and B.1.1.33, respectively. The delKSF was found in 47 samples, whereas R29H and R43C were found in two samples, one for each mutation. The NSP1 structures carrying the mutations R43C and R29H on the N-terminal portion (e.g. residues 10 to 127) showed minor backbone divergence compared to the Wuhan model. However, the NSP1 C-terminal region (residues 145 to 180) was severely affected in the delKSF and R29H mutants. The intermediate variable region (residues 144 to 148) leads to changes in the C-terminal region, particularly in the delKSF structure. New investigations must be carried out to analyze how these changes affect NSP1 activity during the infection. Our results reinforce the need for continuous genomic surveillance of SARS-CoV-2 to better understand virus evolution and assess the potential impact of the viral mutations on the approved vaccines and future therapies.


Subject(s)
COVID-19/epidemiology , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Amino Acid Sequence/genetics , Amino Acid Substitution/genetics , Brazil/epidemiology , Humans , Polymorphism, Genetic/genetics , Sequence Deletion/genetics
18.
Virus Res ; 307: 198618, 2022 01 02.
Article in English | MEDLINE | ID: covidwho-1504602

ABSTRACT

The second wave of COVID-19 caused by severe acute respiratory syndrome virus (SARS-CoV-2) is rapidly spreading over the world. Mechanisms behind the flee from current antivirals are still unclear due to the continuous occurrence of SARS-CoV-2 genetic variants. Brazil is the world's second-most COVID-19 affected country. In the present study, we identified the genomic and proteomic variants of Brazilian SARS-CoV-2 isolates. We identified 16 different genotypic variants were found among the 27 isolates. The genotypes of three isolates such as Bra/1236/2021 (G15), Bra/MASP2C844R2/2020 (G11), and Bra/RJ-DCVN5/2020 (G9) have a unique mutant in NSP4 (S184N), 2'O-Mutase (R216N), membrane protein (A2V) and Envelope protein (V5A). A mutation in RdRp of SARS-CoV-2, particularly the change of Pro-to Leu-at 323 resulted in the stabilization of the structure in BRA/CD1739-P4/2020. NSP4, NSP5 protein mutants are more virulent in genotype 15 and 16. A fast protein folding rate changes the structural stability and leads to escape for current antivirals. Thus, our findings help researchers to develop the best potent antivirals based on the new mutant of Brazilian isolates.


Subject(s)
Coronavirus 3C Proteases/genetics , Protein Folding , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Brazil , COVID-19/pathology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Genetic Variation/genetics , Genome, Viral/genetics , Humans , Phosphoproteins/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Virulence/genetics
19.
Adv Biol (Weinh) ; 5(12): e2101113, 2021 12.
Article in English | MEDLINE | ID: covidwho-1487432

ABSTRACT

Following the entry into the host cell, SARS-CoV-2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B-cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID-19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild-type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication.


Subject(s)
COVID-19 , Single-Domain Antibodies , Antiviral Agents , Humans , Magnetic Resonance Spectroscopy , RNA-Binding Proteins , SARS-CoV-2 , Viral Nonstructural Proteins/genetics
20.
Science ; 374(6571): 1099-1106, 2021 Nov 26.
Article in English | MEDLINE | ID: covidwho-1467657

ABSTRACT

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.


Subject(s)
RNA, Viral/genetics , Replicon/physiology , SARS-CoV-2/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antiviral Agents/pharmacology , Cell Line , Humans , Interferons/pharmacology , Microbial Sensitivity Tests , Mutation , Plasmids , RNA, Viral/metabolism , Replicon/genetics , Reverse Genetics , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Saccharomyces cerevisiae/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virion/genetics , Virion/physiology , Virus Replication
SELECTION OF CITATIONS
SEARCH DETAIL