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1.
Proc Natl Acad Sci U S A ; 118(48)2021 11 30.
Article in English | MEDLINE | ID: covidwho-1517667

ABSTRACT

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of the viral genome into host cells. To understand its detailed entry mechanism and develop a specific entry inhibitor, in situ structural information on the SARS-CoV-2 spike protein in different states is urgent. Here, by using cryo-electron tomography, we observed both prefusion and postfusion spikes in ß-propiolactone-inactivated SARS-CoV-2 virions and solved the in situ structure of the postfusion spike at nanometer resolution. Compared to previous reports, the six-helix bundle fusion core, the glycosylation sites, and the location of the transmembrane domain were clearly resolved. We observed oligomerization patterns of the spikes on the viral membrane, likely suggesting a mechanism of fusion pore formation.


Subject(s)
SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Motifs , Animals , Chlorocebus aethiops , Cryoelectron Microscopy , Electron Microscope Tomography , Glycosylation , Protein Domains , Protein Multimerization , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
2.
J Exp Med ; 218(12)2021 12 06.
Article in English | MEDLINE | ID: covidwho-1462245

ABSTRACT

Broadly protective vaccines against SARS-related coronaviruses that may cause future outbreaks are urgently needed. The SARS-CoV-2 spike receptor-binding domain (RBD) comprises two regions, the core-RBD and the receptor-binding motif (RBM); the former is structurally conserved between SARS-CoV-2 and SARS-CoV. Here, in order to elicit humoral responses to the more conserved core-RBD, we introduced N-linked glycans onto RBM surfaces of the SARS-CoV-2 RBD and used them as immunogens in a mouse model. We found that glycan addition elicited higher proportions of the core-RBD-specific germinal center (GC) B cells and antibody responses, thereby manifesting significant neutralizing activity for SARS-CoV, SARS-CoV-2, and the bat WIV1-CoV. These results have implications for the design of SARS-like virus vaccines.


Subject(s)
Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/immunology , Polysaccharides/immunology , SARS Virus/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Motifs , Animals , COVID-19/genetics , COVID-19/prevention & control , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Female , Humans , Male , Mice , Mice, Inbred BALB C , Polysaccharides/genetics , Protein Domains , SARS Virus/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
3.
Front Immunol ; 12: 707977, 2021.
Article in English | MEDLINE | ID: covidwho-1457901

ABSTRACT

The ongoing COVID-19 pandemic caused by SARS-CoV-2 is a huge public health crisis for the globe. The receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein plays a vital role in viral infection and serves as a major target for developing neutralizing antibodies. In this study, the antibody response to the RBD of SARS-CoV-2 S protein was analyzed by a panel of sera from animals immunized with RBD-based antigens and four linear B-cell epitope peptides (R345, R405, R450 and R465) were revealed. The immunogenicity of three immunodominant peptides (R345, R405, R465) was further accessed by peptide immunization in mice, and all of them could induced potent antibody response to SARS-CoV-2 S protein, indicating that the three determinants in the RBD were immunogenic. We further generated and characterized monoclonal antibodies (15G9, 12C10 and 10D2) binding to these epitope peptides, and finely mapped the three immunodominant epitopes using the corresponding antibodies. Neutralization assays showed that all three monoclonal antibodies had neutralization activity. Results from IFA and western blotting showed that 12C10 was a cross-reactive antibody against both of SARS-CoV-2 and SARS-CoV. Results from conservative and structural analysis showed that 350VYAWN354 was a highly conserved epitope and exposed on the surface of SARS-CoV-2 S trimer, whereas 473YQAGSTP479 located in the receptor binding motif (RBM) was variable among different SARS-CoV-2 strains. 407VRQIAP412 was a highly conserved, but cryptic epitope shared between SARS-CoV-2 and SARS-CoV. These findings provide important information for understanding the humoral antibody response to the RBD of SARS-CoV-2 S protein and may facilitate further efforts to design SARS-CoV-2 vaccines and the target of COVID-19 diagnostic.


Subject(s)
B-Lymphocytes/immunology , Epitopes, B-Lymphocyte/metabolism , Peptides/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Motifs/genetics , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , COVID-19 Vaccines , Conserved Sequence/genetics , Epitope Mapping , Epitopes, B-Lymphocyte/genetics , HEK293 Cells , Humans , Immunity, Humoral , Peptides/genetics , Protein Binding , Spike Glycoprotein, Coronavirus/genetics
4.
Microbiol Spectr ; 9(2): e0131221, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1443363

ABSTRACT

The large (L) polymerase proteins of most nonsegmented, negative-stranded (NNS) RNA viruses have conserved methyltransferase motifs, (G)-G-G-D and K-D-K-E, which are important for the stabilization and translation of mRNA. However, the function of the (G)-G-G-D and K-D-K-E motifs in the NNS RNA virus Newcastle disease virus (NDV) remains unclear. We observed G-G-D and K-D-K-E motifs in all NDV genotypes. By using the infection cloning system of NDV rSG10 strain, recombinant NDVs with a single amino acid mutated to alanine in one motif (G-G-D or K-D-K-E) were rescued. The intracerebral pathogenicity index and mean death time assay results revealed that the G-G-D motif and K-D-K-E motif attenuate the virulence of NDV to various degrees. The replication, transcription, and translation levels of the K-D-K-E motif-mutant strains were significantly higher than those of wild-type virus owing to their altered regulation of the affinity between nucleocapsid protein and eukaryotic translation initiation factor 4E. When the infection dose was changed from a multiplicity of infection (MOI) of 10 to an MOI of 0.01, the cell-to-cell spread abilities of G-G-D- and K-D-K-E-mutant strains were reduced, according to plaque assay and dynamic indirect immunofluorescence assay results. Finally, we found that NDV strains with G-G-D or K-D-K-E motif mutations had less pathogenicity in 3-week-old specific-pathogen-free chickens than wild-type NDV. Therefore, these methyltransferase motifs can affect virulence by regulating the translation and cell-to-cell spread abilities of NDV. This work provides a feasible approach for generating vaccine candidates for viruses with methyltransferase motifs. IMPORTANCE Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts. Our study found that a deficiency in its methyltransferase motifs (G-G-D and K-D-K-E motifs) can attenuate NDV and revealed the molecular mechanism by which these motifs affect pathogenicity, which provides a new direction for the development of NDV vaccines. In addition to the (G)-G-G-D and K-D-K-E motifs of many nonsegmented, negative-stranded RNA viruses, similar motifs have been found in dengue virus, Zika virus, Japanese encephalitis virus (JEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This suggests that such motifs may be present in more viruses. Our finding also provides a molecular basis for the discovery and functional study of (G)-G-G-D and K-D-K-E motifs of other viruses.


Subject(s)
Amino Acid Motifs/genetics , Methyltransferases/genetics , Newcastle Disease/transmission , Newcastle disease virus/growth & development , Newcastle disease virus/genetics , Viral Proteins/genetics , Animals , Cell Line , Chickens , Chlorocebus aethiops , Cricetinae , Genome, Viral/genetics , Newcastle disease virus/pathogenicity , Poultry Diseases/transmission , Poultry Diseases/virology , RNA, Viral/biosynthesis , RNA, Viral/genetics , Vero Cells , Virulence/genetics , Virus Replication/genetics
5.
Glycobiology ; 31(9): 1080-1092, 2021 09 20.
Article in English | MEDLINE | ID: covidwho-1434394

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Protein Processing, Post-Translational/physiology , SARS-CoV-2/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Coronavirus Nucleocapsid Proteins/chemistry , Glycosylation , HEK293 Cells , Humans , Phosphorylation , SARS-CoV-2/chemistry
6.
J Biol Chem ; 297(4): 101175, 2021 10.
Article in English | MEDLINE | ID: covidwho-1401575

ABSTRACT

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.


Subject(s)
SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Motifs , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Humans , Hydrogen-Ion Concentration , Interferometry , Kinetics , Protein Binding , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry
7.
PLoS One ; 16(9): e0252849, 2021.
Article in English | MEDLINE | ID: covidwho-1403295

ABSTRACT

Reverse vaccinology is an evolving approach for improving vaccine effectiveness and minimizing adverse responses by limiting immunizations to critical epitopes. Towards this goal, we sought to identify immunogenic amino acid motifs and linear epitopes of the SARS-CoV-2 spike protein that elicit IgG in COVID-19 mRNA vaccine recipients. Paired pre/post vaccination samples from N = 20 healthy adults, and post-vaccine samples from an additional N = 13 individuals were used to immunoprecipitate IgG targets expressed by a bacterial display random peptide library, and preferentially recognized peptides were mapped to the spike primary sequence. The data identify several distinct amino acid motifs recognized by vaccine-induced IgG, a subset of those targeted by IgG from natural infection, which may mimic 3-dimensional conformation (mimotopes). Dominant linear epitopes were identified in the C-terminal domains of the S1 and S2 subunits (aa 558-569, 627-638, and 1148-1159) which have been previously associated with SARS-CoV-2 neutralization in vitro and demonstrate identity to bat coronavirus and SARS-CoV, but limited homology to non-pathogenic human coronavirus. The identified COVID-19 mRNA vaccine epitopes should be considered in the context of variants, immune escape and vaccine and therapy design moving forward.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , Epitope Mapping , Amino Acid Motifs , Amino Acid Sequence , Coronavirus Infections/immunology , Humans , Immunoglobulin G/blood , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
9.
Med Hypotheses ; 145: 110342, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1386307

ABSTRACT

This study aimed at identifying human neural proteins that can be attacked by cross-reacting SARS-COV-2 antibodies causing Guillain-Barré syndrome. These markers can be used for the diagnosis of Guillain-Barré syndrome (GBS). To achieve this goal, proteins implicated in the development of GBS were retrieved from literature. These human proteins were compared to SARS-COV-2 surface proteins to identify homologous sequences using Blastp. Then, MHC-I and MHC-II epitopes were determined in the homologous sequences and used for further analysis. Similar human and SARS-COV-2 epitopes were docked to the corresponding MHC molecule to compare the binding pattern of human and SARS-COV-2 proteins to the MHC molecule. Neural cell adhesion molecule is the only neural protein that showed homologous sequence to SARS-COV-2 envelope protein. The homologous sequence was part of HLA-A68 and HLA-DQA/HLA-DQB epitopes had a similar binding pattern to SARS-COV-2 envelope protein. Based on these results, the study suggests that NCAM may play a significant role in the immunopathogenesis of GBS. NCAM antibodies can be used as a marker for Guillain-Barré syndrome. However, more experimental studies are needed to prove these results.


Subject(s)
CD56 Antigen/chemistry , Coronavirus Envelope Proteins/chemistry , Guillain-Barre Syndrome/immunology , SARS-CoV-2 , Viral Proteins/chemistry , Amino Acid Motifs , COVID-19/immunology , Computational Biology , Computer Simulation , Crystallography, X-Ray , Epitopes/chemistry , HLA-A Antigens/chemistry , HLA-DQ alpha-Chains/chemistry , HLA-DQ beta-Chains/chemistry , Humans , Major Histocompatibility Complex , Models, Theoretical , Peptides/chemistry , Protein Binding
10.
Biomolecules ; 11(9)2021 08 25.
Article in English | MEDLINE | ID: covidwho-1374290

ABSTRACT

The receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates the viral-host interaction and is a target for most neutralizing antibodies. Nevertheless, SARS-CoV-2 RBD mutations pose a threat due to their role in host cell entry via the human angiotensin-converting enzyme 2 receptor that might strengthen SARS-CoV-2 infectivity, viral load, or resistance against neutralizing antibodies. To understand the molecular structural link between RBD mutations and infectivity, the top five mutant RBDs (i.e., N501Y, E484K L452R, S477N, and N439K) were selected based on their recorded case numbers. These mutants along with wild-type (WT) RBD were studied through all-atom molecular dynamics (MD) simulations of 100 ns. The principal component analysis and the free energy landscape were used too. Interestingly, N501Y, N439K, and E484K mutations were observed to increase the rigidity in some RBD regions while increasing the flexibility of the receptor-binding motif (RBM) region, suggesting a compensation of the entropy penalty. However, S477N and L452R RBDs were observed to increase the flexibility of the RBM region while maintaining similar flexibility in other RBD regions in comparison to WT RBD. Therefore, both mutations (especially S477N) might destabilize the RBD structure, as loose conformation compactness was observed. The destabilizing effect of S477N RBD was consistent with previous work on S477N mutation. Finally, the free energy landscape results showed that mutations changed WT RBD conformation while local minima were maintained for all mutant RBDs. In conclusion, RBD mutations definitely impact the WT RBD structure and conformation as well as increase the binding affinity to angiotensin-converting enzyme receptor.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , COVID-19 , Mutation, Missense , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Amino Acid Motifs , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Protein Domains , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
11.
Immunity ; 54(10): 2399-2416.e6, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1364126

ABSTRACT

With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here, we developed a panel of neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) that bound the receptor binding domain of the spike protein at distinct epitopes and blocked virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Although several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by ancestral SARS-CoV-2 strains, others induced escape variants in vivo or lost neutralizing activity against emerging strains. One mAb, SARS2-38, potently neutralized all tested SARS-CoV-2 variants of concern and protected mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engaged a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of neutralizing antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.


Subject(s)
Antibodies, Neutralizing/immunology , Epitopes/immunology , SARS-CoV-2/immunology , Amino Acid Motifs , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/therapeutic use , COVID-19/prevention & control , COVID-19/virology , Epitopes/chemistry , Epitopes/metabolism , Humans , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/metabolism , Mice , Neutralization Tests , Protein Domains , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
12.
J Zhejiang Univ Sci B ; 21(5): 343-360, 2020 May.
Article in English | MEDLINE | ID: covidwho-1352746

ABSTRACT

Pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection emerged in Wuhan City, Hubei Province, China in December 2019. By Feb. 11, 2020, the World Health Organization (WHO) officially named the disease resulting from infection with SARS-CoV-2 as coronavirus disease 2019 (COVID-19). COVID-19 represents a spectrum of clinical manifestations that typically include fever, dry cough, and fatigue, often with pulmonary involvement. SARS-CoV-2 is highly contagious and most individuals within the population at large are susceptible to infection. Wild animal hosts and infected patients are currently the main sources of disease which is transmitted via respiratory droplets and direct contact. Since the outbreak, the Chinese government and scientific community have acted rapidly to identify the causative agent and promptly shared the viral gene sequence, and have carried out measures to contain the epidemic. Meanwhile, recent research has revealed critical aspects of SARS-CoV-2 biology and disease pathogenesis; other studies have focused on epidemiology, clinical features, diagnosis, management, as well as drug and vaccine development. This review aims to summarize the latest research findings and to provide expert consensus. We will also share ongoing efforts and experience in China, which may provide insight on how to contain the epidemic and improve our understanding of this emerging infectious disease, together with updated guidance for prevention, control, and critical management of this pandemic.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Pneumonia, Viral/epidemiology , Amino Acid Motifs , Animals , Antiviral Agents , COVID-19 , COVID-19 Vaccines , China/epidemiology , Communicable Disease Control/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/physiopathology , Coronavirus Infections/prevention & control , Coronavirus Infections/therapy , Humans , Immunization, Passive , Medicine, Chinese Traditional , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/physiopathology , Pneumonia, Viral/therapy , Protein Domains , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Viral Vaccines
13.
Acta Crystallogr D Struct Biol ; 77(Pt 8): 1040-1049, 2021 Aug 01.
Article in English | MEDLINE | ID: covidwho-1341166

ABSTRACT

The ß-link is a composite protein motif consisting of a G1ß ß-bulge and a type II ß-turn, and is generally found at the end of two adjacent strands of antiparallel ß-sheet. The 1,2-positions of the ß-bulge are also the 3,4-positions of the ß-turn, with the result that the N-terminal portion of the polypeptide chain is orientated at right angles to the ß-sheet. Here, it is reported that the ß-link is frequently found in certain protein folds of the SCOPe structural classification at specific locations where it connects a ß-sheet to another area of a protein. It is found at locations where it connects one ß-sheet to another in the ß-sandwich and related structures, and in small (four-, five- or six-stranded) ß-barrels, where it connects two ß-strands through the polypeptide chain that crosses an open end of the barrel. It is not found in larger (eight-stranded or more) ß-barrels that are straightforward ß-meanders. In some cases it initiates a connection between a single ß-sheet and an α-helix. The ß-link also provides a framework for catalysis in serine proteases, where the catalytic serine is part of a conserved ß-link, and in cysteine proteases, including Mpro of human SARS-CoV-2, in which two residues of the active site are located in a conserved ß-link.


Subject(s)
Protein Structure, Secondary , Serine Proteases/chemistry , Amino Acid Motifs , Animals , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Cysteine Proteases/chemistry , Cysteine Proteases/metabolism , Databases, Protein , Humans , Hydrogen Bonding , Models, Molecular , SARS-CoV-2/chemistry , SARS-CoV-2/enzymology , Serine Proteases/metabolism , Structural Homology, Protein
14.
FEBS Lett ; 595(17): 2248-2256, 2021 09.
Article in English | MEDLINE | ID: covidwho-1326724

ABSTRACT

The endoplasmic reticulum transmembrane protein vesicle-associated membrane protein-associated protein (VAP) plays a central role in the formation and function of membrane contact sites (MCS) through its interactions with proteins. The major sperm protein (MSP) domain of VAP binds to a variety of sequences which are referred to as FFAT-like motifs. In this study, we investigated the interactions of eight peptides containing FFAT-like motifs with the VAP-A MSP domain (VAP-AMSP ) by solution NMR. Six of eight peptides are specifically bound to VAP-A. Furthermore, we found that the RNA-dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2 has an FFAT-like motif which specifically binds to VAP-AMSP as well as other FFAT-like motifs. Our results will contribute to the discovery of new VAP interactors.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/chemistry , Peptides/chemistry , SARS-CoV-2/enzymology , Vesicular Transport Proteins/chemistry , Amino Acid Motifs , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Nuclear Magnetic Resonance, Biomolecular , Peptides/metabolism , Protein Binding , SARS-CoV-2/metabolism , Vesicular Transport Proteins/metabolism
15.
Nat Microbiol ; 6(8): 1094-1101, 2021 08.
Article in English | MEDLINE | ID: covidwho-1294473

ABSTRACT

Here, we report SARS-CoV-2 genomic surveillance from March 2020 until January 2021 in Uganda, a landlocked East African country with a population of approximately 40 million people. We report 322 full SARS-CoV-2 genomes from 39,424 reported SARS-CoV-2 infections, thus representing 0.8% of the reported cases. Phylogenetic analyses of these sequences revealed the emergence of lineage A.23.1 from lineage A.23. Lineage A.23.1 represented 88% of the genomes observed in December 2020, then 100% of the genomes observed in January 2021. The A.23.1 lineage was also reported in 26 other countries. Although the precise changes in A.23.1 differ from those reported in the first three SARS-CoV-2 variants of concern (VOCs), the A.23.1 spike-protein-coding region has changes similar to VOCs including a change at position 613, a change in the furin cleavage site that extends the basic amino acid motif and multiple changes in the immunogenic N-terminal domain. In addition, the A.23.1 lineage has changes in non-spike proteins including nsp6, ORF8 and ORF9 that are also altered in other VOCs. The clinical impact of the A.23.1 variant is not yet clear and it has not been designated as a VOC. However, our findings of emergence and spread of this variant indicate that careful monitoring of this variant, together with assessment of the consequences of the spike protein changes for COVID-19 vaccine performance, are advisable.


Subject(s)
COVID-19/epidemiology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Motifs , Coronavirus Nucleocapsid Proteins/genetics , Genetic Variation/genetics , Genome, Viral/genetics , Humans , Phosphoproteins/genetics , Phylogeny , Uganda/epidemiology , Viral Proteins/genetics
16.
Viruses ; 13(7)2021 06 24.
Article in English | MEDLINE | ID: covidwho-1289017

ABSTRACT

Arenaviruses and coronaviruses include several human pathogenic viruses, such as Lassa virus, Lymphocytic choriomeningitis virus (LCMV), SARS-CoV, MERS-CoV, and SARS-CoV-2. Although these viruses belong to different virus families, they possess a common motif, the DED/EDh motif, known as an exonuclease (ExoN) motif. In this study, proof-of-concept studies, in which the DED/EDh motif in these viral proteins, NP for arenaviruses, and nsp14 for coronaviruses, could be a drug target, were performed. Docking simulation studies between two structurally different chemical compounds, ATA and PV6R, and the DED/EDh motifs in these viral proteins indicated that these compounds target DED/EDh motifs. The concentration which exhibited modest cell toxicity was used with these compounds to treat LCMV and SARS-CoV-2 infections in two different cell lines, A549 and Vero 76 cells. Both ATA and PV6R inhibited the post-entry step of LCMV and SARS-CoV-2 infection. These studies strongly suggest that DED/EDh motifs in these viral proteins could be a drug target to combat two distinct viral families, arenaviruses and coronaviruses.


Subject(s)
Antiviral Agents/pharmacology , Exoribonucleases/antagonists & inhibitors , Lymphocytic choriomeningitis virus/drug effects , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Virus Replication/drug effects , A549 Cells , Amino Acid Motifs , Animals , Chlorocebus aethiops , Drug Discovery , Humans , Molecular Docking Simulation , Vero Cells
17.
Science ; 373(6551): 236-241, 2021 07 09.
Article in English | MEDLINE | ID: covidwho-1266364

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.


Subject(s)
Coenzymes/metabolism , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Cyclic N-Oxides/pharmacology , Iron/metabolism , SARS-CoV-2/drug effects , Sulfur/metabolism , Amino Acid Motifs , Animals , Antiviral Agents/pharmacology , Binding Sites , Catalytic Domain , Chlorocebus aethiops , Coenzymes/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/pharmacology , Iron/chemistry , Protein Domains , RNA Helicases/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Spin Labels , Sulfur/chemistry , Vero Cells , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Zinc/metabolism
18.
J Biol Chem ; 297(1): 100821, 2021 07.
Article in English | MEDLINE | ID: covidwho-1240418

ABSTRACT

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) necessary for the viral life cycle, but it remains unknown whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are methylated. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG motifs, preferred PRMT target sequences. We confirmed arginine methylation of N protein by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated from cell culture. Type I PRMT inhibitor (MS023) or substitution of R95 or R177 with lysine inhibited interaction of N protein with the 5'-UTR of SARS-CoV-2 genomic RNA, a property required for viral packaging. We also defined the N protein interactome in HEK293 cells, which identified PRMT1 and many of its RGG/RG substrates, including the known interacting protein G3BP1 as well as other components of stress granules (SGs), which are part of the host antiviral response. Methylation of R95 regulated the ability of N protein to suppress the formation of SGs, as R95K substitution or MS023 treatment blocked N-mediated suppression of SGs. Also, the coexpression of methylarginine reader Tudor domain-containing protein 3 quenched N protein-mediated suppression of SGs in a dose-dependent manner. Finally, pretreatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. Because type I PRMT inhibitors are already undergoing clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may represent an additional therapeutic application of these drugs.


Subject(s)
Arginine/metabolism , COVID-19/metabolism , COVID-19/virology , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , SARS-CoV-2/physiology , Amino Acid Motifs , COVID-19/genetics , Cytoplasmic Granules/genetics , Cytoplasmic Granules/metabolism , HEK293 Cells , Humans , Methylation , Nucleocapsid Proteins/genetics , RNA Stability , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Virus Replication
19.
Glycobiology ; 31(9): 1080-1092, 2021 09 20.
Article in English | MEDLINE | ID: covidwho-1231033

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Protein Processing, Post-Translational/physiology , SARS-CoV-2/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Coronavirus Nucleocapsid Proteins/chemistry , Glycosylation , HEK293 Cells , Humans , Phosphorylation , SARS-CoV-2/chemistry
20.
FEBS Lett ; 595(13): 1758-1767, 2021 07.
Article in English | MEDLINE | ID: covidwho-1227709

ABSTRACT

The SARS-CoV-2 spike glycoprotein (spike) mediates viral entry by binding ACE2 receptors on host cell surfaces. Spike glycan processing and cleavage, which occur in the Golgi network, are important for fusion at the plasma membrane, promoting both virion infectivity and cell-to-cell viral spreading. We show that a KxHxx motif in the cytosolic tail of spike weakly binds the COPß' subunit of COPI coatomer, which facilitates some recycling of spike within the Golgi, while releasing the remainder to the cell surface. Although histidine (KxHxx) has been proposed to be equivalent to lysine within di-lysine endoplasmic reticulum (ER) retrieval sequences, we show that histidine-to-lysine substitution (KxKxx) retains spike at the ER and prevents glycan processing, protease cleavage, and transport to the plasma membrane.


Subject(s)
Amino Acid Substitution , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Motifs , Binding Sites , Glycosylation , Golgi Apparatus , HEK293 Cells , HeLa Cells , Histidine/genetics , Humans , Lysine/genetics , Protein Domains , Proteolysis , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
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