scholarly journals High-resolution mapping and characterization of epitopes in COVID-19 patients

2020 ◽  
Author(s):  
Winston A. Haynes ◽  
Kathy Kamath ◽  
Joel Bozekowski ◽  
Elisabeth Baum-Jones ◽  
Melissa Campbell ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Resolution ◽  
Antibody Response ◽  
Severe Disease ◽  
Host Protein ◽  
Single Amino Acid ◽  
Furin Cleavage ◽  
Disease Heterogeneity ◽  
Furin Cleavage Site ◽  
Wide Range

AbstractFine scale delineation of epitopes recognized by the antibody response to SARS-CoV-2 infection will be critical to understanding disease heterogeneity and informing development of safe and effective vaccines and therapeutics. The Serum Epitope Repertoire Analysis (SERA) platform leverages a high diversity random bacterial display library to identify epitope binding specificities with single amino acid resolution. We applied SERA broadly, across human, viral and viral strain proteomes in multiple cohorts with a wide range of outcomes from SARS-CoV-2 infection. We identify dominant epitope motifs and profiles which effectively classify COVID-19, distinguish mild from severe disease, and relate to neutralization activity. We identify a repertoire of epitopes shared by SARS-CoV-2 and endemic human coronaviruses and determine that a region of amino acid sequence identity shared by the SARS-CoV-2 furin cleavage site and the host protein ENaC-alpha is a potential cross-reactive epitope. Finally, we observe decreased epitope signal for mutant strains which points to reduced antibody response to mutant SARS-CoV-2. Together, these findings indicate that SERA enables high resolution of antibody epitopes that can inform data-driven design and target selection for COVID-19 diagnostics, therapeutics and vaccines.

scholarly journals Insight towards the effect of the multibasic cleavage site of SARS-CoV-2 spike protein on cellular proteases

2020 ◽  
Author(s):  
Kamal Shokeen ◽  
Shambhavi Pandey ◽  
Manisha Shah ◽  
Sachin Kumar
Keyword(s):  
Amino Acid ◽  
Host Cell ◽  
Cleavage Site ◽  
Decisive Role ◽  
Cell Receptor ◽  
Single Amino Acid ◽  
Furin Cleavage ◽  
Proteolytic Activation ◽  
S Protein ◽  
Furin Cleavage Site

AbstractSevere respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents an immense global health problem. Spike (S) protein of coronavirus is the primary determinant of its entry into the host as it consists of both receptor binding and fusion domain. While tissue tropism, host range, and pathogenesis of coronavirus are primarily controlled by the interaction of S protein with the cell receptor, it is possible that proteolytic activation of S protein by host cell proteases also plays a decisive role. The host-cell proteases have shown to be involved in the proteolysis of S protein and cleaving it into two functional subunits, S1 and S2, during the maturation process. In the present study, the interaction of S protein of SARS-CoV-2 with different host proteases like furin, cathepsin B, and plasmin has been analyzed. Incorporation of the furin cleavage site (R-R-A-R) in the S protein in SARS-CoV-2 has been studied by mutating the individual amino acid. Our results suggest the polytropic nature of the S protein of SARS-CoV-2. Our analysis indicated that a single amino acid substitution in the polybasic cleavage site of S protein perturb the binding of cellular proteases. This mutation study might help to generate an attenuated SARS-CoV-2. Besides, targeting of host proteases by inhibitors may result in a practical approach to stop the cellular spread of SARS-CoV-2 and to develop its antiviral.

scholarly journals The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

2021 ◽  
Vol 22 (12) ◽  
pp. 6490
Author(s):  
Olga A. Postnikova ◽  
Sheetal Uppal ◽  
Weiliang Huang ◽  
Maureen A. Kane ◽  
Rafael Villasmil ◽  
...  
Keyword(s):  
Amino Acid ◽  
Insertion Sequence ◽  
Experimental Studies ◽  
Spike Protein ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
New Host ◽  
S Protein ◽  
Furin Cleavage Site ◽  
Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

scholarly journals Natural variants in SARS-CoV-2 Spike protein pinpoint structural and functional hotspots with implications for prophylaxis and therapeutic strategies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Suman Pokhrel ◽  
Benjamin R. Kraemer ◽  
Scott Burkholz ◽  
Daria Mochly-Rosen
Keyword(s):  
Amino Acid ◽  
Severe Disease ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Severe Respiratory Distress ◽  
S Protein ◽  
Individual Sequences ◽  
Natural Variants ◽  
Novel Coronavirus ◽  
The Individual

AbstractIn December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.

Sorbitol and proline as intracellular osmotic solutes in the green alga Stichococcus bacillaris

1978 ◽  
Vol 56 (6) ◽  
pp. 676-679 ◽  
Author(s):  
Lewis M. Brown ◽  
Johan A. Hellebust
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
High Salinity ◽  
Single Amino Acid ◽  
Salinity Range ◽  
Wide Range ◽  
Osmotic Solutes ◽  
Steady State Levels ◽  
Total Amino Acids ◽  
Stichococcus Bacillaris

A freshwater isolate of Stichococcus bacillaris Naeg. (strain UTEX 314) was evaluated for its ability to grow, photosynthesize, and osmoregulate over a wide range of salinity. The growth and photosynthetic measurements indicate that it is a euryhaline organism. Studies of the soluble organic metabolite pools showed that the steady-state levels of two solutes varied with salinity; sorbitol (a polyol) and proline (an amino acid). Intracellular proline levels increased from 0.002 to 0.28 M over the salinity range of 0 to40%c whereas the sorbitol level increased from 0.10 to 0.52 M. The level of total amino acids (excepting proline) remained relatively constant. No single amino acid of this group exceeded an intracellular concentration of 0.04 M. The changes in the concentrations of these solutes accounted for at least 75% of the required increase in intracellular osmolality in cells following adaptation to high salinity media. Sorbitol and proline are very soluble, nontoxic, and are efficient osmotic solutes. These properties make them ideal solutes for osmoregulation.

scholarly journals Sequence Analysis Indicates that 2019-nCoV Virus Contains a Putative Furin Cleavage Site at the Boundary of S1 and S2 Domains of Spike Protein

2020 ◽  
Author(s):  
Z. Galen WO
Keyword(s):  
Amino Acid ◽  
Cleavage Site ◽  
Spike Protein ◽  
Sequence Motif ◽  
Furin Cleavage ◽  
Host Cell Membrane ◽  
Virus Family ◽  
Prediction Program ◽  
Furin Cleavage Site ◽  
Novel Coronavirus

The infectious 2019-nCoV virus, which caused the current novel coronavirus pneumonia epidemic outbreak, possesses a unique 4-Amino Acid insert at the boundary of the two subdomains (S1 and S2) of Spike protein based on multiple protein sequence alignment with the large SARS and SARS-related virus family. Using Bat CoV_RaTG13 Spike protein as reference (sharing 97% aa identity) the 4-amino acid insert can be identified as PRRA (AA position 681-684). The effect of the 4-AA insertion is the presence of a furin signature sequence motif (PRRARSV) at the boundary of S1 and S2 domains of spike protein. This sequence motif consists the required Arg residue for P1 and P4 position of Furin site. In addition, it contains Arg at P3 site as well as Ser at P1’ site of furin motif. This sequence motif matches Aerolysin furin site in FurinDB and was predicted to be moderately strong (score 0.62) by ProP, a protease cleavage site prediction program. This finding suggests that the infectious 2019-nCoV virus, unlike SARS viruses, may be processed via cellular furin recognition and cleavage of the spike protein before host cell membrane fusion and entry. This putative furin site in spike protein of 2019-nCoV virus, if proven to be functional, suggests the potential of looking into agents inhibiting furin as therapeutic mean for the treatment of the novel coronavirus pneumonia.

scholarly journals Site-Directed Amino Acid Substitutions in the Hydroxylase α Subunit of Butane Monooxygenase from Pseudomonas butanovora: Implications for Substrates Knocking at the Gate

2006 ◽  
Vol 188 (13) ◽  
pp. 4962-4969 ◽  
Author(s):  
Kimberly H. Halsey ◽  
Luis A. Sayavedra-Soto ◽  
Peter J. Bottomley ◽  
Daniel J. Arp
Keyword(s):  
Amino Acid ◽  
Methane Oxidation ◽  
In Vivo Studies ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Butanol Production ◽  
Α Subunit ◽  
Wide Range ◽  
Pseudomonas Butanovora ◽  
Butane Monooxygenase

ABSTRACT Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase α subunit of butane monooxygenase (BMOH-α) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-α were altered to the corresponding residues from the α subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains G113N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to 1-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 μM methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 μM. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-α.

scholarly journals Primary Structural Variation in Anaplasma marginale Msp2 Efficiently Generates Immune Escape Variants

2015 ◽  
Vol 83 (11) ◽  
pp. 4178-4184 ◽  
Author(s):  
Telmo Graça ◽  
Lydia Paradiso ◽  
Shira L. Broschat ◽  
Susan M. Noh ◽  
Guy H. Palmer
Keyword(s):  
Amino Acid ◽  
Antibody Response ◽  
Gene Conversion ◽  
Structural Variation ◽  
Anaplasma Marginale ◽  
Natural Host ◽  
Single Amino Acid ◽  
Structural Variants ◽  
Content Type ◽  
Expression Site

ABSTRACTAntigenic variation allows microbial pathogens to evade immune clearance and establish persistent infection.Anaplasma marginaleutilizes gene conversion of a repertoire of silentmsp2alleles into a single active expression site to encode unique Msp2 variants. As the genomic complement ofmsp2alleles alone is insufficient to generate the number of variants required for persistence,A. marginaleuses segmental gene conversion, in which oligonucleotide segments from multiple alleles are recombined into the expression site to generate a novelmsp2mosaic not represented elsewhere in the genome. Whether these segmental changes are sufficient to evade a broad antibody response is unknown. We addressed this question by identifying Msp2 variants that differed in primary structure within the immunogenic hypervariable region microdomains and tested whether they represented true antigenic variants. The minimal primary structural difference between variants was a single amino acid resulting from a codon insertion, and overall, the amino acid identity among paired microdomains ranged from 18 to 92%. Collectively, 89% of the expressed structural variants were also antigenic variants across all biological replicates, independent of a specific host major histocompatibility complex haplotype. Biological relevance is supported by the following: (i) all structural variants were expressed during infection of a natural host, (ii) the structural variation observed in the microdomains corresponded to the mean length of variants generated by segmental gene conversion, and (iii) antigenic variants were identified using a broad antibody response that developed during infection of a natural host. The findings demonstrate that segmental gene conversion efficiently generates Msp2 antigenic variants.

scholarly journals Conservation of single amino-acid polymorphisms in Plasmodium falciparum erythrocyte membrane protein 1 and association with severe pathophysiology

2017 ◽  
Author(s):  
Daniel Zinder ◽  
Mary M. Rorick ◽  
Kathryn E. Tiedje ◽  
Shazia Ruybal-Pesántez ◽  
Karen P. Day ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Severe Disease ◽  
Single Amino Acid ◽  
Sequence Motifs ◽  
Parasite Protein ◽  
Erythrocyte Membrane Protein

ABSTRACTPlasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a parasite protein encoded by a multigene family known as var. Expressed on the surface of infected red blood cells, PfEMP1 plays a central role in parasite virulence. The DBLα domain of PfEMP1 contains short sequence motifs termed homology blocks. Variation within homology blocks, at the level of single amino-acid modifications, has not been considered before in association with severe disease. Here we identify a total of 2701 amino-acid polymorphisms within DBLα homology blocks, the majority of which are shared between two geographically distant study populations in existing transcription data from Kenya and in a new genomic dataset sampled in Ghana. Parasitemia levels and the transcription levels of specific polymorphisms are as predictive of severe disease (AUC=0.83) and of the degree of rosetting (forecast skill SS=0.45) as the transcription of classic var groups. 11 newly categorized polymorphisms were strongly correlated with grpA var gene expression (SS=0.93) and a different set of 16 polymorphisms was associated with the H3 subset (SS=0.20). These associations provide the basis for a novel method of relating pathophysiology to parasite gene expression levels—one that, being site-specific, has more molecular detail than previous models based on var groups or homology blocks. This newly described variation influences disease outcome, and can help develop anti-malarial intervention strategies such as vaccines that target severe disease. Further replication of this analysis in geographically disparate populations and for larger sample sizes can help improve the identification of the molecular causes of severe disease.

scholarly journals Hemoglobin Genotypes Modulate Inflammatory Response to Plasmodium Infection

2020 ◽  
Vol 11 ◽  
Author(s):  
Keri Oxendine Harp ◽  
Felix Botchway ◽  
Yvonne Dei-Adomakoh ◽  
Michael D. Wilson ◽  
Joshua L. Hood ◽  
...  
Keyword(s):  
Amino Acid ◽  
Inflammatory Response ◽  
Glutamic Acid ◽  
Sickle Cell ◽  
Amino Acid Substitution ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Inflammatory Factor ◽  
Hemoglobin S ◽  
Wide Range

In 2018, 228 million cases and 405,000 malaria-associated deaths were reported worldwide with a majority being in Africa. A wide range of factors, including parasitemia, host immunity, inflammatory responses to infection, and host hemoglobin genotype, mediate the severity of malaria. Among the hemoglobinopathies, hemoglobin S (HbS) is caused by a single amino acid substitution of Glutamic Acid replaced by Valine at the sixth position of the beta-globin chain (E6V). Hemoglobin C (HbC) on the other hand, involves a single amino acid substitution of Glutamic Acid by a Lysine (E6K), which has received the most attention. These substitutions alter the stability of Hb leading to wide-ranging hematological disorders. The homozygous state of hemoglobin S (HbSS) results in sickle cell anemia (SCA) whereas the heterozygous state (HbAS) results in sickle cell trait (SCT). Both mutations are reported to mediate the reduction in the severity and fatality of Plasmodium falciparum malaria. The mechanism underlying this protection is poorly understood. Since both malaria and sickle cell disease (SCD) are associated with the destruction of erythrocytes and widespread systemic inflammation, identifying which inflammatory factor(s) mediate susceptibility of individuals with different hemoglobin genotypes to Plasmodium infection could result in the discovery of new predictive markers and interventions against malaria or SCD severity. We hypothesized that hemoglobin genotypes modulate the inflammatory response to Plasmodium infection. We conducted a cross-sectional study in Ghana, West Africa, between 2014 and 2019 to ascertain the relationships between blood inflammatory cytokines, Plasmodium infection, and hemoglobin genotype. A total of 923 volunteers were enrolled in the study. A total of 74, age and sex-matched subjects were identified with various genotypes including HbAS, HbAC, HbSS, HbSC, HbCC, or HbAA. Complete blood counts and serum inflammatory cytokine expression levels were assessed. The results indicate that differential expression of CXCL10, TNF-α, CCL2, IL-8, and IL-6 were tightly linked to hemoglobin genotype and severity of Plasmodium infection and that these cytokine levels may be predictive for susceptibility to severe malaria or SCD severity.

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