Identification of Conserved Epitopes in SARS-CoV-2 Spike and Nucleocapsid Protein

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel virus that first occurred in Wuhan in December 2019. The spike glycoproteins and nucleocapsid proteins are the most common targets for the development of vaccines and antiviral drugs. Objective: We herein analyze the rate of evolution along with the sequences of spike and nucleocapsid proteins in relation to the spatial locations of their epitopes, previously suggested to contribute to the immune response caused by SARS-CoV-2 infections. Methods: We compare homologous proteins of seven human coronaviruses: HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, and SARS-CoV-2. We then focus on the local, structural order-disorder propensity of the protein regions where the SARS-CoV-2 epitopes are located. Results : We show that most of nucleocapsid protein epitopes overlap the RNA-binding and dimerization domains, and some of them are characterized by a low rate of evolutions. Similarly, spike protein epitopes are preferentially located in regions that are predicted to be ordered and well-conserved, in correspondence of the heptad repeats 1 and 2. Interestingly, both the receptor-binding motif to ACE2 and the fusion peptide of spike protein are characterized by a high rate of evolution. Conclusion: Our results provide evidence for conserved epitopes that might help develop broad-spectrum SARS-CoV-2 vaccines.

Long Term Immune Response Produced by the SputnikV Vaccine

SputnikV is a vaccine against SARS-CoV-2 developed by the Gamaleya National Research Centre for Epidemiology and Microbiology. The vaccine has been shown to induce both humoral and cellular immune responses, yet the mechanisms remain largely unknown. Forty SputnikV vaccinated individuals were included in this study which aimed to demonstrate the location of immunogenic domains of the SARS-CoV-2 S protein using an overlapping peptide library. Additionally, cytokines in the serum of vaccinated and convalescent COVID-19 patients were analyzed. We have found antibodies from both vaccinated and convalescent sera bind to immunogenic regions located in multiple domains of SARS-CoV-2 S protein, including Receptor Binding Domain (RBD), N-terminal Domain (NTD), Fusion Protein (FP) and Heptad Repeats (HRs). Interestingly, many peptides were recognized by immunized and convalescent serum antibodies and correspond to conserved regions in circulating variants of SARS-CoV-2. This breadth of reactivity was still evident 90 days after the first dose of the vaccine, showing that the vaccine has induced a prolonged response. As evidenced by the activation of T cells, cellular immunity strongly suggests the high potency of the SputnikV vaccine against SARS-CoV-2 infection.

Spike Glycoprotein Is Central to Coronavirus Pathogenesis-Parallel Between m-CoV and SARS-CoV-2

Coronaviruses (CoVs) are single-stranded, polyadenylated, enveloped RNA of positive polarity with a unique potential to alter host tropism. This has been exceptionally demonstrated by the emergence of deadly virus outbreaks of the past: Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and Middle East Respiratory Syndrome (MERS-CoV) in 2012. The 2019 outbreak by the new cross-species transmission of SARS-CoV-2 has put the world on alert. CoV infection is triggered by receptor recognition, membrane fusion, and successive viral entry mediated by the surface Spike (S) glycoprotein. S protein is one of the major antigenic determinants and the target for neutralizing antibodies. It is a valuable target in antiviral therapies because of its central role in cell-cell fusion, viral antigen spread, and host immune responses leading to immunopathogenesis. The receptor-binding domain of S protein has received greater attention as it initiates host attachment and contains major antigenic determinants. However, investigating the therapeutic potential of fusion peptide as a part of the fusion core complex assembled by the heptad repeats 1 and 2 (HR1 and HR2) is also warranted. Along with receptor attachment and entry, fusion mechanisms should also be explored for designing inhibitors as a therapeutic intervention. In this article, we review the S protein function and its role in mediating membrane fusion, spread, tropism, and its associated pathogenesis with notable therapeutic strategies focusing on results obtained from studies on a murine β-Coronavirus (m-CoV) and its associated disease process.

H-NS and ToxT inversely control cholera toxin production by binding to overlapping DNA sequences

Vibrio cholerae infects human hosts following ingestion of contaminated food or water, resulting in the severe diarrheal disease cholera. The watery diarrhea that is characteristic of the disease is directly caused by production of cholera toxin (CT). A complex regulatory cascade controls production of CT and other virulence factors. However, ultimately a single protein, ToxT, directly binds to virulence gene promoters and activates their transcription. Previously, we identified two ToxT binding sites, or toxboxes, within the cholera toxin promoter (P ctxAB ). The toxboxes overlap with the two promoter-proximal GATTTTT heptad repeats found within P ctxAB in classical biotype V. cholerae strain O395. These heptad repeats were previously found to be within a large DNA region bound by H-NS, a global transcriptional repressor present in Gram-negative bacteria. The current model for control of P ctxAB transcription proposed complete H-NS displacement from the DNA by ToxT, followed by direct activation by ToxT-RNAP contacts. The goal of this study was to determine more precisely where H-NS binds to P ctxAB and test the hypothesis that ToxT completely displaces H-NS from the P ctxAB promoter before activating transcription. Results suggest that H-NS binds only to the region of P ctxAB encompassing the heptad repeats and ToxT only displaces H-NS from its most promoter proximal binding sites, calling for a revision of the current model involving H-NS and ToxT at P ctxAB . Importance H-NS is a global negative regulator of transcription in Gram negative bacteria, particularly in horizontally acquired genetic islands. Previous work in Vibrio cholerae suggested that H-NS represses transcription of cholera toxin genes by binding to a large region upstream of its promoter, and that virulence activator ToxT derepresses transcription by removing H-NS from the promoter. Here, new data support a revised model in which ToxT only displaces H-NS bound to the most promoter proximal DNA sites that overlap the ToxT binding sites, leaving the upstream sites occupied by H-NS. This introduces a higher resolution mechanism for antirepression of H-NS in control of cholera toxin production.

Elucidating the correlation between the number of TTTTGAT heptamer repeats and cholera toxin promoter activity in Vibrio cholerae O1 pandemic strains

A complex regulatory cascade controls expression of the cholera toxin genes (ctxAB) in Vibrio cholerae; which eventually leads to choleragen (CT) production and secretion, resulting in rice watery diarrhoea. The cholera toxin promoter (PctxAB) contains a series of heptad repeats (5′-TTTTGAT-3′); which have been previously shown to play crucial role in ctxAB transcriptional regulation by recruiting the transcriptional activators ToxT, ToxR, and the nucleoid-associated protein H-NS along the ctx promoter. The numbers of these repeats vary between the two biotypes of V. cholerae O1 strains, and even among strains of the same biotype. In this study, we examined PctxAB activation of V. cholerae O1 pandemic strains to understand the significance of the distal heptad repeats in regulating ctx expression. Interestingly, we found that ctx activation may depend on the number of TTTTGAT heptad repeats within PctxAB, and we posit that the occupation of the distal repeats by H-NS could further prevent transcriptional activation of ctx genes in V. cholerae. We hypothesize that ToxT-dependent transcriptional activation may not require entire displacement of H-NS and propose a revision in the currently accepted model of ToxT dependent PctxAB transcriptional activation.

A live imaging system to analyze spatiotemporal dynamics of RNA polymerase II modification in Arabidopsis thaliana

Spatiotemporal changes in general transcription levels play a vital role in the dynamic regulation of various critical activities. Phosphorylation levels at Ser2 in heptad repeats within the C-terminal domain of RNA polymerase II, representing the elongation form, is an indicator of transcription. However, rapid transcriptional changes during tissue development and cellular phenomena are difficult to capture in living organisms. We introduced a genetically encoded system termed modification-specific intracellular antibody (mintbody) into Arabidopsis thaliana. We developed a protein processing- and 2A peptide-mediated two-component system for real-time quantitative measurement of endogenous modification level. This system enables quantitative tracking of the spatiotemporal dynamics of transcription. Using this method, we observed that the transcription level varies among tissues in the root and changes dynamically during the mitotic phase. The approach is effective for achieving live visualization of the transcription level in a single cell and facilitates an improved understanding of spatiotemporal transcription dynamics.

Brief Bioinformatics characterization of Cotton bZIP transcription factors family from Gossypium hirsutum, Gossypium arboreum and Gossypium raimondii

Cotton is an important commodity in the world economy. In this study we have carried out genome-wide identification and bioinformatics characterization of basic leucine zipper domain proteins (bZIPs) from cultivated cotton species G. hirsutum along with two sub-genome species of allotetraploid cotton, G. arboreum and G. raimondii. A total of 228 bZIP genes of G. hirsutum, 91 bZIP genes of G. arboreum and 86 bZIP genes of G. raimondii were identified from CottonGen database. Cotton bZIP genes were annotated in standard pattern according to their match with Arabidopsis bZIPs. Multiple genes with similar bZIP designations were observed in cotton, linked to the gene duplication. Cotton bZIPs are distributed across all 13 chromosomes with varied density. Phylogenetic characterization of all three cotton species bZIPs classified them into 12 subfamilies, namely A B, C, D, E, F, G, H, I, J, K and S and further into eight subgroups according to their predicted functional similarities, viz., A1, A2, A3, C1, C2, S1, S2 and S3. Subfamily A and S are having maximum number of bZIP genes, subfamily B, H, J and K are single member families. Cotton bZIP protein functions were predicted from identified motifs and orthologs from varied species. BRLZ domain analysis of G. raimondii bZIPs revealed the presence of conserved basic region motif N-X7-R/K in almost all subfamily members, variants are GrbZIP62 with N-X7-I motif and GrbZIP76 with K-X7-R motif. Leucine heptad repeats motif, are also present in variant numbers from two to nine with leucine or other hydrophobic amino acid at designated position among 12 subfamily members. STRING protein interaction network analysis of G. raimondii bZIPs observed strong interaction between A-D, B-K and C-S subfamily members.

Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism

Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the “CTD code” that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing.Saccharomyces cerevisiaeEss1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS)26of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4–5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity.

Probing Protein Folding with Sequence-Reversed α-Helical Bundles

Recurrent protein folding motifs include various types of helical bundles formed by α-helices that supercoil around each other. While specific patterns of amino acid residues (heptad repeats) characterize the highly versatile folding motif of four-α-helical bundles, the significance of the polypeptide chain directionality is not sufficiently understood, although it determines sequence patterns, helical dipoles, and other parameters for the folding and oligomerization processes of bundles. To investigate directionality aspects in sequence-structure relationships, we reversed the amino acid sequences of two well-characterized, highly regular four-α-helical bundle proteins and studied the folding, oligomerization, and structural properties of the retro-proteins, using Circular Dichroism Spectroscopy (CD), Size Exclusion Chromatography combined with Multi-Angle Laser Light Scattering (SEC-MALS), and Small Angle X-ray Scattering (SAXS). The comparison of the parent proteins with their retro-counterparts reveals that while the α-helical character of the parents is affected to varying degrees by sequence reversal, the folding states, oligomerization propensities, structural stabilities, and shapes of the new molecules strongly depend on the characteristics of the heptad repeat patterns. The highest similarities between parent and retro-proteins are associated with the presence of uninterrupted heptad patterns in helical bundles sequences.