SARS-CoV-2 Envelope Protein Forms Clustered Pentamers in Lipid Bilayers.

The SARS-CoV-2 envelope (E) protein is a viroporin associated with the acute respiratory symptoms of COVID-19. E forms cation-selective ion channels that assemble in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment. The channel activity of E is linked to the inflammatory response of the host cell to the virus. Like many viroporins, E is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the E stoichiometry have led to inconclusive results and suggested mixtures of oligomers whose exact nature might vary with the detergent used. Here, we employ 19F solid-state nuclear magnetic resonance and the centerband-only detection of exchange (CODEX) technique to determine the oligomeric number of E's transmembrane domain (ETM) in lipid bilayers. The CODEX equilibrium value, which corresponds to the inverse of the oligomeric number, indicates that ETM assembles into pentamers in lipid bilayers, without any detectable fraction of low-molecular-weight oligomers. Unexpectedly, at high peptide concentrations and in the presence of the lipid phosphatidylinositol, the CODEX data indicate that more than five 19F spins are within a detectable distance of about 2 nm, suggesting that the ETM pentamers cluster in the lipid bilayer. Monte Carlo simulations that take into account peptide-peptide and peptide-lipid interactions yielded pentamer clusters that reproduced the CODEX data. This supramolecular organization is likely important for E-mediated virus assembly and budding and for the channel function of the protein.

Effect of an Amyloidogenic SARS-COV-2 Protein Fragment on α-Synuclein Monomers and Fibrils.

Aggregates of α-synuclein are thought to be the disease-causing agent in Parkinson's disease. Various case studies have hinted at a correlation between COVID-19 and the onset of Parkinson's disease. For this reason, we use molecular dynamics simulations to study whether amyloidogenic regions in SARS-COV-2 proteins can initiate and modulate aggregation of α-synuclein. As an example, we choose the nine-residue fragment SFYVYSRVK (SK9), located on the C-terminal of the envelope protein of SARS-COV-2. We probe how the presence of SK9 affects the conformational ensemble of α-synuclein monomers and the stability of two resolved fibril polymorphs. We find that the viral protein fragment SK9 may alter α-synuclein amyloid formation by shifting the ensemble toward aggregation-prone and preferentially rod-like fibril seeding conformations. However, SK9 has only a small effect on the stability of pre-existing or newly formed fibrils. A potential mechanism and key residues for potential virus-induced amyloid formation are described.

Structure and dynamics of the SARS-CoV-2 envelope protein monomer.

Coronaviruses, especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), present an ongoing threat to human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with the host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic α-helical transmembrane domain (TMD) and amphiphilic α-helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43, and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the monomeric E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane; the protein in the pentameric form displays these properties as well. Localization to curved regions may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate the budding of the viral particles. The presented results may be helpful for a better understanding of the function of the coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.

Elucidation of SARS-Cov-2 Budding Mechanisms through Molecular Dynamics Simulations of M and E Protein Complexes.

SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratory's refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics that inhibit coronavirus budding.

Immunodominant linear B cell epitopes in the spike and membrane proteins of SARS-CoV-2 identified by immunoinformatics prediction and immunoassay.

SARS-CoV-2 continues to infect an ever-expanding number of people, resulting in an increase in the number of deaths globally. With the emergence of new variants, there is a corresponding decrease in the currently available vaccine efficacy, highlighting the need for greater insights into the viral epitope profile for both vaccine design and assessment. In this study, three immunodominant linear B cell epitopes in the SARS-CoV-2 spike receptor-binding domain (RBD) were identified by immunoinformatics prediction, and confirmed by ELISA with sera from Macaca fascicularis vaccinated with a SARS-CoV-2 RBD subunit vaccine. Further immunoinformatics analyses of these three epitopes gave rise to a method of linear B cell epitope prediction and selection. B cell epitopes in the spike (S), membrane (M), and envelope (E) proteins were subsequently predicted and confirmed using convalescent sera from COVID-19 infected patients. Immunodominant epitopes were identified in three regions of the S2 domain, one region at the S1/S2 cleavage site and one region at the C-terminus of the M protein. Epitope mapping revealed that most of the amino acid changes found in variants of concern are located within B cell epitopes in the NTD, RBD, and S1/S2 cleavage site. This work provides insights into B cell epitopes of SARS-CoV-2 as well as immunoinformatics methods for B cell epitope prediction, which will improve and enhance SARS-CoV-2 vaccine development against emergent variants.

SARS-CoV-2 structural coverage map reveals viral protein assembly, mimicry, and hijacking mechanisms.

We modeled 3D structures of all SARS-CoV-2 proteins, generating 2,060 models that span 69% of the viral proteome and provide details not available elsewhere. We found that ˜6% of the proteome mimicked human proteins, while ˜7% was implicated in hijacking mechanisms that reverse post-translational modifications, block host translation, and disable host defenses; a further ˜29% self-assembled into heteromeric states that provided insight into how the viral replication and translation complex forms. To make these 3D models more accessible, we devised a structural coverage map, a novel visualization method to show what is-and is not-known about the 3D structure of the viral proteome. We integrated the coverage map into an accompanying online resource (https://aquaria.ws/covid) that can be used to find and explore models corresponding to the 79 structural states identified in this work. The resulting Aquaria-COVID resource helps scientists use emerging structural data to understand the mechanisms underlying coronavirus infection and draws attention to the 31% of the viral proteome that remains structurally unknown or dark.

Computational Study on the Function of Palmitoylation on the Envelope Protein in SARS-CoV-2.

SARS-CoV-2 that caused COVID-19 has spread since the end of 2019. Its major effects resulted in over four million deaths around the whole world by August 2021. Therefore, understanding virulence mechanisms is important to prevent future outbreaks and for COVID-19 drug development. The envelope (E) protein is an important structural protein, affecting virus assembly and budding. The E protein pentamer is a viroporin, serving as an ion transferring channel in cells. In this work, we applied molecular dynamic simulations and topological and electrostatic analyses to study the effects of palmitoylation on the E protein pentamer. The results indicate that the cation transferring direction is more from the lumen to the cytosol. The structure of the palmitoylated E protein pentamer is more stable while the loss of palmitoylation caused the pore radius to reduce and even collapse. The electrostatic forces on the two sides of the palmitoylated E protein pentamer are more beneficial to attract cations in the lumen and to release cations into the cytosol. The results indicate the importance of palmitoylation, which can help the drug design for the treatment of COVID-19.

Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers.

An essential protein of the SARS-CoV-2 virus, the envelope protein E, forms a homopentameric cation channel that is important for virus pathogenicity. Here we report a 2.1-Å structure and the drug-binding site of E's transmembrane domain (ETM), determined using solid-state NMR spectroscopy. In lipid bilayers that mimic the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane, ETM forms a five-helix bundle surrounding a narrow pore. The protein deviates from the ideal α-helical geometry due to three phenylalanine residues, which stack within each helix and between helices. Together with valine and leucine interdigitation, these cause a dehydrated pore compared with the viroporins of influenza viruses and HIV. Hexamethylene amiloride binds the polar amino-terminal lumen, whereas acidic pH affects the carboxy-terminal conformation. Thus, the N- and C-terminal halves of this bipartite channel may interact with other viral and host proteins semi-independently. The structure sets the stage for designing E inhibitors as antiviral drugs.

NCAM protein and SARS-COV-2 surface proteins: In-silico hypothetical evidence for the immunopathogenesis of Guillain-Barré syndrome.

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.

Structural basis of coronavirus E protein interactions with human PALS1 PDZ domain.

SARS-CoV-2 infection leads to coronavirus disease 2019 (COVID-19), which is associated with severe and life-threatening pneumonia and respiratory failure. However, the molecular basis of these symptoms remains unclear. SARS-CoV-1 E protein interferes with control of cell polarity and cell-cell junction integrity in human epithelial cells by binding to the PALS1 PDZ domain, a key component of the Crumbs polarity complex. We show that C-terminal PDZ binding motifs of SARS-CoV-1 and SARS-CoV-2 E proteins bind the PALS1 PDZ domain with 29.6 and 22.8 µM affinity, whereas the related sequence from MERS-CoV did not bind. We then determined crystal structures of PALS1 PDZ domain bound to both SARS-CoV-1 and SARS-CoV-2 E protein PDZ binding motifs. Our findings establish the structural basis for SARS-CoV-1/2 mediated subversion of Crumbs polarity signalling and serve as a platform for the development of small molecule inhibitors to suppress SARS-CoV-1/2 mediated disruption of polarity signalling in epithelial cells.

Structural basis for SARS-CoV-2 envelope protein recognition of human cell junction protein PALS1.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has created global health and economic emergencies. SARS-CoV-2 viruses promote their own spread and virulence by hijacking human proteins, which occurs through viral protein recognition of human targets. To understand the structural basis for SARS-CoV-2 viral-host protein recognition, here we use cryo-electron microscopy (cryo-EM) to determine a complex structure of the human cell junction protein PALS1 and SARS-CoV-2 viral envelope (E) protein. Our reported structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains of PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce E-mediated virulence.

Quasispecies of SARS-CoV-2 revealed by single nucleotide polymorphisms (SNPs) analysis.

New SARS-CoV-2 mutants have been continuously indentified with enhanced transmission ever since its outbreak in early 2020. As an RNA virus, SARS-CoV-2 has a high mutation rate due to the low fidelity of RNA polymerase. To study the single nucleotide polymorphisms (SNPs) dynamics of SARS-CoV-2, 158 SNPs with high confidence were identified by deep meta-transcriptomic sequencing, and the most common SNP type was C > T. Analyses of intra-host population diversity revealed that intra-host quasispecies' composition varies with time during the early onset of symptoms, which implicates viral evolution during infection. Network analysis of co-occurring SNPs revealed the most abundant non-synonymous SNP 22,638 in the S glycoprotein RBD region and 28,144 in the ORF8 region. Furthermore, SARS-CoV-2 variations differ in an individual's respiratory tissue (nose, throat, BALF, or sputum), suggesting independent compartmentalization of SARS-CoV-2 populations in patients. The positive selection analysis of the SARS-CoV-2 genome uncovered the positive selected amino acid G251V on ORF3a. Alternative allele frequency spectrum (AAFS) of all variants revealed that ORF8 could bear alternate alleles with high frequency. Overall, the results show the quasispecies' profile of SARS-CoV-2 in the respiratory tract in the first two months after the outbreak.

Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity.

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.

An update on the origin of SARS-CoV-2: Despite closest identity, bat (RaTG13) and pangolin derived coronaviruses varied in the critical binding site and O-linked glycan residues.

The initial cases of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) occurred in Wuhan, China, in December 2019 and swept the world by 23 June 2020 with 8 993 659 active cases, 469 587 deaths across 216 countries, areas or territories. This strongly implies global transmission occurred before the lockdown of China. However, the initial source's transmission routes of SARS-CoV-2 remain obscure and controversial. Research data suggest bat (RaTG13) and pangolin carried CoV were the proximal source of SARS-CoV-2. In this study, we used systematic phylogenetic analysis of Coronavirinae subfamily along with wild type human SARS-CoV, MERS-CoV, and SARS-CoV-2 strains. The key residues of the receptor-binding domain (RBD) and O-linked glycan were compared. SARS-CoV-2 strains were clustered with RaTG13 (97.41% identity), Pangolin-CoV (92.22% identity) and Bat-SL-CoV (80.36% identity), forms a new clade-2 in lineage B of beta-CoV. The alignments of RBD contact residues to ACE2 justified? Those SARS-CoV-2 strains sequences were 100% identical by each other, significantly varied in RaTG13 and pangolin-CoV. SARS-CoV-2 has a polybasic cleavage site with an inserted sequence of PRRA compared to RaTG13 and only PRR to pangolin. Only serine (Ser) in pangolin and both threonine (Thr) and serine (Ser) O-linked glycans were seen in RaTG13, suggesting that a detailed study needed in pangolin (Manis javanica) and bat (Rhinolophus affinis) related CoV.

Characterization of the SARS-CoV-2 E Protein: Sequence, Structure, Viroporin, and Inhibitors.

The COVID-19 epidemic is one of the most influential epidemics in history. Understanding the impact of coronaviruses (CoVs) on host cells is very important for disease treatment. The SARS-CoV-2 envelope (E) protein is a small structural protein involved in many aspects of the viral life cycle. The E protein promotes the packaging and reproduction of the virus, and deletion of this protein weakens or even abolishes the virulence. This review aims to establish new knowledge by combining recent advances in the study of the SARS-CoV-2 E protein and by comparing it with the SARS-CoV E protein. The E protein amino acid sequence, structure, self-assembly characteristics, viroporin mechanisms and inhibitors are summarized and analyzed herein. Although the mechanisms of the SARS-CoV-2 and SARS-CoV E proteins are similar in many respects, specific studies on the SARS-CoV-2 E protein, for both monomers and oligomers, are still lacking. A comprehensive understanding of this protein should prompt further studies on the design and characterization of effective targeted therapeutic measures.

A study on non-synonymous mutational patterns in structural proteins of SARS-CoV-2.

SARS-CoV-2 is mutating and creating divergent variants across the world. An in-depth investigation of the amino acid substitutions in the genomic signature of SARS-CoV-2 proteins is highly essential for understanding its host adaptation and infection biology. A total of 9587 SARS-CoV-2 structural protein sequences collected from 49 different countries are used to characterize protein-wise variants, substitution patterns (type and location), and major substitution changes. The majority of the substitutions are distinct, mostly in a particular location, and lead to a change in an amino acid's biochemical properties. In terms of mutational changes, envelope (E) and membrane (M) proteins are relatively more stable than nucleocapsid (N) and spike (S) proteins. Several co-occurrence substitutions are observed, particularly in S and N proteins. Substitution specific to active sub-domains reveals that heptapeptide repeat, fusion peptides, transmembrane in S protein, and N-terminal and C-terminal domains in the N protein are remarkably mutated. We also observe a few deleterious mutations in the above domains. The overall study on non-synonymous mutation in structural proteins of SARS-CoV-2 at the start of the pandemic indicates a diversity amongst virus sequences.

Decoding SARS-CoV-2 Transmission and Evolution and Ramifications for COVID-19 Diagnosis, Vaccine, and Medicine.

Tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much of this development has been based on the reference genome collected on January 5, 2020. Based on the genotyping of 15 140 genome samples collected up to June 1, 2020, we report that SARS-CoV-2 has undergone 8309 single mutations which can be clustered into six subtypes. We introduce mutation ratio and mutation h-index to characterize the protein conservativeness and unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively nonconservative. In particular, we have identified mutations on 40% of nucleotides in the nucleocapsid gene in the population level, signaling potential impacts on the ongoing development of COVID-19 diagnosis, vaccines, and antibody and small-molecular drugs.

Evolution of SARS-CoV-2 Envelope, Membrane, Nucleocapsid, and Spike Structural Proteins from the Beginning of the Pandemic to September 2020: A Global and Regional Approach by Epidemiological Week.

Monitoring acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity and emerging mutations in this ongoing pandemic is crucial for understanding its evolution and assuring the performance of diagnostic tests, vaccines, and therapies against coronavirus disease (COVID-19). This study reports on the amino acid (aa) conservation degree and the global and regional temporal evolution by epidemiological week for each residue of the following four structural SARS-CoV-2 proteins: spike, envelope, membrane, and nucleocapsid. All, 105,276 worldwide SARS-CoV-2 complete and partial sequences from 117 countries available in the Global Initiative on Sharing All Influenza Data (GISAID) from 29 December 2019 to 12 September 2020 were downloaded and processed using an in-house bioinformatics tool. Despite the extremely high conservation of SARS-CoV-2 structural proteins (>99%), all presented aa changes, i.e., 142 aa changes in 65 of the 75 envelope aa, 291 aa changes in 165 of the 222 membrane aa, 890 aa changes in 359 of the 419 nucleocapsid aa, and 2671 changes in 1132 of the 1273 spike aa. Mutations evolution differed across geographic regions and epidemiological weeks (epiweeks). The most prevalent aa changes were D614G (81.5%) in the spike protein, followed by the R203K and G204R combination (37%) in the nucleocapsid protein. The presented data provide insight into the genetic variability of SARS-CoV-2 structural proteins during the pandemic and highlights local and worldwide emerging aa changes of interest for further SARS-CoV-2 structural and functional analysis.

Development of new vaccine target against SARS-CoV2 using envelope (E) protein: An evolutionary, molecular modeling and docking based study.

COVID-19 is one of the fatal pandemic throughout the world. For cellular fusion, its antigenic peptides are presented by major histocompatibility complex (MHC) in humans. Therefore, exploration into residual interaction details of CoV2 with MHCs shall be a promising point for instigating the vaccine development. Envelope (E) protein, the smallest outer surface protein from SARS-CoV2 genome was found to possess the highest antigenicity and is therefore used to identify B-cell and T-cell epitopes. Four novel mutations (T55S, V56F, E69R and G70del) were observed in E-protein of SARS-CoV2 after evolutionary analysis. It showed a coil➔helix transition in the protein conformation. Antigenic variability of the epitopes was also checked to explore the novel mutations in the epitope region. It was found that the interactions were more when SARS-CoV2 E-protein interacted with MHC-I than with MHC-II through several ionic and H-bonds. Tyr42 and Tyr57 played a predominant role upon interaction with MHC-I. The higher ΔG values with lesser dissociation constant values also affirm the stronger and spontaneous interaction by SARS-CoV2 proteins with MHCs. On comparison with the consensus E-protein, SARS-CoV2 E-protein showed stronger interaction with the MHCs with lesser solvent accessibility. E-protein can therefore be targeted as a potential vaccine target against SARS-CoV2.