Distinct motifs in the E protein are required for SARS-CoV-2 virus particle formation and lysosomal deacidification in host cells.

IMPORTANCE: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has caused a global public health crisis. The E protein, a structural protein found in this virus particle, is also known to be a viroporin. As such, it forms oligomeric ion channels or pores in the host cell membrane. However, the relationship between these two functions is poorly understood. In this study, we showed that the roles of E protein in virus particle and viroporin formation are distinct. This study contributes to the development of drugs that inhibit SARS-CoV-2 virus particle formation. Additionally, we designed a highly sensitive and high-throughput virus-like particle detection system using the HiBiT tag, which is a useful tool for studying the release of SARS-CoV-2.

Identification of Host PDZ-Based Interactions with the SARS-CoV-2 E Protein in Human Monocytes.

Proteins containing PDZ (post-synaptic density, PSD-95/disc large, Dlg/zonula occludens, ZO-1) domains assemble signaling complexes that orchestrate cell responses. Viral pathogens target host PDZ proteins by coding proteins containing a PDZ-binding motif (PBM). The presence of a PBM in the SARS-CoV-2 E protein contributes to the virus's pathogenicity. SARS-CoV-2 infects epithelia, but also cells from the innate immune response, including monocytes and alveolar macrophages. This process is critical for alterations of the immune response that are related to the deaths caused by SARS-CoV-2. Identification of E-protein targets in immune cells might offer clues to understanding how SARS-CoV-2 alters the immune response. We analyzed the interactome of the SARS-CoV-2 E protein in human monocytes. The E protein was expressed fused to a GFP tag at the amino terminal in THP-1 monocytes, and associated proteins were identified using a proteomic approach. The E-protein interactome provided 372 partners; only 8 of these harbored PDZ domains, including the cell polarity protein ZO-2, the chemoattractant IL-16, and syntenin. We addressed the expression and localization of the identified PDZ proteins along the differentiation of primary and THP-1 monocytes towards macrophages and dendritic cells. Our data highlight the importance of identifying the functions of PDZ proteins in the maintenance of immune fitness and the viral alteration of inflammatory response.

Glycosylation of Zika Virus is Important in Host-Virus Interaction and Pathogenic Potential.

Zika virus (ZIKV) is a global public health issue due to its association with severe developmental disorders in infants and neurological disorders in adults. ZIKV uses glycosylation of its envelope (E) protein to interact with host cell receptors to facilitate entry; these interactions could also be important for designing therapeutics and vaccines. Due to a lack of proper information about Asn-linked (N-glycans) on ZIKV E, we analyzed ZIKV E of various strains derived from different cells. We found ZIKV E proteins being extensively modified with oligomannose, hybrid and complex N-glycans of a highly heterogeneous nature. Host cell surface glycans correlated strongly with the glycomic features of ZIKV E. Mechanistically, we observed that ZIKV N-glycans might play a role in viral pathogenesis, as mannose-specific C-type lectins DC-SIGN and L-SIGN mediate host cell entry of ZIKV. Our findings represent the first detailed mapping of N-glycans on ZIKV E of various strains and their functional significance.

Electrophysiological properties and structural prediction of the SARS-CoV-2 viroprotein E.

COVID-19, the infectious disease caused by the most recently discovered coronavirus SARS- CoV-2, has caused millions of sick people and thousands of deaths all over the world. The viral positive-sense single-stranded RNA encodes 31 proteins among which the spike (S) is undoubtedly the best known. Recently, protein E has been reputed as a potential pharmacological target as well. It is essential for the assembly and release of the virions in the cell. Literature describes protein E as a voltage-dependent channel with preference towards monovalent cations whose intracellular expression, though, alters Ca2+ homeostasis and promotes the activation of the proinflammatory cascades. Due to the extremely high sequence identity of SARS-CoV-2 protein E (E-2) with the previously characterized E-1 (i.e., protein E from SARS-CoV) many data obtained for E-1 were simply adapted to the other. Recent solid state NMR structure revealed that the transmembrane domain (TMD) of E-2 self-assembles into a homo-pentamer, albeit the oligomeric status has not been validated with the full-length protein. Prompted by the lack of a common agreement on the proper structural and functional features of E-2, we investigated the specific mechanism/s of pore-gating and the detailed molecular structure of the most cryptic protein of SARS-CoV-2 by means of MD simulations of the E-2 structure and by expressing, refolding and analyzing the electrophysiological activity of the transmembrane moiety of the protein E-2, in its full length. Our results show a clear agreement between experimental and predictive studies and foresee a mechanism of activity based on Ca2+ affinity.

SARS-CoV-2-Mediated Lung Edema and Replication Are Diminished by Cystic Fibrosis Transmembrane Conductance Regulator Modulators.

Coronaviruses (CoVs) of genera α, ß, γ, and δ encode proteins that have a PDZ-binding motif (PBM) consisting of the last four residues of the envelope (E) protein (PBM core). PBMs may bind over 400 cellular proteins containing PDZ domains (an acronym formed by the combination of the first letter of the names of the three first proteins where this domain was identified), making them relevant for the control of cell function. Three highly pathogenic human CoVs have been identified to date: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The PBMs of the three CoVs were virulence factors. SARS-CoV mutants in which the E protein PBM core was replaced by the E protein PBM core from virulent or attenuated CoVs were constructed. These mutants showed a gradient of virulence, depending on whether the alternative PBM core introduced was derived from a virulent or an attenuated CoV. Gene expression patterns in the lungs of mice infected with SARS-CoVs encoding each of the different PBMs were analyzed by RNA sequencing of infected lung tissues. E protein PBM of SARS-CoV and SARS-CoV-2 dysregulated gene expression related to ion transport and cell homeostasis. Decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, essential for alveolar edema resolution, was shown. Reduced CFTR mRNA levels were associated with edema accumulation in the alveoli of mice infected with SARS-CoV and SARS-CoV-2. Compounds that increased CFTR expression and activity, significantly reduced SARS-CoV-2 growth in cultured cells and protected against mouse infection, suggesting that E protein virulence is mediated by a decreased CFTR expression. IMPORTANCE Three highly pathogenic human CoVs have been identified: SARS-CoV, MERS-CoV, and SARS-CoV-2. The E protein PBMs of these three CoVs were virulence factors. Gene expression patterns associated with the different PBM motifs in the lungs of infected mice were analyzed by deep sequencing. E protein PBM motif of SARS-CoV and SARS-CoV-2 dysregulated the expression of genes related to ion transport and cell homeostasis. A decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is essential for edema resolution, was observed. The reduction of CFTR mRNA levels was associated with edema accumulation in the lungs of mice infected with SARS-CoV-2. Compounds that increased the expression and activity of CFTR drastically reduced the production of SARS-CoV-2 and protected against its infection in a mice model. These results allowed the identification of cellular targets for the selection of antivirals.

Phospholipid binding of the dengue virus envelope E protein segment containing the conserved His residue.

Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.

Binding of the SARS-CoV-2 envelope E protein to human BRD4 is essential for infection.

Emerging new variants of SARS-CoV-2 and inevitable acquired drug resistance call for the continued search of new pharmacological targets to fight the potentially fatal infection. Here, we describe the mechanisms by which the E protein of SARS-CoV-2 hijacks the human transcriptional regulator BRD4. We found that SARS-CoV-2 E is acetylated in vivo and co-immunoprecipitates with BRD4 in human cells. Bromodomains (BDs) of BRD4 bind to the C-terminus of the E protein, acetylated by human acetyltransferase p300, whereas the ET domain of BRD4 recognizes the unmodified motif of the E protein. Inhibitors of BRD4 BDs, JQ1 or OTX015, decrease SARS-CoV-2 infectivity in lung bronchial epithelial cells, indicating that the acetyllysine binding function of BDs is necessary for the virus fitness and that BRD4 represents a potential anti-COVID-19 target. Our findings provide insight into molecular mechanisms that contribute to SARS-CoV-2 pathogenesis and shed light on a new strategy to block SARS-CoV-2 infection.

Refinement of SARS-CoV-2 envelope protein structure in a native-like environment by molecular dynamics simulations.

COVID-19 has become an unprecedented threat to human health. The SARS-CoV-2 envelope (E) protein plays a critical role in the viral maturation process and pathogenesis. Despite intensive investigation, its structure in physiological conditions remains mysterious: no high-resolution full-length structure is available and only an NMR structure of the transmembrane (TM) region has been determined. Here, we present a refined E protein structure, using molecular dynamics (MD) simulations to investigate its structure and dynamics in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer system. Our initial homology model based upon the SARS-CoV E protein structure is shown to be unstable in the lipid bilayer, and the H3 helices tend to move away from the membrane center to the membrane-water interface. A more stable model was developed by replacing all H3 helices with the fully equilibrated H3 structure sampled in the MD simulations. This refined model exhibited more favorable contacts with lipids and water than the original homology model and induced local membrane curvature, decreasing local lipid order. Interestingly, the pore radius profiles showed that the channel in both homology and refined models remained in a closed state throughout the simulations. We also demonstrated the utility of this structure to develop anti-SARS-CoV-2 drugs by docking a library of FDA-approved, investigational, and experimental drugs to the refined E protein structure, identifying 20 potential channel blockers. This highlights the power of MD simulations to refine low-resolution structures of membrane proteins in a native-like membrane environment, shedding light on the structural features of the E protein and providing a platform for the development of novel antiviral treatments.

Envelope E protein of dengue virus and phospholipid binding to the late endosomal membrane.

Flaviviruses include many significant human pathogens, comprising dengue, West Nile, Yellow fever, Japanese encephalitis, Zika and tick-borne encephalitis viruses and many others, affecting millions of people in the world. These viruses have produced important epidemics in the past, they continue to do it and they will undoubtedly continue to do so in the future. Flaviviruses enter into the cells via receptor-mediated endocytosis by fusing its membrane with the endosomal membrane in a pH-dependent manner with the help of the envelope E protein, a prototypical class II membrane fusion protein. The envelope E protein has a conserved fusion peptide at its distal end, which is responsible in the first instance of inserting the protein into the host membrane. Since the participation of other segments of the E protein in the fusion process should not be ruled out, we have used atomistic molecular dynamics to study the binding of the distal end of domain II of the envelope E protein from Dengue virus (DENV) with a complex membrane similar to the late-endosome one. Our work shows that not only the fusion peptide participates directly in the fusion, but also two other sequences of the protein, next to the fusion peptide it in the three-dimensional structure, are jointly wrapped in the fusion process. Overall, these three sequences represent a new target that would make it possible to obtain effective antivirals against DENV in particular and Flaviviruses in general.

The truncated E protein of DTMUV provide protection in young ducks.

Duck Tembusu virus (DTMUV) is a major pathogen of duck industry in China. In the current study, we generated different constructs containing envelope (E) protein, pre-membrane-envelope (prM-E) protein, and C-terminally truncated E protein of the DTMUV. The constructed proteins could induce specific antibody responses in young ducks. When ducklings were immunized with the constructed proteins, they were 100% protected against DTMUV infection. Furthermore, the fluorescent signal of the truncated E protein was stronger than other constructed proteins, when Bac-to-Bac baculovirus expression system was applied. Our data demonstrated that the truncated E protein used in the current study could be applied as a potential vaccine candidate to control DTMUV infection in young ducks.

Identification of the phospholipid binding regions of the envelope E protein of flaviviruses by molecular dynamics.

The Flavivirus genus comprise several important human pathogens, including dengue, West Nile, Yellow fever, Japanese encephalitis, Zika, and tick-borne encephalitis viruses. These enveloped viruses affect more than 2 billion people in the world, mainly in less developed countries. Although some vaccines exist for some flaviviruses, these vaccines are not universally available due to many factors and since their infections are a world-wide public health issue, the development of antiviral molecules is fundamental. Flavivirus membranes, through the help of the envelope E glycoprotein, fuse with endosomal compartments in a pH-dependent way to release their genome into the cytoplasm and require specific lipids, such as bis(monoacylglycero)phosphate (BMP), for efficient fusion. The fundamental role the envelope E protein has on viral entry and membrane fusion suggest that it is an essential antiviral target. In this work, we have used atomistic molecular dynamics simulations to study the binding of the head-group of BMP to the tip of the envelope E proteins of ZIKV, DENV, TBEV and JEV viruses whose three-dimensional structures are known. Our results indicate that, apart from the fusion loop, there are different amino acid residues in different regions of the envelope E proteins of flaviviruses capable of binding the head-group of BMP. These regions should work together to accomplish the binding and fusion of the envelope and endosomal membranes and represent a new target to develop and design potent and effective antiviral agents capable of blocking flavivirus-endosome membrane fusion. [Formula: see text].

Computational Study of the Ion and Water Permeation and Transport Mechanisms of the SARS-CoV-2 Pentameric E Protein Channel.

Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus (SARS-CoV-2) and represents the causative agent of a potentially fatal disease that is a public health emergency of international concern. Coronaviruses, including SARS-CoV-2, encode an envelope (E) protein, which is a small, hydrophobic membrane protein; the E protein of SARS-CoV-2 shares a high level of homology with severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, we provide insights into the function of the SARS-CoV-2 E protein channel and the ion and water permeation mechanisms using a combination of in silico methods. Based on our results, the pentameric E protein promotes the penetration of cation ions through the channel. An analysis of the potential mean force (PMF), pore radius and diffusion coefficient reveals that Leu10 and Phe19 are the hydrophobic gates of the channel. In addition, the pore exhibits a clear wetting/dewetting transition with cation selectivity under transmembrane voltage, indicating that it is a hydrophobic voltage-dependent channel. Overall, these results provide structure-based insights and molecular dynamic information that are needed to understand the regulatory mechanisms of ion permeability in the pentameric SARS-CoV-2 E protein channel.

An in-silico approach to develop of a multi-epitope vaccine candidate against SARS-CoV-2 envelope (E) protein.

Since the first appearance of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS- CoV-2) in China on December 2019, the world has now witnessed the emergence of the SARS- CoV-2 outbreak. Therefore, due to the high transmissibility rate of virus, there is an urgent need to design and develop vaccines against SARS-CoV-2 to prevent more cases affected by the virus. In this study, a computational approach is proposed for vaccine design against the envelope (E) protein of SARS-CoV-2, which contains a conserved sequence feature. First, we sought to gain potential B-cell and T-cell epitopes for vaccine designing against SARS-CoV-2. Second, we attempted to develop a multi-epitope vaccine. Immune targeting of such epitopes could theoretically provide defense against SARS-CoV-2. Finally, we evaluated the affinity of the vaccine to major histocompatibility complex (MHC) molecules to stimulate the immune system response to this vaccine. We also identified a collection of B-cell and T-cell epitopes derived from E proteins that correspond identically to SARS-CoV-2 E proteins. The in-silico design of our potential vaccine against E protein of SARS-CoV-2 demonstrated a high affinity to MHC molecules, and it can be a candidate to make a protection against this pandemic event.

SARS-CoV-2 Envelope (E) Protein Interacts with PDZ-Domain-2 of Host Tight Junction Protein ZO1.

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2 induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe respiratory dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway barrier damage and mitigate virus spread.