Immunoinformatic analysis of the SARS-CoV-2 envelope protein as a strategy to assess cross-protection against COVID-19

Envelope protein of coronaviruses is a structural protein existing in both monomeric and homo-pentameric form. It has been related to a multitude of roles including virus infection, replication, dissemination and immune response stimulation. In the present study, we employed an immunoinformatic approach to investigate the major immunogenic domains of the SARS-CoV-2 envelope protein and map them among the homologue proteins of coronaviruses with tropism for animal species that are closely inter-related with the human beings population all over the world. Also, when not available, we predicted the envelope protein structural folding and mapped SARS-CoV-2 epitopes. Envelope sequences alignment provides evidence of high sequence homology for some of the investigated virus specimens; while the structural mapping of epitopes resulted in the interesting maintenance of the structural folding and epitope sequence localization also in the envelope proteins scoring a lower alignment score. In line with the One-Health approach, our evidences provide a molecular structural rationale for a potential role of taxonomically related coronaviruses in conferring protection from SARS-CoV-2 infection and identifying potential candidates for the development of diagnostic tools and prophylactic-oriented strategies.     

Probing effects of the SARS-CoV-2 E protein on membrane curvature and intracellular calcium

SARS-CoV-2 contains four structural proteins in its genome. These proteins aid in the assembly and budding of new virions at the ER-Golgi intermediate compartment (ERGIC). Current fundamental research efforts largely focus on one of these proteins – the spike (S) protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other structural proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length envelope (E) protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca²⁺, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.     

Peptides derived from a distinct region of GB virus C glycoprotein E2 mediate strain-specific HIV-1 entry inhibition

The nonpathogenic human GB virus C (GBV-C), a member of the Flaviviridae, is highly prevalent in individuals with HIV-1 infections or with parenteral and sexual risk factors. Long-term GBV-C viremia has been associated with better survival or improved diagnosis in several epidemiological studies. In a previous study we reported that the E2 glycoprotein of GBV-C interferes with HIV-1 entry in vitro. To address the question what region of the E2 protein is involved in suppression of HIV-1 replication, we performed an E2-derived peptide scanning and determined the HIV-inhibitory activity of each peptide in HIV replication assays. We demonstrate here that peptides representing the N-terminal part of the E2 protein from amino acids (aa) 29 to 72 are able to inhibit efficiently HIV-1 replication in vitro. In particular, the peptides P6-2 (representing the E2-region from aa 45 to 64) and P4762 (aa 37 to 64) showed the highest potency in HIV replication assays performed on TZM-bl cells with 50% inhibitory concentrations between 0.1 and 2 μM. However, primary HIV-1 isolates representing clades A to H showed a high variability in their sensitivity to E2 peptides. Pseudovirus inhibition assays revealed that the sensitivity is determined by the gp120/gp41 envelope proteins. Using HIV-1 BlaM-Vpr-based fusion assays, we demonstrate that the E2-derived peptides prevent HIV-1 binding or fusion, presumably via interaction with the HIV-1 particle. Together, these findings reveal a new mechanism of viral interference, suggesting that the envelope protein E2 of GBV-C target directly HIV-1 particles to avoid entry of these virions.     

Regulation of epithelial sodium channel activity by SARS-CoV-1 and SARS-CoV-2 proteins

Severe acute respiratory syndrome (SARS) coronavirus (CoV) 2 (SARS-CoV-2), which causes the coronavirus disease 2019, encodes several proteins whose roles are poorly understood. We tested their ability either to directly form plasma membrane ion channels or to change functions of two mammalian plasma membrane ion channels, the epithelial sodium channel (ENaC) and the α3β4 nicotinic acetylcholine receptor. In mRNA-injected Xenopus oocytes, none of nine SARS-CoV-2 proteins or two SARS-CoV-1 proteins produced conductances, nor did co-injection of several combinations. Immunoblots for ORF8, spike (S), and envelope (E) proteins revealed that the proteins are expressed at appropriate molecular weights. In experiments on coexpression with ENaC, three tested SARS proteins (SARS-CoV-1 E, SARS-CoV-2 E, and SARS-CoV-2 S) markedly decrease ENaC currents. SARS-CoV-1 S protein decreases ENaC currents modestly. Coexpressing the E proteins but not the S proteins with α3β4 nicotinic acetylcholine receptors significantly reduces acetylcholine-induced currents. ENaC inhibition does not occur if the SARS-CoV protein mRNAs are injected 24 h after the ENaC mRNAs, suggesting that SARS-CoV proteins affect early step(s) in functional expression of channel proteins. Consistent with the hypothesis that the SARS-CoV-2 S protein-induced ENaC inhibition involves competition for available protease, mutating the furin cleavage site in SARS-CoV-2 S protein partially relieves inhibition of ENaC currents. Extending previous suggestions that SARS proteins affect ENaC currents via protein kinase C (PKC) activation, PKC activation via phorbol 12-myristate 13-acetate decreases ENaC and α3β4 activity. Phorbol 12-myristate 13-acetate application reduced membrane capacitance ～5%, presumably via increased endocytosis, but this decrease is much smaller than the SARS proteins’ effects on conductances. Also, incubating oocytes in Gö-6976, a PKCα and PKCβ inhibitor, did not alter E or S protein-induced channel inhibition. We conclude that SARS-CoV-1 and SARS-CoV-2 proteins alter the function of human plasma membrane channels, via incompletely understood mechanisms. These interactions may play a role in the coronavirus 2019 pathophysiology.     

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.     

SARS-CoV-2 variants reveal features critical for replication in primary human cells

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern.

SARS-CoV-2 variants have appeared throughout the emergence of the current COVID-19 pandemic. In this study, characterization of a panel of infectious SARS-CoV-2 isolates in primary human respiratory cells allows the identification of important sequence features that contribute to virus replication.     

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.Subject terms: Cell death, Molecular biology     

A Dimer Is the Minimal Proton-Conducting Unit of the Influenza A Virus M2 Channel

When influenza A virus infects host cells, its integral matrix protein M2 forms a proton-selective channel in the viral envelope. Although X-ray crystallography and NMR studies using fragment peptides have suggested that M2 stably forms a tetrameric channel irrespective of pH, the oligomeric states of the full-length protein in the living cells have not yet been assessed directly. In the present study, we utilized recently developed stoichiometric analytical methods based on fluorescence resonance energy transfer using coiled-coil labeling technique and spectral imaging, and we examined the relationship between the oligomeric states of full-length M2 and its channel activities in living cells. In contrast to previous models, M2 formed proton-conducting dimers at neutral pH and these dimers were converted to tetramers at acidic pH. The antiviral drug amantadine hydrochloride inhibited both tetramerization and channel activity. The removal of cholesterol resulted in a significant decrease in the activity of the dimer. These results indicate that the minimum functional unit of the M2 protein is a dimer, which forms a complex with cholesterol for its function.     

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.     

Comparative genomic analysis reveals varying levels of mammalian adaptation to coronavirus infections

Severe acute respiratory coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is of zoonotic origin. Evolutionary analyses assessing whether coronaviruses similar to SARS-CoV-2 infected ancestral species of modern-day animal hosts could be useful in identifying additional reservoirs of potentially dangerous coronaviruses. We reasoned that if a clade of species has been repeatedly exposed to a virus, then their proteins relevant for viral entry may exhibit adaptations that affect host susceptibility or response. We perform comparative analyses across the mammalian phylogeny of angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2, in order to uncover evidence for selection acting at its binding interface with the SARS-CoV-2 spike protein. We uncover that in rodents there is evidence for adaptive amino acid substitutions at positions comprising the ACE2-spike interaction interface, whereas the variation within ACE2 proteins in primates and some other mammalian clades is not consistent with evolutionary adaptations. We also analyze aminopeptidase N (APN), the receptor for the human coronavirus 229E, a virus that causes the common cold, and find evidence for adaptation in primates. Altogether, our results suggest that the rodent and primate lineages may have had ancient exposures to viruses similar to SARS-CoV-2 and HCoV-229E, respectively.     

东部马脑脊髓炎病毒的分子生物学进展

东部马脑脊髓炎病毒属虫媒病毒,能引起人和马发生急性脑炎。东马病毒为单股正链RNA病毒,可分为南美型和北美型,包括两个开放读码框架,分别编码结构蛋白（E1,E2,E2,C,6K）和非结构蛋白（nsp1,nsp2,nsp3,nsp4)。其中E1/E2包膜糖蛋白以异二聚体的形式病毒颗粒外刺突。非结构蛋白主要能与负链RNA的合成,近来,随着研究深入,病毒受体越来越受到广泛关注。本介绍东部马脑脊髓炎病毒结构、进化、复制、组装等方面的分子生物学进展。     

Influenza A virus can undergo multiple cycles of replication without M2 ion channel activity

Ion channel proteins are common constituents of cells and have even been identified in some viruses. For example, the M2 protein of influenza A virus has proton ion channel activity that is thought to play an important role in viral replication. Because direct support for this function is lacking, we attempted to generate viruses with defective M2 ion channel activity. Unexpectedly, mutants with apparent loss of M2 ion channel activity by an in vitro assay replicated as efficiently as the wild-type virus in cell culture. We also generated a chimeric mutant containing an M2 protein whose transmembrane domain was replaced with that from the hemagglutinin glycoprotein. This virus replicated reasonably well in cell culture but showed no growth in mice. Finally, a mutant lacking both the transmembrane and cytoplasmic domains of M2 protein grew poorly in cell culture and showed no growth in mice. Thus, influenza A virus can undergo multiple cycles of replication without the M2 transmembrane domain responsible for ion channel activity, although this activity promotes efficient viral replication.     

Characterization of human papillomavirus type 11 E1 and E2 proteins expressed in insect cells.

The study of human papillomavirus replication has been hampered by the lack of an in vitro system which reliably supports virus replication. Recent results from the bovine papillomavirus (BPV) system indicate that the E1 and E2 proteins are the only viral gene products required for replication. By analogy with simian virus 40 large T antigen, E1 is thought to possess ATPase and helicase activity, which may play a direct role in viral DNA replication. The precise role of E2 is unclear, but it may function in part to help localize E1 to the replication origin. We have initiated a study of replication in the human papillomavirus type 11 system which, by analogy to BPV, has focused on the E1 and E2 proteins of this virus. We have expressed the full-length E1 and E2 proteins in Sf9 insect cells by using a baculovirus expression vector. Both the 80-kDa E1 protein and the 42.5-kDa E2 protein are nuclear phosphoproteins. The E1 and E2 proteins form a heteromeric complex within the insect cells, and both proteins localize to a DNA fragment which contains the viral origin of replication. In addition, we have detected an E1-associated ATPase and GTPase activity, which is likely part of an energy-generating system for the helicase activity which is predicted for this protein. The human papillomavirus type 11 E1 and E2 proteins possess the same replication-associated activities exhibited by the corresponding BPV proteins, suggesting that the replication activities of these viruses are tightly conserved.     

The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a β-Sheet Bundle in Lipid Bilayers

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8–65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. ¹³C and ¹⁵N chemical shifts indicate that the cytoplasmic domain adopts a β-sheet-rich conformation that contains three β-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third β-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the β-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt β-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the β-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.     

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, therapeutics that can help manage the disease are still required until immunity has been achieved globally. The identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2-ΔOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in-line with reported proteinuria and liver damage in patients with COVID-19. Using the nano-luciferase as a measure of virus replication we identified 35 drugs that reduced replication in Vero cells and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.     

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards the human respiratory tract. First, the S proteins exhibit an intrinsic temperature preference, corresponding with the temperature of the upper or lower airways. Pseudoviruses bearing the SARS-CoV-2 spike (SARS-2-S) were more infectious when produced at 33°C instead of 37°C, a property shared with the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV and MERS-CoV favored 37°C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not only TMPRSS2, but also the TMPRSS13 protease, thus broadening the cell tropism of SARS-CoV-2. Both proteases proved relevant in the context of authentic virus replication. TMPRSS13 appeared an effective spike activator for the virulent coronaviruses but not the low pathogenic HCoV-229E virus. Activation of SARS-2-S by these surface proteases requires processing of the S1/S2 cleavage loop, in which both the furin recognition motif and extended loop length proved critical. Conversely, entry of loop deletion mutants is significantly increased in cathepsin-rich cells. Finally, we demonstrate that the D614G mutation increases SARS-CoV-2 stability, particularly at 37°C, and, enhances its use of the cathepsin L pathway. This indicates a link between S protein stability and usage of this alternative route for virus entry. Since these spike properties may promote virus spread, they potentially explain why the spike-G614 variant has replaced the early D614 variant to become globally predominant. Collectively, our findings reveal adaptive mechanisms whereby the coronavirus spike protein is adjusted to match the temperature and protease conditions of the airways, to enhance virus transmission and pathology.     

Sulfated Polysaccharide,Curdlan Sulfate,Efficiently Prevents Entry/Fusion and Restricts Antibody-Dependent Enhancement of Dengue Virus Infection In Vitro: A Possible Candidate for Clinical Application

Curdlan sulfate (CRDS), a sulfated 1→3-β-D glucan, previously shown to be a potent HIV entry inhibitor, is characterized in this study as a potent inhibitor of the Dengue virus (DENV). CRDS was identified by in silico blind docking studies to exhibit binding potential to the envelope (E) protein of the DENV. CRDS was shown to inhibit the DENV replication very efficiently in different cells in vitro. Minimal effective concentration of CRDS was as low as 0.1 µg/mL in LLC-MK2 cells, and toxicity was observed only at concentrations over 10 mg/mL. CRDS can also inhibit DENV-1, 3, and 4 efficiently. CRDS did not inhibit the replication of DENV subgenomic replicon. Time of addition experiments demonstrated that the compound not only inhibited viral infection at the host cell binding step, but also at an early post-attachment step of entry (membrane fusion). The direct binding of CRDS to DENV was suggested by an evident reduction in the viral titers after interaction of the virus with CRDS following an ultrafiltration device separation, as well as after virus adsorption to an alkyl CRDS-coated membrane filter. The electron microscopic features also showed that CRDS interacted directly with the viral envelope, and caused changes to the viral surface. CRDS also potently inhibited DENV infection in DC-SIGN expressing cells as well as the antibody-dependent enhancement of DENV-2 infection. Based on these data, a probable binding model of CRDS to DENV E protein was constructed by a flexible receptor and ligand docking study. The binding site of CRDS was predicted to be at the interface between domains II and III of E protein dimer, which is unique to this compound, and is apparently different from the β-OG binding site. Since CRDS has already been tested in humans without serious side effects, its clinical application can be considered.