SARS-CoV-2 infection alkalinizes the ERGIC and lysosomes through the viroporin activity of the viral envelope protein.

The coronavirus SARS-CoV-2, the agent of the deadly COVID-19 pandemic, is an enveloped virus propagating within the endocytic and secretory organelles of host mammalian cells. Enveloped viruses modify the ionic homeostasis of organelles to render their intra-luminal milieu permissive for viral entry, replication and egress. Here, we show that infection of Vero E6 cells with the delta variant of the SARS-CoV-2 alkalinizes the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as well as lysosomes, mimicking the effect of inhibitors of vacuolar proton ATPases. We further show the envelope protein of SARS-CoV-2 accumulates in the ERGIC when expressed in mammalian cells and selectively dissipates the ERGIC pH. This viroporin action is prevented by mutations of Val25 but not Asn15 within the channel pore of the envelope (E) protein. We conclude that the envelope protein acts as a proton channel in the ERGIC to mitigate the acidity of this intermediate compartment. The altered pH homeostasis of the ERGIC likely contributes to the virus fitness and pathogenicity, making the E channel an attractive drug target for the treatment of COVID-19.

SARS-COV-2 viroporins activate the NLRP3-inflammasome by the mitochondrial permeability transition pore.

Background: Compared to healthy controls, severe COVID19 patients display increased levels of activated NLRP3-inflammasome (NLRP3-I) and interleukin (IL)-1ß. SARS-CoV-2 encodes viroporin proteins E and Orf3a(2-E+2-3a) with homologs to SARS-CoV-1, 1-E+1-3a, which elevate NLRP3-I activation; by an unknown mechanism. Thus, we investigated how 2-E+2-3a activates the NLRP3-I to better understand the pathophysiology of severe COVID-19. Methods: We generated a polycistronic expression-vector co-expressing 2-E+2-3a from a single transcript. To elucidate how 2-E+2-3a activates the NLRP3-I, we reconstituted the NLRP3-I in 293T cells and used THP1-derived macrophages to monitor the secretion of mature IL-1ß. Mitochondrial physiology was assessed using fluorescent microscopy and plate reader assays, and the release of mitochondrial DNA (mtDNA) was detected from cytosolic-enriched fractions using Real-Time PCR. Results: Expression of 2-E+2-3a in 293T cells increased cytosolic Ca++ and elevated mitochondrial Ca++, taken up through the MCUi11-sensitive mitochondrial calcium uniporter. Increased mitochondrial Ca++ stimulated NADH, mitochondrial reactive oxygen species (mROS) production and the release of mtDNA into the cytosol. Expression of 2-E+2-3a in NLRP3-I reconstituted 293T cells and THP1-derived macrophages displayed increased secretion of IL-1ß. Increasing mitochondrial antioxidant defenses via treatment with MnTBAP or genetic expression of mCAT abolished 2-E+2-3a elevation of mROS, cytosolic mtDNA levels, and secretion of NLRP3-activated-IL-1ß. The 2-E+2-3a-induced release of mtDNA and the secretion of NLRP3-activated-IL-1ß were absent in cells lacking mtDNA and blocked in cells treated with the mitochondrial-permeability-pore(mtPTP)-specific inhibitor NIM811. Conclusion: Our findings revealed that mROS activates the release of mitochondrial DNA via the NIM811-sensitive mitochondrial-permeability-pore(mtPTP), activating the inflammasome. Hence, interventions targeting mROS and the mtPTP may mitigate the severity of COVID-19 cytokine storms.

Channel activity of SARS-CoV-2 viroporin ORF3a inhibited by adamantanes and phenolic plant metabolites.

SARS-CoV-2 has been responsible for the major worldwide pandemic of COVID-19. Despite the enormous success of vaccination campaigns, virus infections are still prevalent and effective antiviral therapies are urgently needed. Viroporins are essential for virus replication and release, and are thus promising therapeutic targets. Here, we studied the expression and function of recombinant ORF3a viroporin of SARS-CoV-2 using a combination of cell viability assays and patch-clamp electrophysiology. ORF3a was expressed in HEK293 cells and transport to the plasma membrane verified by a dot blot assay. Incorporation of a membrane-directing signal peptide increased plasma membrane expression. Cell viability tests were carried out to measure cell damage associated with ORF3a activity, and voltage-clamp recordings verified its channel activity. The classical viroporin inhibitors amantadine and rimantadine inhibited ORF3a channels. A series of ten flavonoids and polyphenolics were studied. Kaempferol, quercetin, epigallocatechin gallate, nobiletin, resveratrol and curcumin were ORF3a inhibitors, with IC50 values ranging between 1 and 6 µM, while 6-gingerol, apigenin, naringenin and genistein were inactive. For flavonoids, inhibitory activity could be related to the pattern of OH groups on the chromone ring system. Thus, the ORF3a viroporin of SARS-CoV-2 may indeed be a promising target for antiviral drugs.

SARS-CoV-2 ORF3a positively regulates NF-κB activity by enhancing IKKß-NEMO interaction.

Coronavirus disease 2019 (COVID-19) is a global pandemic caused by SARS-CoV-2 infection. Patients with severe COVID-19 exhibit robust induction of proinflammatory cytokines, which are closely associated with the development of acute respiratory distress syndrome. However, the underlying mechanisms of the NF-κB activation mediated by SARS-CoV-2 infection remain poorly understood. Here, we screened SARS-CoV-2 genes and found that ORF3a induces proinflammatory cytokines by activating the NF-κB pathway. Moreover, we found that ORF3a interacts with IKKß and NEMO and enhances the interaction of IKKß-NEMO, thereby positively regulating NF-κB activity. Together, these results suggest ORF3a may play pivotal roles in the pathogenesis of SARS-CoV-2 and provide novel insights into the interaction between host immune responses and SARS-CoV-2 infection.

Predicting the Assembly of the Transmembrane Domains of Viral Channel Forming Proteins and Peptide Drug Screening Using a Docking Approach.

A de novo assembly algorithm is provided to propose the assembly of bitopic transmembrane domains (TMDs) of membrane proteins. The algorithm is probed using, in particular, viral channel forming proteins (VCPs) such as M2 of influenza A virus, E protein of severe acute respiratory syndrome corona virus (SARS-CoV), 6K of Chikungunya virus (CHIKV), SH of human respiratory syncytial virus (hRSV), and Vpu of human immunodeficiency virus type 2 (HIV-2). The generation of the structures is based on screening a 7-dimensional space. Assembly of the TMDs can be achieved either by simultaneously docking the individual TMDs or via a sequential docking. Scoring based on estimated binding energies (EBEs) of the oligomeric structures is obtained by the tilt to decipher the handedness of the bundles. The bundles match especially well for all-atom models of M2 referring to an experimentally reported tetrameric bundle. Docking of helical poly-peptides to experimental structures of M2 and E protein identifies improving EBEs for positively charged (K,R,H) and aromatic amino acids (F,Y,W). Data are improved when using polypeptides for which the coordinates of the amino acids are adapted to the Cα coordinates of the respective experimentally derived structures of the TMDs of the target proteins.

The envelope proteins from SARS-CoV-2 and SARS-CoV potently reduce the infectivity of human immunodeficiency virus type 1 (HIV-1).

BACKGROUND: Viroporins are virally encoded ion channels involved in virus assembly and release. Human immunodeficiency virus type 1 (HIV-1) and influenza A virus encode for viroporins. The human coronavirus SARS-CoV-2 encodes for at least two viroporins, a small 75 amino acid transmembrane protein known as the envelope (E) protein and a larger 275 amino acid protein known as Orf3a. Here, we compared the replication of HIV-1 in the presence of four different ß-coronavirus E proteins. RESULTS: We observed that the SARS-CoV-2 and SARS-CoV E proteins reduced the release of infectious HIV-1 yields by approximately 100-fold while MERS-CoV or HCoV-OC43 E proteins restricted HIV-1 infectivity to a lesser extent. Mechanistically, neither reverse transcription nor mRNA synthesis was involved in the restriction. We also show that all four E proteins caused phosphorylation of eIF2-α at similar levels and that lipidation of LC3-I could not account for the differences in restriction. However, the level of caspase 3 activity in transfected cells correlated with HIV-1 restriction in cells. Finally, we show that unlike the Vpu protein of HIV-1, the four E proteins did not significantly down-regulate bone marrow stromal cell antigen 2 (BST-2). CONCLUSIONS: The results of this study indicate that while viroporins from homologous viruses can enhance virus release, we show that a viroporin from a heterologous virus can suppress HIV-1 protein synthesis and release of infectious virus.

Weak Point of SARS-CoV-2: Human and Viral Ion Channels under External Physical Fields.

The ionic E-nanochannel (viroporin) is the weak point of SARS-CoV-2, the virus responsible for the (still threatening) COVID-19 since it is vital to the virus's budding and propagation. Therefore, targeting it to disable its functions ought to incapacitate, or at least weaken, the virus. The ionic currents inside this channel could be affected and disturbed by direct physical attack via the actions of external fields. The paper presents the first step towards the application of such methods in the fight against the current pandemic, numerical simulations of external fields' impact on ionic currents through viral channels. These simulations-based on the actual, detailed physical nanostructure of ionic channels, measured experimentally and reported in the literature-show that external physical fields can diminish the channel's currents and that the lower the channel's selectivity, the stronger the effect. Simulations suggest that SARS-CoV-2 E-viroporin is almost non-selective, which means that the whole virus ought to be highly vulnerable to the actions of external physical fields, much more vulnerable than the much more selective human cell ionic channels. If corroborated by experiment, this observation may result in an innovative method of dealing with the recent pandemic caused by SARS-CoV-2 and other similar viruses.

A proof-of-concept study of the secondary structure of influenza A, B M2 and MERS- and SARS-CoV E transmembrane peptides using folding molecular dynamics simulations in a membrane mimetic solvent.

Here, we have carried out a proof-of-concept molecular dynamics (MD) simulation with adaptive tempering in a membrane mimetic environment to study the folding of single-pass membrane peptides. We tested the influenza A M2 viroporin, influenza B M2 viroporin, and protein E from coronaviruses MERS-Cov-2 and SARS-CoV-2 peptides with known experimental secondary structures in membrane bilayers. The two influenza-derived peptides are significantly different in the peptide sequence and secondary structure and more polar than the two coronavirus-derived peptides. Through a total of more than 50 µs of simulation time that could be accomplished in trifluoroethanol (TFE), as a membrane model, we characterized comparatively the folding behavior, helical stability, and helical propensity of these transmembrane peptides that match perfectly their experimental secondary structures, and we identified common motifs that reflect their quaternary organization and known (or not) biochemical function. We showed that BM2 is organized into two structurally distinct parts: a significantly more stable N-terminal half, and a fast-converting C-terminal half that continuously folds and unfolds between α-helical structures and non-canonical structures, which are mostly turns. In AM2, both the N-terminal half and C-terminal half are very flexible. In contrast, the two coronavirus-derived transmembrane peptides are much more stable and fast helix-formers when compared with the influenza ones. In particular, the SARS-derived peptide E appears to be the fastest and most stable helix-former of all the four viral peptides studied, with a helical structure that persists almost without disruption for the whole of its 10 µs simulation. By comparing the results with experimental observations, we benchmarked TFE in studying the conformation of membrane and hydrophobic peptides. This work provided accurate results suggesting a methodology to run long MD simulations and predict structural properties of biologically important membrane peptides.

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.

Porcine deltacoronavirus accessory protein NS7a possesses the functional characteristics of a viroporin.

Viroporins are virus-encoded proteins that mediate ion channel (IC) activity, playing critical roles in virus entry, replication, pathogenesis, and immune evasion. Previous studies have shown that some coronavirus accessory proteins have viroporin-like activity. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that encodes three accessory proteins, NS6, NS7, and NS7a. However, whether any of the PDCoV accessory proteins possess viroporin-like activity, and if so which, remains unknown. In this study, we analyzed the biochemical properties of the three PDCoV-encoded accessory proteins and found that NS7a could enhance the membrane permeability of both mammalian cells and Escherichia coli cells. Indirect immunofluorescence assay and co-immunoprecipitation assay results further indicated that NS7a is an integral membrane protein and can form homo-oligomers. A bioinformation analysis revealed that a putative viroporin domain (VPD) is located within amino acids 69-88 (aa69-88) of NS7a. Experiments with truncated mutants and alanine scanning mutagenesis additionally demonstrated that the amino acid residues 69FLR71 of NS7a are essential for its viroporin-like activity. Together, our findings are the first to demonstrate that PDCoV NS7a possesses viroporin-like activity and identify its key amino acid residues associated with viroporin-like activity.

Functions of Viroporins in the Viral Life Cycle and Their Regulation of Host Cell Responses.

Viroporins are virally encoded transmembrane proteins that are essential for viral pathogenicity and can participate in various stages of the viral life cycle, thereby promoting viral proliferation. Viroporins have multifaceted effects on host cell biological functions, including altering cell membrane permeability, triggering inflammasome formation, inducing apoptosis and autophagy, and evading immune responses, thereby ensuring that the virus completes its life cycle. Viroporins are also virulence factors, and their complete or partial deletion often reduces virion release and reduces viral pathogenicity, highlighting the important role of these proteins in the viral life cycle. Thus, viroporins represent a common drug-protein target for inhibiting drugs and the development of antiviral therapies. This article reviews current studies on the functions of viroporins in the viral life cycle and their regulation of host cell responses, with the aim of improving the understanding of this growing family of viral proteins.

Hijacking of Cellular Functions by Severe Acute Respiratory Syndrome Coronavirus-2. Permeabilization and Polarization of the Host Lipid Membrane by Viroporins.

Like all viral infections, SARS-CoV-2 acts at multiple levels, hijacking fundamental cellular functions and assuring its replication and immune system evasion. In particular, the viral 3' Open Reading Frame (ORF3a) codes for a hydrophobic protein, which embeds in the cellular membrane, where it acts as an ion viroporin and is related to strong inflammatory response. Here we report equilibrium and enhanced sampling molecular dynamic simulation of the SARS-CoV-2 ORF3a in a model lipid bilayer, showing how the protein permeabilizes the lipid membrane, via the formation of a water channel, which in turn assures ion transport. We report the free energy profile for both K+ and Cl- transfer from the cytosol to the extracellular domain. The important role of ORF3a in the viral cycle and its high conservation among coronaviruses may also make it a target of choice for future antiviral development, further justifying the elucidation of its mechanism at the atomistic level.

Secretory autophagy maintains proteostasis upon lysosome inhibition.

The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.

A single-shot vaccine approach for the universal influenza A vaccine candidate M2e.

SignificanceAlthough the need for a universal influenza vaccine has long been recognized, only a handful of candidates have been identified so far, with even fewer advancing in the clinical pipeline. The 24-amino acid ectodomain of M2 protein (M2e) has been developed over the past two decades. However, M2e-based vaccine candidates have shortcomings, including the need for several administrations and the lack of sustained antibody titers over time. We report here a vaccine targeting strategy that has the potential to confer sustained and strong protection upon a single shot of a small amount of M2e antigen. The current COVID-19 pandemic has highlighted the importance of developing versatile, powerful platforms for the rapid deployment of vaccines against any incoming threat.

SARS-CoV-2 ORF3a induces RETREG1/FAM134B-dependent reticulophagy and triggers sequential ER stress and inflammatory responses during SARS-CoV-2 infection.

SARS-CoV-2 infections have resulted in a very large number of severe cases of COVID-19 and deaths worldwide. However, knowledge of SARS-CoV-2 infection, pathogenesis and therapy remains limited, emphasizing the urgent need for fundamental studies and drug development. Studies have shown that induction of macroautophagy/autophagy and hijacking of the autophagic machinery are essential for the infection and replication of SARS-CoV-2; however, the mechanism of this manipulation and the function of autophagy during SARS-CoV-2 infection remain unclear. In the present study, we identified ORF3a as an inducer of autophagy (in particular reticulophagy) and revealed that ORF3a localizes to the ER and induces RETREG1/FAM134B-related reticulophagy through the HMGB1-BECN1 (beclin 1) pathway. As a consequence, ORF3a induces ER stress and inflammatory responses through reticulophagy and then sensitizes cells to the acquisition of an ER stress-related early apoptotic phenotype and facilitates SARS-CoV-2 infection, suggesting that SARS-CoV-2 ORF3a hijacks reticulophagy and then disrupts ER homeostasis to induce ER stress and inflammatory responses during SARS-CoV-2 infection. These findings reveal the sequential induction of reticulophagy, ER stress and acute inflammatory responses during SARS-CoV-2 infection and imply the therapeutic potential of reticulophagy and ER stress-related drugs for COVID-19.Abbreviations: CQ: chloroquine; DEGs: differentially expressed genes; ER: endoplasmic reticulum; GSEA: gene set enrichment analysis; HMGB1: high mobility group box 1; HMOX1: heme oxygenase 1; MERS-CoV: Middle East respiratory syndrome coronavirus; RETREG1/FAM134B: reticulophagy regulator 1; RTN4: reticulon 4; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TN: tunicamycin.

The Proteins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19.

The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.

A Gradient pH-Sensitive Polymer-Based Antiviral Strategy via Viroporin-Induced Membrane Acidification.

Lipid-membrane-targeting strategies hold great promise to develop broad-spectrum antivirals. However, it remains a big challenge to identify novel membrane-based targets of viruses and virus-infected cells for development of precision targeted approaches. Here, it is discovered that viroporins, viral-encoded ion channels, which have been reported to mediate release of hydrogen ions, trigger membrane acidification of virus-infected cells. Through development of a fine-scale library of gradient pH-sensitive (GPS) polymeric nanoprobes, the cellular membrane pH transitions are measured from pH 6.8-7.1 (uninfection) to pH 6.5-6.8 (virus-infection). In response to the subtle pH alterations, the GPS polymer with sharp response at pH 6.8 (GPS6.8 ) selectively binds to virus-infected cell membranes or the viral envelope, and even completely disrupts the viral envelope. Accordingly, GPS6.8 treatment exerts suppressive effects on a wide variety of viruses including SARS-CoV-2 through triggering viral-envelope lysis rather than affecting immune pathway or viability of host cells. Murine viral-infection models exhibit that supplementation of GPS6.8 decreases viral titers and ameliorates inflammatory damage. Thus, the gradient pH-sensitive nanotechnology offers a promising strategy for accurate detection of biological pH environments and robust interference with viruses.

Loss of Detection of sgN Precedes Viral Abridged Replication in COVID-19-Affected Patients-A Target for SARS-CoV-2 Propagation.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.