Transport mechanisms of SARS-CoV-E viroporin in calcium solutions: Lipid-dependent Anomalous Mole Fraction Effect and regulation of pore conductance.

The envelope protein E of the SARS-CoV coronavirus is an archetype of viroporin. It is a small hydrophobic protein displaying ion channel activity that has proven highly relevant in virus-host interaction and virulence. Ion transport through E channel was shown to alter Ca2+ homeostasis in the cell and trigger inflammation processes. Here, we study transport properties of the E viroporin in mixed solutions of potassium and calcium chloride that contain a fixed total concentration (mole fraction experiments). The channel is reconstituted in planar membranes of different lipid compositions, including a lipid mixture that mimics the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane where the virus localizes within the cell. We find that the E ion conductance changes non-monotonically with the total ionic concentration displaying an Anomalous Mole Fraction Effect (AMFE) only when charged lipids are present in the membrane. We also observe that E channel insertion in ERGIC-mimic membranes - including lipid with intrinsic negative curvature - enhances ion permeation at physiological concentrations of pure CaCl2 or KCl solutions, with a preferential transport of Ca2+ in mixed KCl-CaCl2 solutions. Altogether, our findings demonstrate that the presence of calcium modulates the transport properties of the E channel by interacting preferentially with charged lipids through different mechanisms including direct Coulombic interactions and possibly inducing changes in membrane morphology.

Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis.

Viroporins are viral proteins with ion channel (IC) activity that play an important role in several processes, including virus replication and pathogenesis. While many coronaviruses (CoVs) encode two viroporins, severe acute respiratory syndrome CoV (SARS-CoV) encodes three: proteins 3a, E, and 8a. Additionally, proteins 3a and E have a PDZ-binding motif (PBM), which can potentially bind over 400 cellular proteins which contain a PDZ domain, making them potentially important for the control of cell function. In the present work, a comparative study of the functional motifs included within the SARS-CoV viroporins was performed, mostly focusing on the roles of the IC and PBM of E and 3a proteins. Our results showed that the full-length E and 3a proteins were required for maximal SARS-CoV replication and virulence, whereas viroporin 8a had only a minor impact on these activities. A virus missing both the E and 3a proteins was not viable, whereas the presence of either protein with a functional PBM restored virus viability. E protein IC activity and the presence of its PBM were necessary for virulence in mice. In contrast, the presence or absence of the homologous motifs in protein 3a did not influence virus pathogenicity. Therefore, dominance of the IC and PBM of protein E over those of protein 3a was demonstrated in the induction of pathogenesis in mice.IMPORTANCE Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines.

Channel-Inactivating Mutations and Their Revertant Mutants in the Envelope Protein of Infectious Bronchitis Virus.

It has been shown previously in the severe acute respiratory syndrome coronavirus (SARS-CoV) that two point mutations, N15A and V25F, in the transmembrane domain (TMD) of the envelope (E) protein abolished channel activity and led to in vivo attenuation. Pathogenicity was recovered in mutants that also regained E protein channel activity. In particular, V25F was rapidly compensated by changes at multiple V25F-facing TMD residues located on a neighboring monomer, consistent with a recovery of oligomerization. Here, we show using infected cells that the same mutations, T16A and A26F, in the gamma-CoV infectious bronchitis virus (IBV) lead to, in principle, similar results. However, IBV E A26F did not abolish oligomer formation and was compensated by mutations at N- and C-terminal extramembrane domains (EMDs). The C-terminal EMD mutations clustered along an insertion sequence specific to gamma-CoVs. Nuclear magnetic resonance data are consistent with the presence of only one TMD in IBV E, suggesting that recovery of channel activity and fitness in these IBV E revertant mutants is through an allosteric interaction between EMDs and TMD. The present results are important for the development of IBV live attenuated vaccines when channel-inactivating mutations are introduced in the E protein.IMPORTANCE The ion channel activity of SARS-CoV E protein is a determinant of virulence, and abolishment of channel activity leads to viral attenuation. E deletion may be a strategy for generating live attenuated vaccines but can trigger undesirable compensatory mechanisms through modifications of other viral proteins to regain virulence. Therefore, a more suitable approach may be to introduce small but critical attenuating mutations. For this, the stability of attenuating mutations should be examined to understand the mechanisms of reversion. Here, we show that channel-inactivating mutations of the avian infectious bronchitis virus E protein introduced in a recombinant virus system are deficient in viral release and fitness and that revertant mutations also restored channel activity. Unexpectedly, most of the revertant mutations appeared at extramembrane domains, particularly along an insertion specific for gammacoronaviruses. Our structural data propose a single transmembrane domain in IBV E, suggesting an allosteric interaction between extramembrane and transmembrane domains.

Ion channel activity of the CSFV p7 viroporin in surrogates of the ER lipid bilayer.

Viroporins comprise a family of non-structural proteins that play significant and diverse roles during the replication cycle of many animal viruses. Consequently, they have become promising targets for inhibitory drug and vaccine development. Structure­function traits common to all members of the family are their small size (ca. 60­120 aa), high hydrophobicity, and the presence of helical domains that transverse the membrane and assemble into oligomeric-permeating structures therein. The possibility that viroporins show in particular conditions any kind of specificity in the transport of ions and small solutes remains a point of contention in the field. Here we have approached this issue using the Classical Swine Fever Virus (CSFV) protein p7 viroporin as a model. We have previously reported that CSFV-p7 induces release of ANTS (MW: 427.33) from lipid vesicles that emulate the Endoplasmic Reticulum (ER) membrane, and that this process is dependent on pH, modulated by the lipid composition, and recreated by a C-terminal transmembrane helix. Here we have assayed CSFV-p7 for its capacity to form ion-conducting channels in ER-like planar lipid membranes, and established whether this activity is subject to regulation by the same factors. The analysis of electrophysiological recordings in ER membrane surrogates suggests that CSFV-p7 forms pores wide enough to allow ANTS release. Moreover, we were able to discriminate between two pore structures with slightly different sizes and opposite ion selectivities. The fact that the relative abundances of each pore type depend crucially on membrane composition strengthens the view that the physicochemical properties of the lipid bilayers present in the cell endomembrane system modulate viroporin activity.

Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome.

Severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein is a viroporin involved in virulence. E protein ion channel (IC) activity is specifically correlated with enhanced pulmonary damage, edema accumulation and death. IL-1ß driven proinflammation is associated with those pathological signatures, however its link to IC activity remains unknown. In this report, we demonstrate that SARS-CoV E protein forms protein-lipid channels in ERGIC/Golgi membranes that are permeable to calcium ions, a highly relevant feature never reported before. Calcium ions together with pH modulated E protein pore charge and selectivity. Interestingly, E protein IC activity boosted the activation of the NLRP3 inflammasome, leading to IL-1ß overproduction. Calcium transport through the E protein IC was the main trigger of this process. These findings strikingly link SARS-CoV E protein IC induced ionic disturbances at the cell level to immunopathological consequences and disease worsening in the infected organism.

Relevance of Viroporin Ion Channel Activity on Viral Replication and Pathogenesis.

Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology.

MERS coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified pathogen able of human transmission that causes a mortality of almost 40%. As in the case of SARS-CoV, MERS virus lacking E protein represents a potential vaccine. In both cases, abolishment of channel activity may be a contributor to the attenuation observed in E-deleted viruses. Herein, we report that purified MERS-CoV E protein, like SARS-CoV E protein, is almost fully α-helical, has a single α-helical transmembrane domain, and forms pentameric ion channels in lipid bilayers. Based on these similarities, and the proposed involvement of channel activity as virulence factor in SARS-CoV E protein, MERS-CoV E protein may constitute a potential drug target.

Entropy-enthalpy compensation at the single protein level: pH sensing in the bacterial channel OmpF.

The pH sensing mechanism of the OmpF channel operates via ligand modification: increasing acidity induces the replacement of cations with protons in critical binding sites decreasing the channel conductance. Aside from the change in enthalpy associated with the binding, there is also a change in the microscopic arrangements of ligands, receptors and the surrounding solvent. We show that the pH-modulation of the single channel conduction involves small free energy changes because large enthalpic and entropic contributions change in opposite ways, demonstrating an approximate enthalpy-entropy compensation for different salts and concentrations.

Inhibition of the human respiratory syncytial virus small hydrophobic protein and structural variations in a bicelle environment.

The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single α-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the α-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. The latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin. Importance: The human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small-molecule inhibitor, pyronin B, can reduce SH channel activity, and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low-pH activation, which we have not been able to reproduce.

Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis.

Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1ß were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1ß was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence.

Amphiphilic COSAN and I2-COSAN crossing synthetic lipid membranes: planar bilayers and liposomes.

The boron-rich cobaltabisdicarbollide (COSAN) and its 8,8'-I2 derivative (I2-COSAN), both of purely inorganic nature, are shown to cross through synthetic lipid membranes. These results reveal unexpected properties at the interface of biological and synthetic membranes.

Lipid charge regulation of non-specific biological ion channels.

Ion channels are specialized proteins that enable the movement of charges through otherwise impermeable lipidic membranes. Their action is essential in living organisms facilitating electric signaling, muscle contraction or osmotic stress response among other effects. The protein and the lipid charges configure a polarized interface that yields local ionic concentrations and electric potentials that are very different from those of the bulk electrolyte. The combined effect of gradients of ionic concentration and electric potential causes the transport of ions through channels. Here we analyze charge regulation effects in different protein-lipid conformations, stressing how important is the role of electrostatic interactions in the ion channel function that traditionally has been rationalized paying attention mainly to changes in pore size. Tuning lipid charge combined with conductance and selectivity measurements is shown to be a complementary method to evidence lipid involvement in the structure of a biological ion channel.

Electrostatic interactions drive the nonsteric directional block of OmpF channel by La3+.

Ion channels regulate the transport of molecules and the electric signal transduction in living cells by means of complex and even highly sophisticated mechanisms. We focus here on the crucial role that polyvalent ions, well-known modulators of many biological nanosystems, play in ion channel function. In particular, we show that trace amounts of lanthanum are able to block the bacterial porin OmpF, a large biological pore of Escherichia coli wide enough to exchange antibiotics and other larger molecules. The underlying mechanism has a strong directional character: it is sensitive to the sign of the applied voltage and to the side of the blocker addition. We explore these channel features by combining planar lipid bilayer electrophysiology at the single channel level, site-directed mutagenesis, and inductively coupled plasma mass spectrometry (ICP-MS). In contrast to other well-described channel blockers, which seem to occlude the narrower part of the pore, we envisage a nonsteric mechanism based on electrostatic interactions.

Analysis of SARS-CoV E protein ion channel activity by tuning the protein and lipid charge.

A partial characterization of the ion channels formed by the SARS coronavirus (CoV) envelope (E) protein was previously reported (C. Verdiá-Báguena et al., 2012 [12]). Here, we provide new significant insights on the involvement of lipids in the structure and function of the CoV E protein channel on the basis of three series of experiments. First, reversal potential measurements over a wide range of pH allow the dissection of the contributions to channel selectivity coming from ionizable residues of the protein transmembrane domain and also from the negatively charged groups of diphytanoyl phosphatidylserine (DPhPS) lipid. The corresponding effective pKas are consistent with the model pKas of the acidic residue candidates for titration. Second, the change of channel conductance with salt concentration reveals two distinct regimes (Donnan-controlled electrodiffusion and bulk-like electrodiffusion) fully compatible with the outcomes of selectivity experiments. Third, by measuring channel conductance in mixtures of neutral diphytanoyl phosphatidylcholine (DPhPC) lipids and negatively charged DPhPS lipids in low and high salt concentrations we conclude that the protein-lipid conformation in the channel is likely the same in charged and neutral lipids. Overall, the whole set of experiments supports the proteolipidic structure of SARS-CoV E channels and explains the large difference in channel conductance observed between neutral and charged membranes.

Coronavirus E protein forms ion channels with functionally and structurally-involved membrane lipids.

Coronavirus (CoV) envelope (E) protein ion channel activity was determined in channels formed in planar lipid bilayers by peptides representing either the transmembrane domain of severe acute respiratory syndrome CoV (SARS-CoV) E protein, or the full-length E protein. Both of them formed a voltage independent ion conductive pore with symmetric ion transport properties. Mutations N15A and V25F located in the transmembrane domain prevented the ion conductivity. E protein derived channels showed no cation preference in non-charged lipid membranes, whereas they behaved as pores with mild cation selectivity in negatively-charged lipid membranes. The ion conductance was also controlled by the lipid composition of the membrane. Lipid charge also regulated the selectivity of a HCoV-229E E protein derived peptide. These results suggested that the lipids are functionally involved in E protein ion channel activity, forming a protein-lipid pore, a novel concept for CoV E protein ion channel entity.