Post-translational modifications of ATG8 proteins - an emerging mechanism of autophagy control.

Autophagy is a recycling mechanism involved in cellular homeostasis with key implications for health and disease. The conjugation of the ATG8 family proteins, which includes LC3B (also known as MAP1LC3B), to autophagosome membranes, constitutes a hallmark of the canonical autophagy process. After ATG8 proteins are conjugated to the autophagosome membranes via lipidation, they orchestrate a plethora of protein-protein interactions that support key steps of the autophagy process. These include binding to cargo receptors to allow cargo recruitment, association with proteins implicated in autophagosome transport and autophagosome-lysosome fusion. How these diverse and critical protein-protein interactions are regulated is still not well understood. Recent reports have highlighted crucial roles for post-translational modifications of ATG8 proteins in the regulation of ATG8 functions and the autophagy process. This Review summarizes the main post-translational regulatory events discovered to date to influence the autophagy process, mostly described in mammalian cells, including ubiquitylation, acetylation, lipidation and phosphorylation, as well as their known contributions to the autophagy process, physiology and disease.

The Autophagy, Inflammation and Metabolism Center international eSymposium - an early-career investigators' seminar series during the COVID-19 pandemic.

The Autophagy, Inflammation and Metabolism (AIM) Center organized a globally accessible, virtual eSymposium during the COVID-19 pandemic in 2020. The conference included presentations from scientific leaders, as well as a career discussion panel, and provided a much-needed platform for early-career investigators (ECIs) to showcase their research in autophagy. This Perspective summarizes the science presented by the ECIs during the event and discusses the lessons learned from a virtual meeting of this kind during the pandemic. The meeting was a learning experience for all involved, and the ECI participants herein offer their thoughts on the pros and cons of virtual meetings as a modality, either as standalone or hybrid events, with a view towards the post-pandemic world.

Macroautophagy and aging: The impact of cellular recycling on health and longevity.

Aging is associated with many deleterious changes at the cellular level, including the accumulation of potentially toxic components that can have devastating effects on health. A key protective mechanism to this end is the cellular recycling process called autophagy. During autophagy, damaged or surplus cellular components are delivered to acidic vesicles called lysosomes, that secure degradation and recycling of the components. Numerous links between autophagy and aging exist. Autophagy declines with age, and increasing evidence suggests that this reduction plays important roles in both physiological aging and the development of age-associated disorders. Studies in pharmacologically and genetically manipulated model organisms indicate that defects in autophagy promote age-related diseases, and conversely, that enhancement of autophagy has beneficial effects on both healthspan and lifespan. Here, we review our current understanding of the role of autophagy in different physiological processes and their molecular links with aging and age-related diseases. We also highlight some recent advances in the field that could accelerate the development of autophagy-based therapeutic interventions.

LC3B phosphorylation: autophagosome's ticket for a ride toward the cell nucleus.

Macroautophagy/autophagy is a multi-step process that leads to cargo degradation via the fusion of hydrolases-containing lysosomes with cargo-loaded autophagosomes. For this process to occur, autophagosomes are directionally transported by molecular motors toward the nucleus, where they fuse with lysosomes for cargo degradation. The molecular basis for this regulation, including the cell machinery required for this directional transport, has not been fully identified. Using a combination of proteomic and live-imaging approaches in mammalian cells, including primary neurons, we describe that the phosphorylation of the autophagosome protein Atg8/LC3B by the Hippo kinase STK4/MST1, an event we previously reported to be required for autophagy completion, reduces the binding of the transport-related protein FYCO1 to MAP1LC3B/LC3B. This event in turn allows the proficient microtubule-based transport of autophagosomes toward the perinuclear area, thus facilitating the contact of autophagosomes with lysosomes. In the absence of LC3B phosphorylation, autophagosomes undergo aberrant transport including increased movement toward the cell periphery resulting in reduced autophagosome-lysosome colocalization. Thus, LC3B phosphorylation modulates the directional transport of autophagosomes to meet with lysosomes in the perinuclear area, a crucial event in ensuring autophagic degradation of cargo.

LC3B phosphorylation regulates FYCO1 binding and directional transport of autophagosomes.

Macroautophagy (hereafter referred to as autophagy) is a conserved process that promotes cellular homeostasis through the degradation of cytosolic components, also known as cargo. During autophagy, cargo is sequestered into double-membrane vesicles called autophagosomes, which are predominantly transported in the retrograde direction to the perinuclear region to fuse with lysosomes, thus ensuring cargo degradation.1 The mechanisms regulating directional autophagosomal transport remain unclear. The ATG8 family of proteins associates with autophagosome membranes2 and plays key roles in autophagy, including the movement of autophagosomes. This is achieved via the association of ATG8 with adaptor proteins like FYCO1, involved in the anterograde transport of autophagosomes toward the cell periphery.1,3-5 We previously reported that phosphorylation of LC3B/ATG8 on threonine 50 (LC3B-T50) by the Hippo kinase STK4/MST1 is required for autophagy through unknown mechanisms.6 Here, we show that STK4-mediated phosphorylation of LC3B-T50 reduces the binding of FYCO1 to LC3B. In turn, impairment of LC3B-T50 phosphorylation decreases starvation-induced perinuclear positioning of autophagosomes as well as their colocalization with lysosomes. Moreover, a significantly higher number of LC3B-T50A-positive autophagosomes undergo aberrant anterograde movement to axonal tips in mammalian neurons and toward the periphery of mammalian cells. Our data support a role of a nutrient-sensitive STK4-LC3B-FYCO1 axis in the regulation of the directional transport of autophagosomes, a key step of the autophagy process, via the post-translational modification of LC3B.

Beyond Autophagy: The Expanding Roles of ATG8 Proteins.

The ATG8 family proteins are critical players in autophagy, a cytoprotective process that mediates degradation of cytosolic cargo. During autophagy, ATG8s conjugate to autophagosome membranes to facilitate cargo recruitment, autophagosome biogenesis, transport, and fusion with lysosomes, for cargo degradation. In addition to these canonical functions, recent reports demonstrate that ATG8s are also delivered to single-membrane organelles, which leads to highly divergent degradative or secretory fates, vesicle maturation, and cargo specification. The association of ATG8s with different vesicles involves complex regulatory mechanisms still to be fully elucidated. Whether individual ATG8 family members play unique canonical or non-canonical roles, also remains unclear. This review summarizes the many open molecular questions regarding ATG8s that are only beginning to be unraveled.

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.

Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.

A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

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.

Severe acute respiratory syndrome coronaviruses with mutations in the E protein are attenuated and promising vaccine candidates.

UNLABELLED: Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouse-adapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo. To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-ΔE attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy-terminal region of E protein, led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx, and increased CD4(+) and CD8(+) T cell counts in the lungs of BALB/c mice, compared to mice infected with the wild-type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E*-infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates. IMPORTANCE: Human coronaviruses are important zoonotic pathogens. SARS-CoV caused a worldwide epidemic infecting more than 8,000 people with a mortality of around 10%. Therefore, understanding the virulence mechanisms of this pathogen and developing efficacious vaccines are of high importance to prevent epidemics from this and other human coronaviruses. Previously, we demonstrated that a SARS-CoV lacking the E protein was attenuated in vivo. Here, we show that small deletions and modifications within the E protein led to virus attenuation, manifested by minimal lung injury, limited neutrophil influx to the lungs, reduced expression of proinflammatory cytokines, increased anti-inflammatory cytokine levels, and enhanced CD4(+) and CD8(+) T cell counts in vivo, suggesting that these phenomena contribute to virus attenuation. The attenuated mutants fully protected mice from challenge with virulent virus. These studies show that mutations in the E protein are not well tolerated and indicate that this protein is an excellent target for vaccine development.

Coronavirus virulence genes with main focus on SARS-CoV envelope gene.

Coronavirus (CoV) infection is usually detected by cellular sensors, which trigger the activation of the innate immune system. Nevertheless, CoVs have evolved viral proteins that target different signaling pathways to counteract innate immune responses. Some CoV proteins act as antagonists of interferon (IFN) by inhibiting IFN production or signaling, aspects that are briefly addressed in this review. After CoV infection, potent cytokines relevant in controlling virus infections and priming adaptive immune responses are also generated. However, an uncontrolled induction of these proinflammatory cytokines can lead to pathogenesis and disease severity as described for SARS-CoV and MERS-CoV. The cellular pathways mediated by interferon regulatory factor (IRF)-3 and -7, activating transcription factor (ATF)-2/jun, activator protein (AP)-1, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NF-AT), are the main drivers of the inflammatory response triggered after viral infections, with NF-κB pathway the most frequently activated. Key CoV proteins involved in the regulation of these pathways and the proinflammatory immune response are revisited in this manuscript. It has been shown that the envelope (E) protein plays a variable role in CoV morphogenesis, depending on the CoV genus, being absolutely essential in some cases (genus α CoVs such as TGEV, and genus ß CoVs such as MERS-CoV), but not in others (genus ß CoVs such as MHV or SARS-CoV). A comprehensive accumulation of data has shown that the relatively small E protein elicits a strong influence on the interaction of SARS-CoV with the host. In fact, after infection with viruses in which this protein has been deleted, increased cellular stress and unfolded protein responses, apoptosis, and augmented host immune responses were observed. In contrast, the presence of E protein activated a pathogenic inflammatory response that may cause death in animal models and in humans. The modification or deletion of different motifs within E protein, including the transmembrane domain that harbors an ion channel activity, small sequences within the middle region of the carboxy-terminus of E protein, and its most carboxy-terminal end, which contains a PDZ domain-binding motif (PBM), is sufficient to attenuate the virus. Interestingly, a comprehensive collection of SARS-CoVs in which these motifs have been modified elicited full and long-term protection even in old mice, making those deletion mutants promising vaccine candidates. These data indicate that despite its small size, E protein drastically influences the replication of CoVs and their pathogenicity. Although E protein is not essential for CoV genome replication or subgenomic mRNA synthesis, it affects virus morphogenesis, budding, assembly, intracellular trafficking, and virulence. In fact, E protein is responsible in a significant proportion of the inflammasome activation and the associated inflammation elicited by SARS-CoV in the lung parenchyma. This exacerbated inflammation causes edema accumulation leading to acute respiratory distress syndrome (ARDS) and, frequently, to the death of infected animal models or human patients.

The PDZ-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis.

A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.

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.

Inhibition of NF-κB-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of a respiratory disease that has a 10% mortality rate. We previously showed that SARS-CoV lacking the E gene (SARS-CoV-ΔE) is attenuated in several animal model systems. Here, we show that absence of the E protein resulted in reduced expression of proinflammatory cytokines, decreased numbers of neutrophils in lung infiltrates, diminished lung pathology, and increased mouse survival, suggesting that lung inflammation contributed to SARS-CoV virulence. Further, infection with SARS-CoV-ΔE resulted in decreased activation of NF-κB compared to levels for the wild-type virus. Most important, treatment with drugs that inhibited NF-κB activation led to a reduction in inflammation and lung pathology in both SARS-CoV-infected cultured cells and mice and significantly increased mouse survival after SARS-CoV infection. These data indicated that activation of the NF-κB signaling pathway represents a major contribution to the inflammation induced after SARS-CoV infection and that NF-κB inhibitors are promising antivirals in infections caused by SARS-CoV and potentially other pathogenic human coronaviruses.

Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate.

UNLABELLED: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE: Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines.

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-ß, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis.

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.