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-1β 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-1β 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

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 SARSCoVs, 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-1b 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-1b 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

Human Coronavirus Virulence Motifs and Virulence

Trabajo presentado en el XIV International Nidovirus Symposium (Nido2017), celebrado en Kansas City, Missouri (Estados Unidos), del 4 al 9 de junio de 2017We have shown that SARS-CoV E protein is a virulence factor that includes at least two virulence motifs: its ion channel (IC) activity encoded within the transmembrane domain and a PDZ binding motif (PBM) located at its carboxy-terminus. We showed that E protein pathogenicity was caused by the activation of different host signaling pathways. One of them was the activation of inflammasome, a process mediated by the conductance of Ca++ byEprotein IC activity, leading to an increased expression of IL-1beta, TNF-alpha and IL-6 levels. Another signaling pathway implied the activation of a proinflammatory response mediated by NF-kB activation. This activation was a consequence of E protein-syntenin binding mediated by PBM-PDZ interactions. This binding caused an increase of p38MAPK phosphorylation promoting the induction of acute respiratory distress syndrome (ARDS), edema and death of mice infected with a mouse adapted SARS-CoV. The relevance of p38 MAPK activation after infection with the mouse adapted SARS-CoV was confirmed by the protection of mice in the presence of an inhibitor of p38 MAPK, but not in its absence. These results illustrated the identification of an efficient coronavirus (CoV) antiviral. The presence of a virulence factor such as the PBM motif in E protein allows the virus to interact with more than 400 cell proteins containing PDZ motifs, conferring the virus the potential to control a high number of cell-signaling pathways increasing its replication and virulence. In fact, we are analyzing the proteome of the viral PBM-cellular PDZ interactions using system biology approaches. Frequently, the ARDS caused by lung infection with mild respiratory viruses is resolved before it evolves to serious edema. In contrast, after SARS-CoV infection frequently this resolution does not take place. We have shown the binding of E protein to a main mediator of edema resolution, the Na+ /K+ ATPase, and proposed that this may be one of the procedures by which edema recovery is prevented after SARS-CoV infection, either by inhibition of Na+ /K+ ATPase activity or by relocating this enzyme to another subcellular compartment. Deadly human CoVs as SARS- and MERS-CoVs have at least two viral proteins with IC activity and PBM motifs. Studies on the relevance of E and 3a SARS-CoV proteins in replication and virulence, and the interdependence among them have shown that the presence in the virus of at least E or 3a proteins was needed for virus viability. In fact, we have shown that the complementation between E and 3a proteins is mediated by the PBM motifs located at the carboxy-terminus of these proteins. Our studies on the interaction of SARS-CoV and MERS-CoV with the host, and the engineering of reverse genetics systems for each of these viruses, led us to the development of genetically stable vaccine candidates that provided full-protection against the challenge with the homologous virulent virus using mice models

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-beta, 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 pathogenesisThis work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16705), the European Community's Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” EC Grant Agreement number 223498, and U.S. National Institutes of Health (NIH) (2P01AI060699-06A1, W000306844). JAR received a fellowship from the Fundacion La Caix

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-1β 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-1β 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.The work done by the authors was supported by Grants from the Government of Spain (BIO2013-42869-R, FIS2013-40473-P), Generalitat Valenciana (Prometeo 2012/069), Fundació Caixa Castelló-Bancaixa (P1-1B2012-03) and a U.S. National Institutes of Health (NIH) project (5P01 AI060699). JLN, JMJ and JAR received contracts from NIH. CCR received a contract from Fundacion La Caixa

Role of SARS-CoV viroporins E, 3a, and 8a in virus replication and virulence: Complementation between the PBMs of E and 3a proteins

Trabajo presentado en el 6th European Congress of Virology, celebrado en Hamburgo (Alemania), del 19 al 22 de octubre de 2016SARS-CoV has three viroporins: 3a, E and 8a. Recombinant SARS-CoV (rSARS-CoV) variants lacking each of these proteins have been engineered. Their analysis has shown that none of these viroporins were essential for virus replication, and that proteins 3a and E were relevant in virulence. Interestingly, a virus missing both 3a and E genes could not be rescued suggesting a complementation between proteins 3a and E for virus viability. At least two activities are shared by these two viroporins: ion channel (IC) and PDZ binding motif (PBM). In the present work, we studied whether 3a and E protein IC activities or PBMs were responsible for this complementation. To this end, rSARS-CoV in which IC activities of 3a protein or E protein were knocked out, were firstly engineered. In order to construct rSARS-CoV without 3a or E protein IC activity (rSARS-CoV-3aIC-, rSARS-CoV-EIC-), the amino acids involved in their IC activity were determined. E protein has a single transmembrane domain (TMD) while 3a protein has three. The conductance of native and mutant E and 3a proteins was measured in artificial membranes and the amino acids responsible for their IC activity were identified. Then, rSARS-CoV-3aIC- and rSARS-CoV-EIC- viruses were constructed. Evaluation of their pathogenicity in BALB/c mice showed that E protein IC activity is a virulence factor in contrast to 3a protein IC activity. Furthermore, E protein IC activity is required for inflammasome activation, which triggers the expression of proinflammatory cytokines leading to edema accumulation and ARDS. Further experiments will clarify if the complementation between proteins E and 3a was due to the PBM present in their carboxy-terminus

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-1β 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-1β 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.This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/2007–2013) under the project ‘‘EMPERIE’’ EC Grant Agreement number 223498, and U.S. National Institutes of Health (NIH) (2P01AI060699 and 0258-3413/HHSN266200700010C). Financial support from Generalitat Valenciana (Prometeu 2012/069) and Fundacion Caixa Castello-Bancaixa (Project No. P1-1B2012-03) is also acknowledged. JLN received a contract from NIH. JAR and CCR received fellowships from Fundacion La Caixa

Human coronaviruses pathogenesis and protection

Trabajo presentado en el International Meeting of the Federation of Korean MicrobiologicaI Societies: Glimpse of microbial world: from basic science to translation and application, celebrado en Gyeonggi-do (Corea del Sur), del 3 al 4 de noviembre de 2016The emergence of viruses causing diseases in humans is constant in History. Most emergent viruses have been transmitted from animal hosts to humans (zoonosis). Coronaviruses (CoVs) have frequently crossed the species barrier and two novel coronaviruses have caused important zoonosis in the twenty-first centwy. Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 in South East China and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 in Saudi Arabia. Both viruses cause acute respiratory distress syndrome and are associated with mortality rates around 10% and 35% respectively

Role of SARS-CoV viroporins E, 3a and 8a in virus replication and virulence: complementation between the PBMs of E and 3a proteins

Trabajo presentado en el XIV Congreso Nacional de Virología, celebrado en Cádiz (España), del 11 al 14 de junio de 2017Severe acute respiratory syndrome coronavirus (SARS-CoV) causes lethal disease in humans, which is characterized by exacerbated inflammatory response and extensive lung pathology. In addition to protein components, viral genomes can also encode non-coding RNAs (ncRNAs) differing in size, biogenesis, and function, which are not necessarily comparable to those produced by host cells. To address the relevance of small non-coding RNAs in SARS-CoV pathology, we deep sequenced RNAs from the lungs of infected mice, which reproduce the pulmonary pathology observed in humans. Recombinant SARS-CoV (rSARS-CoV) variants lacking each of these proteins have been engineered. Analysis of the deletion mutantsshowed that none of themwere essential for virus replication, and that proteins 3a and E were relevant in virulence. In contrast, a virus simultaneously missing 3a and E genes could not be rescued suggesting a complementation between proteins 3a and E for virus viability. At least two activities are shared by these two viroporins: ion channel (IC) and PDZ binding motif (PBM).PBMs can potentially bind over 400 cellular proteins containing PDZ domains conferring high relevance to PBM motifs in the control of cell behaviour. In the present work, we studied whether 3a and E protein IC activities or PBMswere responsible for this complementation. In order to construct rSARS-CoV without 3a or E protein IC activity, the amino acids involved in this activity were determined and rSARS-CoVs missing E or 3a proteins IC conductancewere constructed

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.This work was supported by grants from the Government of Spain (BIO2013-42869-R and BIO2016-75549-R AEI/FEDER, UE), the European Zoonotic Anticipation and Preparedness Initiative (ZAPI) (IMI_JU_115760), and the U.S. National Institutes of Health (NIH) (0258-3413/HHSN266200700010C awarded to L.E., 2P01AI060699 awarded to L.E. and S.P., and R01 AI129269 awarded to S.P.). V.M.A. and M.Q.M. are grateful for the support of the Government of Spain (FIS2013-40473-P and FIS2016-75257-P AEI/FEDER, UE) and Universitat Jaume I (P1.1B2015-28). C.C.R. received a contract from Fundación La Caixa