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.

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 (SARS‐CoV) is capable of inducing a storm of proinflammatory cytokines. In this study, we show that the SARS‐CoV open reading frame 3a (ORF3a) accessory protein activates the NLRP3 inflammasome by promoting TNF receptor‐associated factor 3 (TRAF3)–mediated ubiquitination of apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC). SARS‐CoV and its ORF3a protein were found to be potent activators of pro–IL‐1β gene transcription and protein maturation, the 2 signals required for activation of the NLRP3 inflammasome. ORF3a induced pro–IL‐1β transcription through activation of NF‐κB, which was mediated by TRAF3‐dependent ubiquitination and processing of p105. ORF3a‐induced elevation of IL‐1β secretion was independent of its ion channel activity or absent in melanoma 2 but required NLRP3, ASC, and TRAF3. ORF3a interacted with TRAF3 and ASC, colocalized with them in discrete punctate structures in the cytoplasm, and facilitated ASC speck formation. TRAF3‐dependent K63‐linked ubiquitination of ASC was more pronounced in SARS‐CoV–infected cells or when ORF3a was expressed. Taken together, our findings reveal a new mechanism by which SARS‐CoV ORF3a protein activates NF‐κB and the NLRP3 inflammasome by promoting TRAF3‐dependent ubiquitination of p105 and ASC.—Siu, K.‐L., Yuen, K.‐S., Castano‐Rodriguez, C., Ye, Z.‐W., Yeung, M.‐L., Fung, S.‐Y., Yuan, S., Chan, C.‐P., Yuen, K.‐Y., Enjuanes, L., Jin, D.‐Y. Severe acute respiratory syndrome Coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3‐dependent ubiquitination of ASC. FASEB J. 33, 8865–8877 (2019). http://www.fasebj.org