Abstract
The molecular mechanisms underlying the severe lung pathology that occurs during SARS-CoV infections remain incompletely understood. The largest of the SARS-CoV accessory protein open reading frames (SARS 3a) oligomerizes, dynamically inserting into late endosomal, lysosomal, and trans-Golgi-network membranes. While previously implicated in a non-inflammatory apoptotic cell death pathway, here we extend the range of SARS 3a pathophysiologic targets by examining its effects on necrotic cell death pathways. We show that SARS 3a interacts with Receptor Interacting Protein 3 (Rip3), which augments the oligomerization of SARS 3a helping drive necrotic cell death. In addition, by inserting into lysosomal membranes SARS 3a triggers lysosomal damage and dysfunction. Consequently, Transcription Factor EB (TFEB) translocates to the nucleus increasing the transcription of autophagy- and lysosome-related genes. Finally, SARS 3a activates caspase-1 either directly or via an enhanced potassium efflux, which triggers NLRP3 inflammasome assembly. In summary, Rip3-mediated oligomerization of SARS 3a causes necrotic cell death, lysosomal damage, and caspase-1 activation—all likely contributing to the clinical manifestations of SARS-CoV infection.
Highlights
Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) that at its peak affected more than 8000 people with a 10% mortality rate[1]
To assay necrotic cell death, we concomitantly monitored cellular ATP and the release of cytosolic proteases[27]
After expression of SARS 3a-flag and Receptor Interacting Protein 3 (Rip3)-Myc in 293T cells, immunoprecipitated SARS 3a-flag pulls down Rip3-Myc, whereas SARS 3a and Rip3Myc cannot be detected from immunoprecipitation using mock antibody beads (Fig. 1c)
Summary
Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) that at its peak affected more than 8000 people with a 10% mortality rate[1]. The recent emergence of a SARS-like CoV called Middle East Respiratory Syndrome coronavirus has underscored the need to understand the mechanisms behind CoV pathogenicity[2]. SARS presents with flu-like symptoms that can progress to respiratory failure secondary to immunopathologic injury[3,4]. Pathologic examination of lung tissue from fatal cases shows diffuse alveolar damage, significant. While the contributions of IMMs to disease pathogenesis is understood, the molecular mechanisms behind their aberrant inflammatory state is not. The SARS-CoV genome encodes eight accessory proteins designated open reading frame (ORF)-3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b8. Several ORF functions have been identified: ORF-7a activates NF-κB;[9] ORF3b upregulates the expression of several cytokines and chemokines;[10,11] ORF-6 reduces
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