SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes

Chong-Shan Shi,Neel R Nabar,John H Kehrl,Ning-Na Huang

doi:10.1038/s41420-019-0181-7

Abstract

The SARS (severe acute respiratory syndrome) outbreak was caused by a coronavirus (CoV) named the SARS-CoV. SARS pathology is propagated both by direct cytotoxic effects of the virus and aberrant activation of the innate immune response. Here, we identify several mechanisms by which a SARS-CoV open reading frame (ORF) activates intracellular stress pathways and targets the innate immune response. We show that ORF8b forms insoluble intracellular aggregates dependent on a valine at residue 77. Aggregated ORF8b induces endoplasmic reticulum (ER) stress, lysosomal damage, and subsequent activation of the master regulator of the autophagy and lysosome machinery, Transcription factor EB (TFEB). ORF8b causes cell death in epithelial cells, which is partially rescued by reducing its ability to aggregate. In macrophages, ORF8b robustly activates the NLRP3 inflammasome by providing a potent signal 2 required for activation. Mechanistically, ORF8b interacts directly with the Leucine Rich Repeat domain of NLRP3 and localizes with NLRP3 and ASC in cytosolic dot-like structures. ORF8b triggers cell death consistent with pyroptotic cell death in macrophages. While in those cells lacking NLRP3 accumulating ORF8b cytosolic aggregates cause ER stress, mitochondrial dysfunction, and caspase-independent cell death.

Highlights

In 2002–2003 the SARS-CoV caused a severe respiratory illness affecting more than 8000 individuals with a mortality rate near 10%1,2
Analysis of the ORF8b sequence predicted a tendency to aggregate based on a carboxy-terminal VLVVL motif, while reanalyzing the ORF8b sequence with V77 mutated to K (V77K) reduced its predicted propensity to aggregate (Fig. 1a)
Recent advances in mouse models have shown that SARS pathogenesis is driven by high initial virus titers resulting from a late interferon response, which drives aberrant recruitment of inflammatory monocyte-macrophages (IMMs) and activation of the innate immune response resulting in cytotoxicity[9]

Summary

Introduction

In 2002–2003 the SARS-CoV caused a severe respiratory illness affecting more than 8000 individuals with a mortality rate near 10%1,2. The subsequent identification of a large pool of coronaviruses circulating in bats and other animals portended the appearance of other highly pathogenic CoVs3, and in 2012 the Middle East Respiratory SyndromeCoV caused a similar outbreak with a higher mortality rate[4]. The SARS-CoV is an enveloped, positive-strand RNA virus that encodes a set of accessory proteins, several of which target the innate immune response. Open reading frame (ORF) 8a and ORF9b trigger cellular apoptosis; ORF7a activates nuclear factor-κB (NF-κB); ORF3b upregulates the expression of several cytokines and chemokines; ORF6 limits interferon production; ORF3a induces necrotic cell death; and ORF8b induces cellular DNA synthesis and suppresses the expression of the viral envelope protein[10,11]. We found that ORF9b localizes to mitochondrial membranes and reduces

Methods

Results

Conclusion