The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localises to mitochondria during infection, where it inhibits innate immunity by restricting IFN-{beta} production, but not NF-{kappa}B activation or JAK-STAT signalling downstream of type I IFN stimulation. We find that ORF3c acts after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterised mechanism of innate immune evasion by this important human pathogen.

Increased supraorganization of respiratory complexes is a dynamic multistep remodelling in response to proteostasis stress.

Proteasome-mediated degradation of misfolded proteins prevents aggregation inside and outside mitochondria. But how do cells safeguard the mitochondrial proteome and mitochondrial functions despite increased aggregation during proteasome inactivation? Here, using a novel two-dimensional complexome profiling strategy, we report increased supraorganization of respiratory complexes (RCs) in proteasome-inhibited cells that occurs simultaneously with increased pelletable aggregation of RC subunits inside mitochondria. Complex II (CII) and complex V (CV) subunits are increasingly incorporated into oligomers. Complex I (CI), complex III (CIII) and complex IV (CIV) subunits are engaged in supercomplex formation. We unravel unique quinary states of supercomplexes during early proteostatic stress that exhibit plasticity and inequivalence of constituent RCs. The core stoichiometry of CI and CIII is preserved, whereas the composition of CIV varies. These partially disintegrated supercomplexes remain functionally competent via conformational optimization. Subsequently, increased stepwise integration of RC subunits into holocomplexes and supercomplexes re-establishes steady-state stoichiometry. Overall, the mechanism of increased supraorganization of RCs mimics the cooperative unfolding and folding pathways for protein folding, but is restricted to RCs and is not observed for any other mitochondrial protein complexes.This article has an associated First Person interview with the first author of the paper.

Aggregation of Respiratory Complex Subunits Marks the Onset of Proteotoxicity in Proteasome Inhibited Cells.

Proteostasis is maintained by optimal expression, folding, transport, and clearance of proteins. Deregulation of any of these processes triggers protein aggregation and is implicated in many age-related pathologies. In this study, using quantitative proteomics and microscopy, we show that aggregation of many nuclear-encoded mitochondrial proteins is an early protein destabilization event during short-term proteasome inhibition. Among these, respiratory chain complex (RCC) subunits represent a group of functionally related proteins consistently forming aggregates under multiple proteostasis stresses with varying aggregation propensities. Sequence analysis reveals that several RCC subunits, irrespective of the cleavable mitochondrial targeting sequence, contain low-complexity regions at the N-terminus. Using different chimeric and mutant constructs, we show that these low-complexity regions partially contribute to the intrinsic instability of multiple RCC subunits. Taken together, we propose that physicochemically driven aggregation of unassembled RCC subunits destabilizes their functional assembly inside mitochondria. This eventually deregulates the biogenesis of respiratory complexes and marks the onset of mitochondrial dysfunction.