ABSTRACT
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 localizes to mitochondria, where it inhibits innate immunity by restricting IFN-ß production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory 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 uncharacterized mechanism of innate immune evasion by this important human pathogen.
ABSTRACT
Identifying the sequence polymorphisms underlying complex trait variation is a key goal of genetics research, since knowing the precise causative molecular events allows insight into the pathways governing trait variation. Genetic analysis of complex traits in model systems regularly starts by constructing QTL maps, but generally fails to identify causative sequence polymorphisms. Previously we mapped a series of QTL contributing to resistance to nicotine in a Drosophila melanogaster multiparental mapping resource and here use a battery of functional tests to resolve QTL to the molecular level. One large-effect QTL resided over a cluster of UDP-glucuronosyltransferases, and quantitative complementation tests using deficiencies eliminating subsets of these detoxification genes revealed allelic variation impacting resistance. RNAseq showed that Ugt86Dd had significantly higher expression in genotypes that are more resistant to nicotine, and anterior midgut-specific RNA interference (RNAi) of this gene reduced resistance. We discovered a segregating 22-bp frameshift deletion in Ugt86Dd, and accounting for the InDel during mapping largely eliminates the QTL, implying the event explains the bulk of the effect of the mapped locus. CRISPR/Cas9 editing of a relatively resistant genotype to generate lesions in Ugt86Dd that recapitulate the naturally occurring putative loss-of-function allele, leads to a large reduction in resistance. Despite this major effect of the deletion, the allele appears to be very rare in wild-caught populations and likely explains only a small fraction of the natural variation for the trait. Nonetheless, this putatively causative coding InDel can be a launchpad for future mechanistic exploration of xenobiotic detoxification.
