The effect of ORF3c, a SARS-CoV-2 accessory protein, on cellular innate immune responses, autophagy, and lung cell mitochondrial metabolism

By Neha MathurOct 7 2022Reviewed by Danielle Ellis, B.Sc. In a recent study posted to the bioRxiv* server, researchers explored the role of the open reading frame 3c , a severe acute respiratory syndrome coronavirus 2 accessory protein not so extensively studied as yet. They determined the cellular localization of ORF3c, its role in modulating the host's antiviral response , and its effects on mitochondrial metabolism.

SARS-CoV-2 has a single-stranded ribonucleic acid genome of 30kilobase. Its positive-strand genomic RNA translates two overlapping ORFs, ORF1a, and ORF1b, which, in turn, generates 16 non-structural proteins . Genomic studies have also identified several unannotated alternative open reading frames within ORF3a, such as ORF3b, ORF3c, and ORF3d.

Further, the researchers investigated ORF3c mitochondrial localization by confocal microscopy, for which they transfected HeLa cells with the pDsRed2-Mito vector. They also confirmed the mitochondrial localization of ORF3c using the two lung cell lines A549 and HSAEC1. Furthermore, the team investigated how the ORF3c protein modified mitochondrial metabolism through Agilent Seahorse XF Mito Stress analysis.

The real-time oxygen consumption rate showed that ORF3c protein increased basal and maximal respiration and mitochondrial ATP synthesis. However, extra-cellular acidification rate profiles showed no increase in glycolysis. The overexpression of each ORF led to an increased proton efflux rate derived from mitochondria and a decrease in glycolytic PER.

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