Activity and cellular distribution of ORF3a mutants of SARS-CoV-2 variants of concern.

Infection with SARS-CoV-2 continues to be a threat to human health. Despite successful immunization campaigns, effective treatment of COVID-19 remains an essential need to help patients and prevent the spread of new virus strains. Viroporins are intracellular ion channels that are essential for virus replication and release, thus presenting promising pharmaceutical targets. Mutations found in variants of concern (VOC) are expected to increase the virulence of the new virus strains. Recognizing the effects of these mutations at the molecular level is essential for the development of improved therapies. Here, we characterized the putative viroporin ORF3a found in VOCs of SARS-CoV-2, using expression constructs containing a myc-tag for identification, and an optional membrane-directing signal peptide. Additionally, constructs containing N-terminal fluorescence protein tags were prepared. Expression and cell surface transport in HEK-293 cells were studied using Western blot and dot blot assays, and the cellular distribution of fluorescent-marked ORF3a was studied using subcellular organelle markers and high-resolution fluorescence microscopy. Viroporin activity of all ORF3a constructs was assessed using cell viability and metabolic assays, as well as patch-clamp recordings of recombinant ORF3a. All ORF3a mutants were expressed well in the recombinant system, and the presence of a signal peptide increased expression on the cellular surface. Intracellular distribution was similar for all variants. The VOC mutants ORF3a-S171L and ORF3a-Q57H showed reduced cytotoxic activity and sensitivity to the viroporin inhibitor rimantadine, respectively, suggesting these positions to be relevant for ORF3a function and a starting point for the search of novel antiviral drugs.