Impact of ORF3a-Q57H variant of SARS-CoV-2 on apoptotic signaling cascades

Introduction: Direct infection and viral protein expression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the respiratory system, and other organs including the heart, contribute to multi-organ dysfunction during COVID-19. We previously showed that the protein encoded by the open reading frame 3a (ORF3a) gene of SARS-CoV-2, which is critical for viral replication, can be localized in the mitochondria and potentially increase oxidative stress and apoptotic signaling. Importantly, a clinical report suggests that one of the ORF3a variants, ORF3a-Q57H, is associated with increased transmission, but lower patient mortality. Computational structural modeling predicts that the Q57H variant destabilizes the ORF3a protein structure. Hypothesis: The SARS-CoV-2-ORF3a-Q57H variant produces less protein expression in host cells compared to the original Wuhan-type (WT) virus due to its unstable structure, thus exhibits less oxidative stress and apoptosis, which may contribute to decreased mortality. Aim: To investigate the impact of ORF3a-Q57H on apoptotic signaling cascades. Methods: Wuhan-type (WT) ORF3a, mutant ORF3a-Q57H, or their control vector were transiently introduced in HEK293T cells and H9c2 cardiac myoblasts, which were used for biochemical assays and live cell imaging by confocal microscopy. Results: Contrary to computational predictions, we found that ORF3a-Q57H exhibited relatively higher protein expression in our cell lines compared to WT ORF3a. Caspase-8, which is mainly regulated by extrinsic pathways, was significantly activated by WT, but not by Q57H ORF3a, assessed by live cell imaging using a fluorescent marker, caspase-8 inhibitor IETD-FMK conjugated to sulfo-rhodamine. Caspase-9, which is mainly regulated by intrinsic and mitochondrial pathways, was activated in both WT- and Q57H-transfected cells, measured by a fluorescent marker, caspase-9 inhibitor LEHD-FMK conjugated to sulfo-rhodamine. Q57H showed equal ability for mitochondrial trafficking as the WT, assessed by the subcellular localization of GFP-tagged WT- and Q57H constructs. Lastly, caspase-3 activity, downstream of both intrinsic and extrinsic apoptotic pathways, was significantly increased in WT compared to Q57H ORF3a, quantified by the number of positively-stained nuclei with a fluorogenic DNA dye coupled to the caspase-3/7 recognition sequence. Conclusion: The SARS-CoV-2-ORF3a-Q57H variant exhibits higher protein expression compared to WT, but induces less apoptosis in host cells due to the lack of extrinsic apoptotic pathway activation. The relatively mild phenotype of the Q57H variant may result from alterations to ORF3a functions by this mutation, rather than its protein expression levels in host cells. IEM Annual Conference Pilot Project Grant (to I.P.), OACA COVID19 Response Grants (to J.O.-U. and B.S.J.), and IEM COVID19 Response Grant (to J.O.-U.) from University of Minnesota This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.