Caspase-8 plays an important role in the extrinsic apoptosis pathway and is an important initiator caspase in the death receptor signaling.

Mechanisms and Key Concepts to Understand Role of ORF3a in Apoptosis: Caspase-8

Caspase-9 is an important initiator caspase in a different apoptosis pathway comparted to caspase-8: the mitochondrion-dependent apoptosis.

Mechanisms and Key Concepts to Understand Role of ORF3a in Apoptosis: Caspase-9

Cytochrome C is responsible for the activation of caspase-9 which occurs when cytochrome C that is released into the cytosol binds with Apaf-1 which cleaves and activates caspase-9. Cytochrome C, Apaf-1, and caspase-9 are together referred to as an “apoptosome”.

Mechanisms and Key Concepts to Understand Role of ORF3a in Apoptosis: Cytochrome C

The role of Bid is seen when the death receptor signaling pathway and mitochondrion-dependent apoptosis pathway overlap which begins with the activation/cleavage of Bid to t-Bid (truncated Bid) by caspase 9 and this leads to the release of Cytochrome C into the cytosol (which leads to caspase-9 activation).

Mechanisms and Key Concepts to Understand Role of ORF3a in Apoptosis: Bid/t-Bid

Bcl-2 plays a role in the intrinsic apoptosis pathway by affecting mitochondrial membrane integrity and thereby either triggers or inhibits apoptosis. 

Mechanisms and Key Concepts to Understand Role of ORF3a in Apoptosis: Bcl-2

The presence of specific molecules that play a role in triggering apoptosis was analyzed based on the expression of ORF3a in transfected HEK293T cells. 

Method of Determining Role of ORF3a of SARS-CoV2 in apoptosis

While many molecules and pathways play a role in apoptosis, in regard to ORF3a’s role in apoptosis, five key molecules need to be explored: Caspase-8, Caspase-9, Cytochrome C, Bid/t-Bid, and Bcl-2.

Ohio State University, Columbus

The ORF3a accessory protein has the ability to trigger apoptosis in SARS-CoV, prompting investigations into its role in SARS-CoV-2. The pro-apoptotic effect of the SARS-CoV-2 ORF3a protein was confirmed in Vero E6, HepG2, and HEK293T cells using staining methods and by tracking key molecules involved in apoptosis pathways.

Role of SARS-CoV-2 ORF3a in Inducing Apoptosis

Chen, M., & Wang, J. (2002). Initiator caspases in apoptosis signaling pathways. Apoptosis, 7(4), 313-319.

Initiator caspases in apoptosis signaling pathways

Mechanisms and Key Concepts to Understand Role of ORF3a in Apoptosis

The effect of the ORF3a protein on apoptosis was investigated by using an apoptosis staining method that involved double staining a culture of cells comprising of Vero E6, HepG2, and HEK293T cells with PI (propidium iodide) and FITC (annexin V-fluorescein 5-isothiocyanate (FITC) where increased staining indicates greater pro-apoptotic activity.

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