Deshpande, Rushikesh Harish
(2024)
Cytotoxic role of viral accessory protein Orf7b in lung injury and T-cell lymphopenia.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Corona virus disease 2019 (COVID-19), caused by severe acute respiratory distress syndrome corona virus 2 (SARS-CoV-2), has resulted in a global pandemic since January 2020. Pulmonary tissue damage and significant decrease in lymphocyte count are the two important clinical outcomes of SARS-CoV-2 infection. However, a comprehensive understanding of which protein(s) might be responsible for these outcomes is still lacking. SARS-CoV Orf7b, a non-accessory protein, causes apoptosis in lung and kidney cells. However, the function of SARS-CoV-2 Orf7b has not been fully explored. Orf7b in SARS-CoV-2 has several non-synonymous substitution type mutations and a truncated sequence. Thus, it is important to study its function to determine any potential alterations.
In the first part of the thesis, we screened the majority of the viral proteins for their potential cytotoxicity using overexpression model and found Orf7b to be one of the top-ranked proteins. We generated RNA seq datasets using Orf7b-transduced lung epithelial cells and analyzed them using IPA and ENRICHR, which predicted the involvement of extrinsic apoptosis and ferroptosis, with potential role of transcription regulator c-Myc in the cell death pathways. Using immunoblotting, cell death inhibitors, CRISPR/CAS9 knockout and in-vivo mouse model we confirmed the RNA seq findings.
In the second part of the thesis, we expanded our study to T-lymphocytes, and assessed the potential role of Orf7b in T-cell lymphopenia. In the in-vitro model, significant cell death was observed in Orf7b-transfected jurkat cells. We analyzed publicly available CD4+ and CD8+ T cell datasets from patients with COVID-19 and recovering patients for pathway analysis. IPA predicted the involvement of NRF2-Keap1 signaling pathway and extrinsic apoptosis signaling. Gene enrichment analysis suggested the role of c-Myc as a top transcription regulator. Immunoblotting of Orf7b-transduced cells confirmed the IPA findings Finally, we expressed Orf7b in mouse spleen using adeno-associated virus vectors (AAV) and assessed the cytotoxicity using flow cytometry and immunoblotting. Orf7b induced significant cell death in both CD4+ and CD8+ T cells via c-Myc mediated apoptosis and ferroptosis. Thus, we elucidated a novel role of accessory protein Orf7b in activating multiple cell death pathways in the context of two important hallmarks of SARS-CoV-2 infection.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
14 May 2024 |
| Date Type: |
Publication |
| Defense Date: |
6 April 2024 |
| Approval Date: |
14 May 2024 |
| Submission Date: |
24 April 2024 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
138 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Public Health > Environmental and Occupational Health |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
SARS-CoV-2; COVID-19; apoptosis; ferroptosis |
| Date Deposited: |
14 May 2024 18:56 |
| Last Modified: |
14 May 2024 18:56 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/46253 |
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