NYMC Student Theses and Dissertations

Date of Award

5-2-2025

Document Type

Doctoral Dissertation - Restricted (NYMC/Touro only) Access

Degree Name

Doctor of Philosophy

Department

Microbiology and Immunology

First Advisor

Christopher Whitehurst, Ph.D.

Abstract

Epstein-Barr Virus (EBV) is an oncogenic gamma-herpesvirus that infects over 95% of the population worldwide. It serves as the etiological agent of infectious mononucleosis and drives several lethal immunoblastic lymphomas in immunocompromised individuals, including Burkitt lymphoma, Hodgkin lymphoma, and nasopharyngeal carcinoma. EBV encodes a deubiquitinating (DUB) enzyme, BamHI P fragment left open reading frame-1 (BPLF1) which plays a critical role in EBV-induced infectious virus production, B-cell immortalization, and tumorigenesis. To explore the molecular interactions of BPLF1, we conducted an affinity-based mass spectrometry screen, which showed that BPLF1 directly interacts with the mechanistic target of rapamycin (mTOR). mTOR, a major regulator of cell growth and proliferation, is present in the cell in two complexes: mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). Dysregulation of the mTOR pathway is a well-established contributor to carcinogenesis. Notably, a shift from predominant mTORC1 activity to an increased mTORC2 activity has been implicated in the tumorigenic process.

To investigate BPLF1’s deubiquitinating activity on its mTOR complex interacting partners, we overexpressed constructs of a truncated wild-type (WT) BPLF1 and an enzymatically inactive (C61S) BPLF1 mutant in cultured cells. Immunoprecipitations followed by western blot analysis were conducted to assess the ubiquitination status of mTOR. Previous studies have demonstrated that the lysosomal localization of mTORC1, which is crucial for its activation, is dependent on K63-linked ubiquitination of mTOR. To further evaluate BPLF1’s impact on mTOR localization, we utilized lysosomal fractionation to isolate and analyze mTOR’s subcellular distribution. The phosphorylation states of downstream effectors of mTORC1 were also assessed as markers of mTORC1 activation. Additionally, the effects of the mTORC1 inhibitor rapamycin and the mTORC2 inhibitor JR-AB2-011 on infectious virus production were evaluated by flow cytometry using Green Fluorescent Protein (GFP)-tagged virus and quantitative PCR.

We found that BPLF1 exhibits direct DUB activity on mTOR, specifically cleaving both K48- and K63-linked ubiquitin chains. Additionally, WT BPLF1 diminished the lysosomal localization of mTORC1 and reduced the phosphorylation of mTORC1 downstream effectors, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase beta-1 (S6K1). An immunoprecipitation analysis of mTOR revealed a decrease in mTORC1 complex formation in the presence of WT BPLF1 without any apparent effect on the formation of mTORC2. Notably, the inhibition of mTORC1 resulted in an increase in infectious virus production, whereas inhibition of mTORC2 led to a decrease in viral production. These findings highlight the differential roles of mTORC1 and mTORC2 in modulating EBV infectious virus production.

We also observed that BPLF1’s deubiquitinating activity disrupts mTORC1 formation and activation. Specifically, BPLF1 reduces mTORC1’s localization to the lysosome and inhibits its activation, thereby preventing the downstream phosphorylation of effectors essential for regulating cell growth and proliferation. It appears that EBV exploits BPLF1 to disrupt mTORC1 activity, a mechanism that supports the production of infectious virus. Additionally, mTORC2 activity also plays a crucial role in viral production, suggesting that other viral factors may cooperate with BPLF1 to optimize the mTOR pathway in favor of the virus. Given BPLF1’s critical involvement in infectious virus production, its potential contribution to EBV’s oncogenic properties through modulation of the mTOR signaling pathway warrants further study.

Keywords

Epstein-Barr Virus; BPLF1; deubiquitinating enzyme; mTOR; mTORC1; DUB; EBV

Disciplines

Cancer Biology | Immunology of Infectious Disease | Laboratory and Basic Science Research | Medical Microbiology | Virus Diseases

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