Epstein – Barr Virus Evasion of Intrinsic and Innate Immune Pathways During the Viral Lytic Cycle

Abstract

Epstein-Barr virus (EBV) persistently infects most people worldwide and causes infectious mononucleosis. It is associated with 200,000 cancers per year and a major multiple sclerosis trigger. EBV colonizes the B-cell compartment and periodically reactivates, inducing expression of 80 viral proteins. Yet much remains unknown about how EBV remodels host cells, dismantles key antiviral responses and contributes to disease development during the lytic cycle. Increase understanding on these pathways could lead to the development of novel therapeutic strategies against EBV-associated disease. In Chapter 2, we assembled a proteomic map of EBV-host and EBV-EBV interactions in B-cells undergoing EBV replication, uncovering conserved herpesvirus versus EBV-specific host cell targets. Based on this viral protein interaction network resource, we revealed that the EBV-encoded G-protein coupled receptor BILF1 serves as an inhibitor for NLRP3 inflammasome and interferon responses during the viral lytic cycle. Specifically, we demonstrated that BILF1 associated with MAVS and the UFM1 E3 ligase UFL1. Whereas UFMylation of 14-3-3 proteins drives RIG-I/MAVS signaling, BILF1-directed MAVS UFMylation instead triggered MAVS packaging into mitochondrial-derived vesicles and lysosomal proteolysis. In the absence of BILF1, EBV replication and MAVS activated the NLRP3 inflammasome and interferon responses, which strongly reduced viral replication and caused cell death. This work provided a viral protein interaction network resource, revealed a UFM1-dependent pathway for selective degradation of mitochondrial cargo and highlighted BILF1 as a novel therapeutic target. In Chapter 3, we uncovered the molecular mechanism employed by EBV to evade intrinsic antiviral responses during viral DNA replication within the nuclear membrane-less replication compartments (RC). Proteomic analysis identified that upon B-cell infection or lytic reactivation, EBV depletes the chromosome maintenance cohesin SMC5/6, which has major roles in chromosome maintenance and DNA damage repair. The major tegument protein BNRF1 targeted SMC5/6 complexes by a ubiquitin proteasome pathway dependent on calpain proteolysis and cullin-7. In the absence of BNRF1, SMC5/6 associated with R-loop structures, including at the viral lytic origin of replication, and interfered with RC formation and encapsidation. CRISPR analysis highlighted RC restriction roles of SMC5/6 components involved in DNA entrapment and SUMOylation. This work highlighted SMC5/6 as a key intrinsic immune sensor and restriction factor for a human herpesvirus RC and have implications for the pathogenesis of EBV-associated cancers. In Chapter 4, we revealed the mode of action of EBV in mediating the degradation of the B-cell receptor (BCR). Proteomics analysis suggested that EBV depletes the cell surface BCR upon EBV lytic reactivation. Notably, the EBV early lytic protein BALF1/0 recognizes the C terminus of the immunoglobulin heavy chain and drives caveolin-dependent BCR endocytosis. The endocytosed BCR subsequently underwent proteolysis through the ER-associated degradation pathway. This work unraveled key biological processes redirected by EBV to dismantle major cell surface receptors. Taken together, this work has shed light on how EBV suppresses intrinsic and innate immune pathways, and contributes to disease development during the viral lytic replication which was otherwise not well understood. The identification of several novel therapeutic targets might encourage the development of new therapeutic strategies against EBV-associated diseases. Furthermore, the presentation of the viral host protein interaction map could serve to support future hypothesis-driven investigations on the functions of EBV-specific and herpesviral-conserved protein or protein complexes.