Kaposi’ sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). PEL cell lines require expression of the cellular FLICE inhibitory protein (cFLIP) for survival, although KSHV encodes a viral homolog of this protein (vFLIP). Cellular and viral FLIP proteins have several functions, including, most importantly, the inhibition of pro-apoptotic caspase 8 (CASP8) and modulation of nuclear factor kappa B (NF-kB) signaling.Here I show that knockout (KO) of cFLIP is lethal in PEL cells, while vFLIP expression varies significantly across different PEL cell lines and that vFLIP knockdown (KD) is largely only lethal in the small subset of PEL cell lines where vFLIP levels are detectable. To investigate the essential role of cFLIP and its potential redundancy with vFLIP in PEL cells, I first performed rescue experiments with human or viral FLIP proteins known to affect FLIP target pathways differently. The long and short isoforms of cFLIP and molluscum contagiosum virus (MCV) MC159L, which are all strong CASP8 inhibitors, efficiently rescued the loss of endogenous cFLIP activity in PEL cells. KSHV vFLIP was unable to fully rescue the loss of endogenous cFLIP and is therefore functionally distinct. Next, I employed genome-wide CRISPR/Cas9 synthetic rescue screens to identify loss of function perturbations that can compensate for cFLIP KO. Results from these screens and my validation experiments implicate the canonical cFLIP target CASP8 and TRAIL receptor 1 (TRAIL-R1 or TNFRSF10A) in promoting constitutive death signaling in PEL cells. However, this process was independent of TRAIL receptor 2 or TRAIL, the latter of which is not detectable in PEL cell cultures. The requirement for cFLIP is also overcome by inactivation of the ER/Golgi resident chondroitin sulfate proteoglycan synthesis and UFMylation pathways, Jagunal homolog 1 (JAGN1) or CXCR4. UFMylation and JAGN1, but not chondroitin sulfate proteoglycan synthesis or chemokine receptor 4 (CXCR4), contribute to TRAIL-R1 expression. In sum, my work shows that cFLIP is required in PEL cells to inhibit ligand-independent TRAIL-R1 cell death signaling downstream of a complex set of ER/Golgi-associated processes that have not previously been implicated in cFLIP or TRAIL-R1 function.

Creator

• Kuehnle, Neil Joseph

DOI

• https://doi.org/10.21985/n2-bwfs-gh71

Subject

• Microbiology
• Biology

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:16705
• etdadmin_upload_1010776

Keyword

• cFLIP
• DR4
• TRAIL-R1
• cell death
• HHV-8
• primary effusion lymphoma

Date created

• 2023-01-01

Resource type

• Dissertation

Rights statement

• In Copyright