Kaposi's sarcoma-associated herpesvirus（KSHV）可导致原发性浆液性淋巴瘤（PEL）。PEL细胞系需要表达细胞FLICE抑制蛋白（cFLIP）以维持生存，尽管KSHV编码这种蛋白的病毒同源物（vFLIP）。细胞和病毒FLIP蛋白有几个功能，包括最重要的是抑制原始性凋亡半胱氨酸蛋白酶8和调节NF-κB信号传导。为了研究cFLIP在PEL细胞中的重要作用及其与vFLIP的潜在冗余性，我们先用已知能以不同方式影响FLIP靶向途径的人类或病毒FLIP蛋白进行抢救实验。cFLIP的长短同功異构体和软疣病毒MC159L均是强的caspase 8抑制剂，有效地挽救了PEL细胞內源性cFLIP活性丧失的情况。KSHV vFLIP无法完全挽救内源性cFLIP丧失，因此在功能上是不同的。接下来，我们采用基因组广泛的CRISPR / Cas9合成救援筛选，以识别可弥补cFLIP敲除的功能丧失的失活干扰。来自这些筛选和我们的验证实验的结果暗示了经典的cFLIP靶向caspase 8和TRAIL受体1（TRAIL-R1或TNFRSF10A）在促进PEL细胞中的常规死亡信号中的作用。然而，这个过程不依赖TRAIL受体2或TRAIL，后者在PEL细胞培养物中无法检测到。cFLIP的要求也可以通过失活ER / Golgi驻留的软骨素硫酸酯蛋白聚糖合成和UFMylation途径，Jagunal同源物1（JAGN1）或CXCR4来克服。UFMylation和JAGN1，但不是软骨素硫酸聚糖合成或CXCR4对TRAIL-R1表达做出贡献。总的来说，我们的工作表明，在ER / Golgi相关的一组复杂过程下，cFLIP在PEL细胞中是必需的，以抑制配体无关的TRAIL-R1细胞死亡信号传导，这些过程以前没有涉及到cFLIP或TRAIL-R1功能。©2023。作者（在ADMC Associazione Differenziamento e Morte Cellulare的独家许可下）。

Kaposi's 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 and modulation of NF-κB signaling. To investigate the essential role of cFLIP and its potential redundancy with vFLIP in PEL cells, we 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 MC159L, which are all strong caspase 8 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, we employed genome-wide CRISPR/Cas9 synthetic rescue screens to identify loss of function perturbations that can compensate for cFLIP knockout. Results from these screens and our validation experiments implicate the canonical cFLIP target caspase 8 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 CXCR4, contribute to TRAIL-R1 expression. In sum, our 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.© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.