Convergence of KSHV Reactivation with EBV latency and cellular growth mediated by the Notch signaling pathway in co-infected cells.. Homo sapiens

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL). All PEL cell lines are infected with KSHV, and 70% are co-infected with Epstein-Barr Virus (EBV). KSHV reactivation from latency requires promoter-specific transactivation by the KSHV Rta protein through interactions with RBP-Jk (CSL), the cellular DNA binding component of the Notch signal transduction pathway. EBV transformation of primary B cells requires EBV nuclear antigen (EBNA)-2 to interact with RBP-Jk to direct the latent viral and cellular gene expression program. Although KSHV Rta and EBV EBNA-2 both require RBP-Jk for transactivation, previous studies have suggested that RBP-Jk-dependent transactivators do not function identically. We have found that the EBV latent protein LMP-1 is expressed in less than 5% of KSHV+/EBV+ PEL cells, but is induced in an Rta-dependent fashion when KSHV reactivates. KSHV Rta transactivates the EBV latency promoters in an RBP-Jk-dependent fashion and forms a ternary complex with RBP-Jk on the promoters. In B cells that are conditionally transformed by EBV alone, we show that KSHV Rta complements a short-term EBNA2 growth deficiency in an autocrine/paracrine manner. Complementaton of EBNA2-deficiency by Rta depends on RBP-Jk and LMP-1, and Rta transactivation is required for optimal growth of KSHV+/EBV+ PEL lines. Our data suggest that Rta can contribute to EBV-driven cellular growth by transactivating RBP-Jk-dependent EBV latency genes. However, our data also suggest that EBNA2 and Rta induce distinct alterations in the cellular proteomes that contribute to growth of infected cells. Overall design: EREB2-5 cells were transfected and grown in the presence or absence of β-estradiol, as described. Seven days post-transfection, protein extracts were prepared, and 200 ugs. of each were analyzed using the RayBio Human Apoptosis Antibody Array Kit (RayBiotech) as per manufacturers suggestions. The membranes were exposed to autoradiography film for different times to detect the chemiluminescent signals. Images with signals in linear range were quantitated using the program ImageJ [59]. For each membrane, signals from the negative control spots were averaged, and then subtracted from each of the other spots. A signal was considered valid if its value exceeded both its average local background, and the average of all valid negative control values. Valid signals were normalized using the positive control spots (for cellular BID protein). Fold change in signals for each spot were quantitated by dividing by the valid signals for each corresponding spot on the minus β-estradiol membrane. Average fold change, and standard deviation, were calculated for each protein.

卡波西肉瘤相关疱疹病毒（Kaposi’s sarcoma-associated herpesvirus, KSHV）是原发性渗出性淋巴瘤（primary effusion lymphoma, PEL）的致病原。所有PEL细胞系均感染KSHV，其中70%同时感染EB病毒（Epstein-Barr Virus, EBV）。KSHV从潜伏状态激活的过程，需要KSHV的Rta蛋白通过与RBP-Jκ（CSL）结合发挥启动子特异性反式激活作用——RBP-Jκ是Notch信号转导通路的细胞DNA结合组分。EB病毒对原代B细胞的转化过程，需要EB病毒核抗原（EBV nuclear antigen, EBNA）-2与RBP-Jκ结合，以调控潜伏状态下的病毒及细胞基因表达程序。尽管KSHV Rta与EBV EBNA-2均依赖RBP-Jκ实现反式激活，但既往研究提示，依赖RBP-Jκ的反式激活因子功能并不完全一致。本研究发现，EB病毒潜伏膜蛋白1（latent membrane protein 1, LMP-1）在不足5%的KSHV阳性/EBV阳性PEL细胞中表达，但当KSHV激活时，其表达会以Rta依赖的方式被诱导。KSHV Rta可通过RBP-Jκ依赖的方式反式激活EB病毒潜伏启动子，并与RBP-Jκ在启动子区域形成三元复合物。在仅被EB病毒条件性转化的B细胞中，本研究证实KSHV Rta可通过自分泌/旁分泌方式弥补EBNA2短期缺失导致的生长缺陷。Rta对EBNA2缺失的弥补作用依赖于RBP-Jκ与LMP-1，且Rta的反式激活功能对于KSHV阳性/EBV阳性PEL细胞系的最优生长不可或缺。本研究数据表明，Rta可通过反式激活依赖RBP-Jκ的EB病毒潜伏基因，参与EB病毒驱动的细胞生长过程。但同时数据也提示，EBNA2与Rta会在细胞蛋白质组中诱导不同的改变，这些改变可促进受感染细胞的生长。实验总体设计：如前文所述，将EREB2-5细胞进行转染，并在添加或不添加β-雌二醇的条件下培养。转染7天后制备蛋白质提取物，取每份200 μg蛋白样品，按照RayBio人细胞凋亡抗体芯片试剂盒（RayBio Human Apoptosis Antibody Array Kit）的制造商说明书进行分析。将膜片置于放射自显影胶片中曝光不同时长以检测化学发光信号。选取信号处于线性范围内的图像，使用ImageJ软件[59]进行定量分析。对于每张膜片，先取阴性对照斑点的信号平均值，再将其从其余所有斑点的信号值中扣除。若某斑点的信号值同时高于其局部平均背景值与所有有效阴性对照值的平均值，则判定该信号有效。使用阳性对照斑点（对应细胞BID蛋白）对有效信号进行标准化处理。通过将每个斑点的信号值除以不含β-雌二醇的膜片上对应斑点的有效信号值，计算得到各斑点的信号变化倍数。对每种蛋白质的信号变化倍数计算平均值与标准差。