Kaposi's Sarcoma-Associated Herpesvirus-Induced Angiogenin Plays Roles in Latency via the Phospholipase Cγ Pathway: Blocking Angiogenin Inhibits Latent Gene Expression and Induces the Lytic Cycle
Kaposi's Sarcoma-Associated Herpesvirus-Induced Angiogenin Plays Roles in Latency via the Phospholipase Cγ Pathway: Blocking Angiogenin Inhibits Latent Gene Expression and Induces the Lytic Cycle
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DOI：
10.1128/jvi.01532-10
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发表时间：
2011-03-01
影响因子：
5.4
通讯作者：
Chandran, Bala
中科院分区：
文献类型：
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作者：
Sadagopan, Sathish;Veettil, Mohanan Valiya;Chandran, Bala
卡波西肉瘤相关疱疹病毒（Kaposi's sarcoma-associated herpesvirus，KSHV）在人真皮微血管内皮细胞（human dermal microvascular endothelial cells，HMVEC-d）的新生感染过程中，诱导多功能血管生成素（angiogenin，ANG）蛋白进入细胞核和核仁，刺激45 S rRNA基因转录、增殖和管形成，而用抗生素新霉素（S. Sadagopan等人，J. Virol. 83：3342-3364，2009）。ANG由KSHV潜伏蛋白拉娜-1（开放阅读框73 [ORF 73]）诱导。在这里，我们研究了血管紧张素在KSHV阳性（KSHV+）原发性渗出性淋巴瘤（PEL/BCBL）细胞中的存在和功能。在KSHV+细胞中观察到显著的ANG基因表达和分泌（BCBL-1和BC-3）和KSHV+和EB病毒阳性（KSHV+ EBV+）（JSC-1）PEL细胞和BJAB-KSHV细胞中，但不存在于EBV-KSHV-淋巴瘤细胞中（Akata、Loukes、拉莫斯和BJAB），EBV+淋巴瘤细胞（Akata-EBV和Raji）和来自EBV+淋巴母细胞样细胞系的细胞，从而表明ANG在KSHV生物学中的特异性关联。抑制ANG的核转位导致BCBL-1和TIVE-LTC（潜伏感染的内皮细胞）细胞存活和增殖减少，而EBV-和EBV+ Akata细胞不受影响。阻断ANG的核转运抑制了潜伏ORF 73基因的表达，并增加了裂解开关ORF 50基因的表达，无论是在从头感染和潜伏感染的细胞。在新霉素处理的BCBL-1细胞的上清液中检测到比12-O-十四烷酰佛波醇-13-乙酸酯（TPA）处理的细胞更大量的感染性KSHV。新霉素处理和ANG沉默抑制磷脂酶C γ（PLC-γ）和AKT磷酸化，相反，ANG诱导ORF 73表达和PLC-γ和AKT磷酸化。进一步的研究提供了新霉素阻断PLC-γ激活似乎介导了对潜在基因表达的抑制的证据，因为用常规PLC-γ抑制剂U 73122治疗也显示出类似的结果。ANG的沉默也导致BCBL-1和TIVE-LTC细胞中和从头感染期间细胞存活减少、ORF 73基因表达减少和裂解基因活化。综上所述，这些研究表明，KSHV已经进化到利用ANG的优势，通过迄今尚未探索的PLC-γ途径维持其潜伏期。
During de novo infection of human dermal microvascular endothelial cells (HMVEC-d), Kaposi's sarcoma-associated herpesvirus (KSHV) induced the multifunctional angiogenin (ANG) protein, which entered the nuclei and nucleoli of infected cells and stimulated 45S rRNA gene transcription, proliferation, and tube formation, which were inhibited by blocking ANG nuclear translocation with the antibiotic neomycin (S. Sadagopan et al., J. Virol. 83: 3342-3364, 2009). ANG was induced by KSHV latency protein LANA-1 (open reading frame 73 [ORF73]). Here we examined the presence and functions of ANG in KSHV-positive (KSHV+) primary effusion lymphoma (PEL/BCBL) cells. Significant ANG gene expression and secretion were observed in KSHV+ (BCBL-1 and BC-3) and KSHV+ and Epstein-Barr virus-positive (KSHV+ EBV+) (JSC-1) PEL cells and in BJAB-KSHV cells but not in EBV- KSHV- lymphoma cells (Akata, Loukes, Ramos, and BJAB), EBV+ lymphoma cells (Akata-EBV and Raji), and cells from an EBV+ lymphoblastoid cell line, thus suggesting a specific association of ANG in KSHV biology. Inhibition of nuclear translocation of ANG resulted in reduced BCBL-1 and TIVE-LTC (latently infected endothelial) cell survival and proliferation, while EBV- and EBV+ Akata cells were unaffected. Blocking nuclear transport of ANG inhibited latent ORF73 gene expression and increased lytic switch ORF50 gene expression, both during de novo infection and in latently infected cells. A greater quantity of infectious KSHV was detected in the supernatants of neomycin-treated BCBL-1 cells than 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells. Neomycin treatment and ANG silencing inhibited phospholipase C gamma (PLC-gamma) and AKT phosphorylation, and in contrast, ANG induced ORF73 expression and PLC-gamma and AKT phosphorylation. Further studies provided evidence that blockage of PLC-gamma activation by neomycin appears to be mediating the inhibition of latent gene expression, since treatment with the conventional PLC-gamma inhibitor U73122 also showed similar results. Silencing of ANG also resulted in reduced cell survival, reduced ORF73 gene expression, and lytic gene activation in BCBL-1 and TIVE-LTC cells and during de novo infection. Taken together, these studies suggest that KSHV has evolved to exploit ANG for its advantage via a so-far-unexplored PLC-gamma pathway for maintaining its latency.