Anti-nucleolin aptamer AS1411: Applications in Kaposi's Sarcoma Associated Herpes Virus (KSHV) biology

卡波西肉瘤相关疱疹病毒（KSHV），人γ-疱疹病毒家族的成员，也被称为 人类疱疹病毒8型（HHV-8）。KSHV是一种称为卡波西氏的内皮肿瘤的病原体 肉瘤（KS）和一种称为原发性渗出性淋巴瘤的高度侵袭性淋巴增生性B细胞淋巴瘤 （PEL）。KS是HIV感染者中最常见的血管恶性肿瘤，发病率和死亡率高。 PEL是一种罕见的造血和淋巴组织肿瘤，预后不良。PEL案例 自从联合抗逆转录病毒疗法（cART）的广泛使用以来， 但KSHV仍然在发展中国家造成严重死亡。在KSHV目标定位方面， 特异性抗病毒药物，耐受性非常好，免疫功能低下患者无严重不良事件 KSHV相关恶性肿瘤患者。KSHV在其生命周期中表现出两个不同的阶段。在等待期间， 最小数量的潜伏基因，如vFLIP（ORF 71）、vCyclin（ORF 72）、潜伏相关的核转录因子（NF-κ B）、核转录因子（NF-κ B）、核转录因子（NF-κ B）、核转录因子（NF-κ B）和核转录因子（NF-κ B）。 抗原-1（拉娜-1; ORF 73）和卡泊辛表达。相反，在裂解期，KSHV表达 一系列的立即早期（IE），延迟早期（DE）和晚期基因;进行积极的复制和生产 病毒体后代。KSHV已被证明利用多种宿主生长因子、细胞因子、血管生成因子， 细胞信号传导和代谢相关蛋白，用于为其复制，存活， 和延迟。我们令人兴奋的研究发现：1）KS皮肤病变表达非常强烈的表达， 与健康皮肤组织相比，核仁素; 2）KSHV在原代内皮细胞中的新生感染诱导了高水平的核仁素表达。 核仁素和磷酸-核仁素水平，更重要的是，3）将KSHV感染的细胞与G- 形成四链体的抗核仁素适体AS 1411减少KSHV潜伏（ORF 73）并增加裂解（ORF 50） 基因表达，我们在核仁素沉默的KSHV感染的PEL细胞中获得了类似的结果。4)AS1411 处理在诱导KSHV感染的PEL细胞系中的细胞死亡方面是有效的。根据初步结果， 我们假设KSHV诱导宿主因子核仁素以支持其潜伏期、生命周期和靶向 核仁素与AS 1411联合治疗KSHV相关恶性肿瘤具有治疗潜力。为了验证这一 假设，我们制定了两个具体的目标，我们将1）确定调节机制 KSHV感染后核仁素表达及其在KSHV潜伏期中的作用，以及2）评估 使用AS 1411治疗KS和PEL的化疗潜力。我们的研究意义重大， 通过推进KSHV生物学中适配体的未探索和新型靶向治疗诊断领域， 了解它们的抗病毒和抗癌潜力也可以应用于其他病毒性恶性肿瘤。

Kaposi’s sarcoma-associated herpesvirus (KSHV), a member of the human γ-herpesvirus family is also termed as human herpesvirus type 8 (HHV-8). KSHV is an etiological agent of an endothelial tumor called Kaposi’s sarcoma (KS) and a highly aggressive lymphoproliferative B cell lymphoma called primary effusion lymphoma (PEL). KS is the most common vascular malignancy causing high morbidity and mortality in HIV-infected patients. PEL is a rare tumor of hematopoietic and lymphoid tissues and is associated with a poor prognosis. PEL cases have been dramatically reduced in the US since the widespread use of combined antiretroviral therapy (cART) but KSHV still causes significant mortality in the developing world. There is a critical need in KSHV targeting specific antiviral drugs, which are very well tolerated with no severe adverse events for immunocompromised patients with KSHV associated malignancies. KSHV exhibits two distinct phases in its life cycle. During latency, a minimal number of latency genes such as vFLIP (ORF71), vCyclin (ORF72), latency-associated nuclear antigen-1 (LANA-1; ORF73), and Kaposin are expressed. By contrast, during the lytic phase, KSHV expresses a wide array of immediate-early (IE), delayed-early (DE), and late genes; undergo active replication and produce virion progeny. KSHV has been shown to utilize multiple host growth factors, cytokines, angiogenic factors, and cell signaling and metabolism-related proteins for creating a beneficial environment for its replication, survival, and latency. Our exciting studies discovered that: 1) KS skin lesions express remarkably robust expression of nucleolin compared to healthy skin tissue; 2) KSHV de novo infection in primary endothelial cells induced a high level of nucleolin and phospho-nucleolin, and more importantly, 3) Incubation of KSHV infected cells with G- quadruplex forming anti-nucleolin aptamer AS1411 reduced KSHV latent (ORF73) and increased lytic (ORF50) gene expression and we obtained similar results in nucleolin silenced KSHV infected PEL cells. 4) AS1411 treatment was efficacious in inducing cell death in KSHV infected PEL cell lines. Based on our preliminary results, we hypothesize that KSHV induces host factor nucleolin to support its latency and life cycle and targeting nucleolin with AS1411 would have therapeutic potential in KSHV associated malignancies. To test this hypothesis, we have formulated two specific aims in which we will 1) To determine the regulatory mechanisms of nucleolin expression upon KSHV infection and its role in KSHV latency, and 2) To evaluate the chemotherapeutic potential of using AS1411 to treat KS and PEL. Our studies are significant and will have a positive impact by advancing the unexplored and novel targeted theranostic field of aptamers in KSHV biology and understanding their antiviral and anticancer potential can also be applied to other viral malignancies.