Mechanism and Therapeutic Implication of Host Cell Telomerase Modulation by Human Cytomegalovirus

项目摘要 人巨细胞病毒（HCMV）是一种疱疹病毒，其血清阳性率和疾病负担仍然很高 国际吧虽然疾病在免疫功能正常的个体中通常是轻微的，但对于免疫功能正常的个体来说，疾病通常是严重的。 然而，免疫功能低下的个体和先天性感染仍然是出生缺陷的主要原因。有 目前没有针对HCMV的疫苗或永久有效的抗病毒疗法。宿主端粒酶， 维持端粒以保护DNA的完整性，是细胞存活的关键， 包括癌症在内的几种疾病。端粒酶在生命周期中起着重要的作用 和几种疱疹病毒的潜在致癌活性，最近已被证明在 对活动性HCMV感染的反应。此外，端粒酶抑制已经与显著的 降低HCMV实验室和临床毒株的病毒感染性。然而，端粒酶的重要性 在HCMV病毒生命周期中的作用，以及这种关系的潜在治疗意义，仍然未被探索。 我们假设宿主端粒酶对HCMV病毒的生命周期是重要的。我们将首先建立端粒酶 HCMV感染后活性上调，端粒酶抑制后HCMV复制减少， 多个菌株和细胞系使用几种方法来抑制端粒酶。端粒酶检测 在药物端粒酶抑制剂BIBR 1532和MST-312的存在下， 在实验室和临床菌株中抑制端粒酶后降低病毒感染性。后续 使用诱导型遗传抑制剂，特别是针对hTERT的siRNA和诱导型shRNA构建体的测定， 将测试病毒复制的减少是否是端粒酶抑制的结果，而不是脱靶效应。到 为了明确HCMV上调端粒酶活性的作用机制，我们将评估端粒酶 关键病毒基因和相关基因的异位表达和shRNA介导的敲低后的活性 产品将在感染和端粒酶抑制的感染条件下进行结合试验，以评估 通过ChiP和QTIP测定与DNA结合，并通过IP和质谱法与其他蛋白质结合。芯片 在感染和端粒酶抑制的感染条件下的测定也将在宿主蛋白上进行 以前显示影响端粒酶活性。为了明确端粒酶在病毒生命周期中的作用，我们 将对端粒酶进行不同时间的抑制，并评估病毒基因类别， 受到端粒酶缺失的限制。总的来说，这些实验将建立潜在的临床 端粒酶在HCMV感染中的相关性，并确定HCMV 上调端粒酶活性和端粒酶在病毒生命周期中的功能。进一步认识 这些关系可以提供对其他疱疹病毒的深入了解，为临床HCMV管理提供信息，并且 导致新的抗病毒疗法。

Project Summary Human Cytomegalovirus (HCMV) is a herpesvirus whose seroprevalence and disease burden remain high worldwide. While disease is often mild in immunocompetent individuals, it is often severe for immunocompromised individuals, and congenital infection remains a leading cause of birth defects. There is currently no vaccine or permanent effective anti-viral therapy against HCMV. The host enzyme telomerase, which maintains telomeres to protect the integrity of DNA, is key for cell survival and has been implicated in several diseases including cancer. Telomerase has been implicated as an important agent in the life cycles and potential oncogenic activity of several herpesviruses, and recently has been shown to be hyperactive in response to active HCMV infection. Furthermore, telomerase inhibition has been associated with a significant reduction of viral infectivity in laboratory and clinical strains of HCMV. However, the significance of telomerase in the HCMV viral life cycle, and the potential therapeutic implications of this relationship, remain unexplored. We hypothesize host telomerase is important for the HCMV viral life cycle. We will first establish telomerase activity upregulation upon HCMV infection, and reduced HCMV replication following telomerase inhibition, in multiple strains and cell lines using several approaches to telomerase repression. Assays of telomerase activity in the presence of pharmaceutical telomerase inhibitors BIBR1532 and MST-312 will establish a reduction of viral infectivity following telomerase inhibition in both laboratory and clinical strains. Subsequent assays using inducible genetic inhibitors, specifically siRNA and inducible shRNA constructs against hTERT, will test whether reduction of viral replication is a result of telomerase inhibition and not off-target effects. To define the mechanism of action by which HCMV upregulates telomerase activity, we will assess telomerase activity following ectopic expression and shRNA-mediated knockdown of key viral genes, and implicated gene products will be subjected to binding assays in infected and telomerase-inhibited infected conditions to assess association with DNA via ChiP and QTIP assays, and with other proteins via IP and mass spectrometry. ChiP assays in infected and telomerase-inhibited infected conditions will also be performed on host proteins previously shown to influence telomerase activity. To define the function of telomerase in the viral life cycle, we will conduct differentially-timed inhibition of telomerase and assess the viral gene class(es) the activity of which is constrained by the absence of telomerase. Overall, these experiments will establish the potential clinical relevance of telomerase in HCMV infection and define both the mechanism of action by which HCMV upregulates telomerase activity and the function of telomerase in the viral life cycle. Increased understanding of these relationships may provide insight into other herpesviruses, inform clinical HCMV management, and lead to new anti-viral therapies.