EPITRANSCRIPTOMIC REGULATION OF CYTOMEGALOVIRUS INFECTION

7.项目摘要/摘要 对mRNA的化学修饰为动态改变真核细胞中的基因表达提供了一种强有力的手段 通过表位转录的改变。特别是，N6位的腺苷甲基化(M6A)构成了 最广泛的对mRNA的内部碱基修饰。M6A对mRNA的修饰影响了许多人 生物过程包括发育、分化、重新编程、昼夜节律、细胞周期、 疾病发病机制，以及包括病毒感染在内的应激反应。值得注意的是，病毒编码的mRNA 也被M6A化学修饰，并且M6A在人类巨细胞病毒(HCMV)感染生物学中的作用是 正在浮现。作为典型的TORCH(弓形虫、其他、风疹病毒、巨细胞病毒、单纯疱疹病毒)病原体，原发巨细胞病毒 孕期感染仍然是导致出生缺陷的主要病毒原因。而人巨细胞病毒感染导致轻微 如果任何产妇发病，并在健康人中以无症状为主，就会导致生命危险 免疫受损人群中的疾病，包括实体器官或干细胞移植的接受者，是一种 发达国家新生儿先天发病率和死亡率的重要来源。 解决人巨细胞病毒先天性感染仍然是一个严重的未得到满足的医疗需求，因为目前还没有人巨细胞病毒疫苗 预防妊娠期间的原发感染，目前没有预防母婴传播的治疗方法 胎儿。我们的长期目标是了解宿主和/或病毒RNA的化学修饰是如何 M6A感染会影响人巨细胞病毒的繁殖，这是一种常见的感染，仍然是引起 先天畸形。初步结果表明，细胞内的m6A甲基转移酶亚基 METTL3/14，M6A去甲基酶ALKBH5，和M6A识别蛋白调节HCMV的复制和 未感染细胞对双链DNA(DsDNA)的反应。这在一定程度上是通过改变 干扰素b基因(IFNB1)表达。这些发现证实了m6A RNA修饰酶调节 细胞对巨细胞病毒和dsDNA感应的反应，它塑造宿主免疫并促进自身免疫 疾病。这进一步表明，m6A表位转录改变在细胞固有的先天发育中起着重要的作用。 对TORCH病原体HCMV的免疫应答。根据我们的初步结果，我们假设 HCMV的复制是由宿主M6A修饰机制差异控制的。在这里，这个假设 将在三个特定目标中进行测试，旨在：(I)确定主机M6A修改机械的情况 对巨细胞病毒感染的调控；(Ii)确定细胞内M6A修饰酶如何调控IFNB1 HCMV感染细胞中的mRNA积累；以及(Iii)确定HCMV基因的表达是如何受 差异M6A修饰。这个项目意义重大，因为它研究了表位转录如何变化 影响人巨细胞病毒的繁殖和先天免疫。了解人类巨细胞病毒感染是如何通过 表位转录RNA修饰可能为治疗干预带来新的机会，并可能 疫苗开发的新战略。

7. PROJECT SUMMARY / ABSTRACT Chemical modification of mRNA provides a powerful means to dynamically alter gene expression in eukaryotes via epitranscriptomic changes. In particular, methylation of adenosine at the N6 position (m6A) constitutes the most widespread internal base modification to mRNA. Modification of mRNA by m6A influences numerous biological processes including development, differentiation, reprogramming, circadian rhythm, cell cycle, disease pathogenesis, and stress responses including virus infection. Significantly, virus-encoded mRNAs are also chemically modified by m6A, and a role for m6A in Human Cytomegalovirus (HCMV) infection biology is emerging. As a canonical TORCH (T. gondii, other, rubella virus, HCMV, HSV) pathogen, primary HCMV infection during pregnancy remains the leading viral cause of birth defects. While HCMV infection causes mild if any maternal morbidity and is predominately asymptomatic in healthy individuals, it results in life-threatening disease among the immunocompromised, including solid-organ or stem cell transplant recipients, and is a significant source of congenital morbidity and mortality among newborn infants in the developed world. Addressing HCMV congenital infection remains a serious unmet medical need as there is no HCMV vaccine to prevent primary infection during pregnancy and no current treatment to prevent transmission from mother to fetus. Our long-term objective is to understand how the chemical modification of host and/or viral RNA by m6A impacts reproduction of HCMV, a common infection that remains the leading viral cause of congenital abnormalities. Preliminary results demonstrate that cellular m6A methyltransferase subunits METTL3/14, the m6A demethylase ALKBH5, and m6A recognition proteins regulate HCMV reproduction and responses to double strand DNA (dsDNA) in uninfected cells. This is achieved in part through changes in interferon b gene (IFNB1) expression. These findings establish that m6A RNA modification enzymes regulate cellular responses to HCMV and dsDNA sensing, which shapes host immunity and contributes to autoimmune disease. It further suggests that m6A epitranscriptomic changes play a fundamental role in cell-intrinsic innate immune responses to the TORCH pathogen HCMV. Based upon our preliminary results, we hypothesize that HCMV reproduction is differentially controlled by the host m6A modification machinery. Here, this hypothesis will be tested in three specific aims designed to: (i) identify how the host m6A modification machinery is regulated in response to HCMV infection; (ii) determine how cellular m6A modification enzymes regulate IFNB1 mRNA accumulation in HCMV-infected cells; and (iii) identify how HCMV gene expression is impacted by differential m6A modification. The project is significant because it investigates how epitranscriptomic changes impact HCMV reproduction and innate immunity. Understanding how HCMV infection is regulated by epitranscriptomic RNA modification could lead to new opportunities for therapeutic intervention and possibly new strategies for vaccine development.