Viral Chromatin Methylation as a Host Defense Against Geminiviruses

在植物中，RNA沉默是一种重要的抗病毒适应性防御。植物的几种沉默途径包括由小干扰rna （sirna）介导的转录后基因沉默（PTGS）、通过microRNAs （miRNA）调控基因表达、sirna介导的DNA和相关组蛋白甲基化导致转录基因沉默（TGS）和异染色质的建立。大多数植物病毒具有RNA基因组，并被PTGS靶向，导致病毒基因组和mrna的降解。DNA病毒（如双病毒）的转录本也受到PTGS的影响。此外，由于双病毒通过与细胞组蛋白相关的dsDNA中间体在细胞核中复制，它们的基因组受到甲基化和TGS的影响。本项目主要研究双病毒染色质甲基化。提出的研究是基于Bisaro实验室最近的几项发现，这些发现暗示了双子星病毒基因组甲基化是一种重要的宿主防御，并表明双子星病毒编码的蛋白质通过抑制甲基化来对抗这种防御。首先，利用遗传和生化方法的结合，甲基化的抗病毒性质将得到证实。其次，将研究相关双病毒蛋白AL2和L2抑制和逆转TGS的机制，并研究这些蛋白改变胞嘧啶和组蛋白甲基化状态的能力。最后，由于双病毒提供了一种独特的rna定向甲基化途径，双病毒将被用作模型系统来鉴定参与甲基化途径的新细胞基因。甲基化控制正常发育过程中的细胞基因表达，导致异染色质的形成。它还通过沉默内源性侵入性dna（如重复序列和转座子）来维持基因组的完整性。因此，研究双病毒染色质甲基化有望增加我们对病毒发病机制和控制细胞染色质组织和活动的基本过程的理解。在植物中，RNA沉默是一种重要的抗病毒防御手段，可导致病毒mRNA的特异性降解。就DNA病毒而言，一个相关的过程（rna定向甲基化）导致病毒DNA和相关组蛋白的甲基化，这些组蛋白共同构成病毒染色体。在这个项目中，rna定向甲基化作为一种防御含有dna的双病毒的作用将被确定。病毒蛋白对抗这种防御和抑制甲基化的能力也将被研究。最后，将利用双病毒作为模型系统来鉴定参与甲基化的新宿主基因。甲基化控制正常发育过程中的细胞基因表达，对组织细胞染色体的非活性区域很重要。因此，研究双病毒染色质甲基化有望增加我们对病毒发病机制和控制细胞染色质活性的基本过程的理解。更好地了解宿主抗病毒防御和病毒反防御将最终导致植物的发展，能够更好地抵御或抵抗病毒的攻击。此外，从植物系统中学到的知识也可能适用于哺乳动物病毒与宿主的相互作用。该项目的其他好处包括培养本科生、研究生和博士后（包括代表性不足的少数民族）了解现代分子生物学和病毒学的概念和方法。

In plants, RNA silencing is an important adaptive defense against viruses. The several silencing pathways in plants include post-transcriptional gene silencing (PTGS) mediated by small interfering RNAs (siRNAs), regulation of gene expression by microRNAs (miRNA), and siRNA-directed methylation of DNA and associated histones leading to transcriptional gene silencing (TGS) and the establishment of heterochromatin. Most plant viruses have RNA genomes and are targeted by PTGS, which results in degradation of viral genomes and mRNAs. The transcripts of DNA viruses (e.g. geminiviruses) are also subject to PTGS. In addition, because geminiviruses replicate in the nucleus through dsDNA intermediates that associate with cellular histones, their genomes are subject to methylation and TGS. This project is focused on geminivirus chromatin methylation. The studies proposed are based on several recent discoveries from the Bisaro lab which implicate geminivirus genome methylation as an important host defense and suggest that geminiviruses encode proteins that counter this defense by inhibiting methylation. First, using a combination of genetic and biochemical methods, the antiviral nature of methylation will be confirmed. Second, mechanisms by which the related geminivirus proteins AL2 and L2 suppress and reverse TGS will be studied, and the ability of these proteins to alter cytosine and histone methylation status examined. Finally, because geminiviruses offer a unique access to RNA-directed methylation, geminiviruses will be used as model systems to identify novel cellular genes that participate in the methylation pathway. Methylation controls cellular gene expression during normal development and leads to the establishment of heterochromatin. It also maintains genome integrity by silencing endogenous invasive DNAs such as repeated sequences and transposons. Thus studying geminivirus chromatin methylation promises to increase our understanding of viral pathogenesis and a fundamental process that governs the organization and activity of cellular chromatin.Non-technical SummaryIn plants, RNA silencing is an important antiviral defense that results in specific degradation of viral mRNA. In the case of DNA viruses, a related process (RNA-directed methylation) leads to the methylation of viral DNA and associated histone proteins that together constitute the viral chromosome. In this project, the role of RNA-directed methylation as a defense against DNA-containing geminiviruses will be determined. The ability of viral proteins to counter this defense and inhibit methylation will also be examined. Finally, geminiviruses will be used as model systems to identify new host genes involved in methylation. Methylation controls cellular gene expression during normal development and is important for organizing inactive regions of cellular chromosomes. Thus studying geminivirus chromatin methylation promises to increase our understanding of viral pathogenesis and a fundamental process that governs the activity of cellular chromatin. A better understanding of host antiviral defenses and viral counterdefense will eventually lead to the development of plants that are better able to withstand or resist virus attack. Further, what is learned in plant systems may also prove applicable to mammalian virus-host interactions. Additional benefits of this project include the training of undergraduate students, graduate students, and postdoctoral fellows (including under-represented minorities) in the concepts and methods of modern molecular biology and virology.