EBV reactivation causes widespread host de novo promoter transcription and transcriptional interference

摘要 爱泼斯坦-巴尔病毒(EBV)是一种DNA肿瘤病毒，它会导致淋巴瘤在 艾滋病毒/艾滋病人口。虽然世界上90%以上的人口携带EBV，但这种病毒通常存在于 “潜伏”状态，对寄主影响不大。响应于某些刺激或局部微环境提示， 然而，EBV进入裂解病毒复制程序，导致病毒在宿主之间和宿主内传播。 除了已知的病毒潜伏蛋白在EBV相关癌症中的作用外，还有一些公认的 裂解复制与EBV相关癌症之间的联系；艾滋病毒联合感染中EBV裂解复制的升高 感染(+或-ART)可能导致HIV感染者对EBV的易感性增加 伴发淋巴瘤。 由于遗传含量极低，病毒的复制和复制无情地依赖于宿主细胞资源 它们进化出调节宿主细胞代谢过程的机制，以促进有效的病毒生产。一 疱疹病毒复制中最保守的病毒-宿主相互作用之一是病毒编码的“宿主关闭” 因子降解宿主细胞的mRNAs，释放翻译资源用于专门生产病毒结构蛋白 蛋白质。最近，格劳辛格实验室发现，小鼠γ疱疹病毒MHV68也能抑制POL II 加载细胞基因。这说明了分层主机关闭机制的利用 克服病毒结构蛋白生产的限速步骤。 使用EBV再激活模型促进评估纯再激活细胞的转录组变化 我们对EBV与宿主细胞转录组的相互作用有了新的和意想不到的见解。 在观察到的许多转录组变化中，我们发现EBV导致转录起始于 超过25,000个以前没有启动子活性证据的细胞基因组位点。这些“新人” 启动子异常简单，只由一个或两个短病毒转录因子结合组成 基序(S)，由于它们的短暂性质，在整个细胞基因组中随机出现的频率很高。我们的 初步数据表明，其中一个基序是由病毒预起始复合体vPIC识别的，它 结合一个类似TATA的基序(TATT(TA)AA)，并在某些情况下被证明可以激活病毒晚期基因 没有其他转录因子的明显支持。我们假设EBV利用vPIC的能力 通过这些简单的基序单枪匹马地激活转录，以产生数千个新宿主 启动子和实质上改变细胞转录组和染色质结构。 我们的初步研究还表明，最高活性的从头启动子丰富了本地化 在1.5kb的典型细胞启动子范围内，转录方向为各自的典型 推动者。我们假设，虽然EBV利用晚期病毒启动子基序的原始性质来靶向 在整个细胞基因组中有数千个位置，它利用常规启动子附近的染色质上下文来 驱动高水平的从头启动子转录，导致宿主的靶向转录干扰(TI) 推动者。这代表了一种新的病毒-宿主相互作用的机械概念，这种相互作用会导致中断 细胞基因表达和病毒结构蛋白的专属生产。 在这个提案中，我们将1)研究从头启动子规范和局部 染色质上下文驱动现有正则启动子附近的高水平转录，以及2)研究 从头转录和转录干扰在下调细胞mrna表达中的影响。

Summary The Epstein Barr virus (EBV) is a DNA tumor virus that causes an elevated incidence of lymphoma in the HIV/AIDS population. While more than 90% of the world's population carries EBV, the virus typically exists in a “latent” state with little impact on the host. In response to certain stimuli or local microenvironmental cues, however, EBV enters the lytic viral replication program, leading to viral spread both between and within hosts. In addition to the known role of viral latency proteins in EBV associated cancers, there are well-established links between lytic replication and EBV associated cancers; and elevated EBV lytic replication in HIV co- infection (+ or – ART) likely contributes to the increased susceptibility of HIV infected individuals to EBV associated lymphomas. With minimal genetic content, viruses are inexorably dependent on host cell resources for their replication and they evolve mechanisms to modulate host cell metabolic processes to facilitate efficient virus production. One of the most conserved virus-host interactions in herpesvirus replication is “host shut off” where virus encoded factors degrade host cell mRNAs, freeing up translation resources for dedicated production of viral structural proteins. Recently, the Glaunsinger lab showed that the murine γ-herpesvirus, MHV68 also inhibits Pol II loading on cell genes. This illustrates the utilization of layered host shut off mechanisms that together overcome the rate-limiting step of viral structural protein production. Using EBV reactivation models that facilitate assessment of transcriptome changes in pure reactivating cell populations, we gained new and unexpected insights into EBV's interactions with the host cell transcriptome. Among a number of observed transcriptome alterations is our finding that EBV causes transcription initiation at more than 25,000 cell genomic sites that have no previous evidence of promoter activity. These “de novo” promoters are unusually simple, being composed of only one or two short viral transcription factor binding motif(s) that due to their short nature, occur randomly at a high frequency across the cell genome. Our preliminary data suggests that one of these motifs is recognized by the viral preinitiation complex, vPIC, which binds a “TATA”-like motif (TATT(TA)AA) and has been shown in some cases to activate viral late genes without apparent support from other transcription factors. We hypothesize that EBV utilizes the ability of vPIC to single-handedly activate transcription through these simple motifs to spawn thousands of host de novo promoters and substantially alter the cell transcriptome and chromatin architecture. Our preliminary studies also show that the most highly active de novo promoters have enriched localization within 1.5 kb of canonical cell promoters, with transcriptional orientations toward the respective canonical promoters. We hypothesize that while EBV utilizes the primal nature of late viral promoter motifs to target thousands of sites throughout the cell genome, it leverages chromatin context near canonical promoters to drive high-level de novo promoter transcription, leading to targeted transcriptional interference (TI) of host promoters. This represents a new mechanistic concept for virus-host interactions that contributes to disrupted cell gene expression and dedicated production of viral structural proteins. In this proposal, we will 1) investigate the underlying mechanisms of de novo promoter specification and local chromatin context driving high level transcription near existing canonical promoters, and 2) investigate the impact of de novo transcription and transcriptional interference in downregulating cell mRNA expression.