Vaccinia virus DNA binding proteins and their role in virion morphogenesis

痘病毒仍然是一个令人着迷的生物学难题，天花被根除35年后，天花不再是一个医学问题。原型痘病毒，牛痘病毒（VAC），在~200 kbp双链体DNA基因组内编码>200个基因，并且病毒体合成需要许多精心安排的生物化学步骤。在这个过程中，病毒必须复制其基因组并将其包装成成熟的病毒颗粒，同时试图避免触发或积极抑制先天免疫系统，否则会阻止感染。 VAC编码许多DNA结合蛋白，其中许多是催化转录和DNA复制所需的酶。有趣的是，文献和生物信息学确定了至少七个病毒基因，编码DNA结合蛋白，其中许多是必需的，其中一些功能尚不清楚。我们将使用我们目前的NSERC奖开发的RNA干扰方法，结合先进的显微镜，病毒反向遗传学和其他技术，进一步探索这些特征不佳的蛋白质的功能。我们预测它们支持病毒复制、基因组包装和/或dsDNA触发的宿主应答的病毒减毒。 作为第一步，我们已经建立了一组siRNA，其通过逆转录酶PCR判断选择性抑制VAC基因表达。噬菌斑试验已用于测量对病毒生长的影响。迄今为止，我们已经建立了靶向I3 L、I6 L或E8 R的siRNA，其明显降低了人类细胞中的基因表达和病毒复制。针对E5 R和J1 R的siRNA也观察到了类似但不那么强烈的渗透作用，我们正在筛选其他siRNA以改进方法。 使用超分辨率荧光显微镜，我们还观察到一种新的病毒DNA结构，可能代表了早期的包装中间体。电子显微镜显示成熟的病毒颗粒包裹着由DNA包裹蛋白质核心组成的管状结构。我们观察到的管状病毒DNA结构，在病毒粒子组装和包装，是第一次发现这样的结构以外的成熟颗粒。在设计了一种方法来可视化这些假定的包装中间体，我们将确定这些结构的组成和测试VAC DNA结合蛋白在其组装或加工中发挥作用。* 最后，我们对以下事实感兴趣：在24小时的复制周期中，感染细胞产生的病毒DNA几乎与细胞核中发现的病毒DNA一样多，尽管存在越来越多的细胞DNA传感器（例如cGAS，DAI，Pol-III，STING和TLR 9）。已知VAC阻断某些病原体相关分子模式（例如dsRNA和5 '-三磷酸加帽RNA）的检测，并且还抑制否则将诱导炎症、凋亡和/或干扰素的下游信号传导。我们假设一个或多个VAC编码的DNA结合蛋白也可能作为细胞质DNA传感器的抑制剂发挥尚未确定的作用。特别令人感兴趣的是细胞的DNA损伤传感系统，因为病毒复制产生DNA中间体，这些中间体应该触发损伤反应。这可以将细胞周期阻滞在G2-M，这对病毒复制的支持很差。事实上，我们已经观察到VAC感染不触发DNA损伤反应，尽管存在片段化的dsDNA，如通过TUNEL测定所指示的。* 这些初步的数据提供了一个研究计划的基础，旨在更好地定义这些VAC DNA结合蛋白的生物学。 这些研究还将提供高级病毒学，分子生物学和最先进的显微成像方面的高质量早期职业培训。

Poxviruses remain a fascinating biological puzzle, 35 years after variola was eradicated and smallpox ceased to be a medical problem. The prototypic poxvirus, vaccinia virus (VAC), encodes >200 genes within a ~200 kbp duplex DNA genome, and virion synthesis requires many carefully orchestrated biochemical steps. During this process the virus must replicate its genome and package it into mature virus particles, whilst trying to avoid triggering, or actively inhibiting, the innate immune system that would otherwise block infection.* VAC encodes numerous DNA binding proteins, of which many are enzymes required to catalyze transcription and DNA replication. Interestingly, the literature and bioinformatics identify at least seven more virus genes, encoding DNA binding proteins, many of which are essential and where the function(s) of some are still unclear. We will use the RNA interference methods developed with our current NSERC award, in conjunction with advanced microscopy, virus-reverse genetics, and other technologies to further explore the functions served by these poorly characterized proteins. We predict that they support viral replication, genome packaging, and/or viral attenuation of a dsDNA-triggered host response.* As a first step we have established a panel of siRNA's that selectively inhibit VAC gene expression as judged by reverse transcriptase PCR. Plaque assays have been used to measure effects on virus growth. To date we have established siRNA's targeting I3L, I6L, or E8R that clearly decrease both gene expression and virus replication in human cells. Similar, but not so strongly penetrant effects have been seen with siRNAs against E5R, and J1R and we are screening additional siRNAs to improve the methods.* Using super resolution fluorescence microscopy, we have also observed a novel viral DNA structure that may represent an early packaging intermediate. From electron microscopy it has been suggested that mature viral particles enclose tubular structures composed of DNA wrapped around a protein core. Our observation of tubular viral DNA structures, prior to virion assembly and packaging, is the first discovery of such structures outside of the mature particle. Having devised a way to visualize these putative packaging intermediates, we will determine the composition of these structures and test which VAC DNA binding proteins play a role in their assembly or processing. * Lastly, we have been intrigued by the fact that over a 24 hr replication cycle, an infected cell makes almost as much viral DNA as is found in the nucleus and does so despite the presence of a growing list of cellular DNA sensors (e.g. cGAS, DAI, Pol-III, STING, and TLR9). VAC is known to block detection of certain pathogen-associated molecular patterns (e.g. dsRNA and 5'-triphosphate capped RNA) and also inhibits the downstream signaling that would otherwise induce inflammation, apoptosis, and/or interferons. We hypothesize that one or more of the VAC-encoded DNA-binding proteins may also serve a still unidentified role as inhibitors of cytoplasmic DNA sensors. Of particular interest is the cell's DNA damage sensing system, since virus replication produces DNA intermediates that should trigger a damage response. This can block the cell cycle at G2-M, which is poorly supportive of virus replication. In fact, we have observed that VAC infection does not trigger a DNA damage response despite the presence of fragmented dsDNA, as indicated by TUNNEL assay. * These preliminary data provide a basis for a program of studies designed to better define the biology of these VAC DNA-binding proteins. These studies will also provide high-quality early-career training in advanced virology, molecular biology, and state-of-the-art microscopic imaging.