Functional Genomics of Insect Baculoviruses

昆虫杆状病毒的功能基因组学

• 批准号: RGPIN-2014-05472
• 负责人: Krell, Peter
• 金额: $ 5.17万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=567196
• 关键词: Functional; Genomics; Insect; Baculoviruses

Insect baculoviruses infect many insect pests of agriculture and forests and nuisance pests like mosquitoes and blackflies giving them a major role as biological control agents. Baculoviruses are also used as efficient expression vectors (e.g. Cervarix vaccine). Its large dsDNA genome encodes some 150 genes important in the virus replication cycle. The functions of some of these baculovirus genes are known, some for initial infection, production of virion proteins, egress from the cell, cell and host regulation and virus transmission. Much less is known about other genes. Among these is the me53 gene, a major early gene encoding a 53 kDa protein. Me53 is transcribed shortly after infection and later. We showed it was an important viral protein because in its absence virus replication is severely impacted. Since it has protein domains consistent with transcription factors, we felt it must work in the nucleus to regulate transcription. We found that not only is ME53 located in the nucleus (as expected of a transcription factor) but surprisingly also in foci at the cell periphery where it colocalizes with a viral envelope protein GP64. From this we surmised that ME53 might help virions bud through the membrane at these ME53/GP64 foci. But we are still stuck with the question, what is the role(s) of ME53? We posited two hypotheses, one on its role in transcriptional regulation and one on virus egress. One of our objectives is to follow differences in the virus replication cycle of ME53 deleted viruses (deltaME53) compared to wild type virus. We will start our search by electron microscopy for an ultrastructural comparison (e.g. looking at virogenic stroma and assembly/transit of virions in the nucleus and at the cell membrane). We will also compare virus (and cellular) transcriptional profiles. To differentiate the nuclear-specific function from the cell membrane one of ME53, a second objective is to map domains of ME53 needed for nuclear translocation (NTD) and those for membrane translocation (MTD). By mutating those translocation domains we hope to assess the roles of ME53 at different intracellular sites. For example mutating NTD will prevent nuclear translocation and any differences observed (e.g. lack of regulation of certain viral, or even host, genes for the NTD mutant), must be due to its nuclear function, and vice versa for the MTD mutants. While ME53 associates with GP64 and the capsid protein VP39, we suspect it also interacts with other viral (or host) proteins, for example to help ME53 translocate to the nucleus or plasma membrane. For this we will be looking for ME53 protein partners (the ME53 interactome) using a genetic, yeast two hybrid screen, and biochemical “pull down” experiments to remove ME53 from a cell preparation but with associated proteins still attached. Binding proteins from those “pull downs” will be identified by very precise spectroscophotometric measures. This would be followed by further mutagenesis to try to identify domains in ME53 important for binding to the different partners (and mutate the partners themselves). ME53 mutations unable to bind to certain partners will then be assessed for their affect on virus replication. Any differences noticed between the binding domain mutant ME53 and normal ME53 must relate to the importance of that particular protein. From this work we should better understand the function of ME53 in the baculovirus life cycle. While such discoveries advance fundamental science we cannot predict if there will be any practical or commercial value to this knowledge. As these viruses are used as biological control agents, perhaps this knowledge might be used to improve their efficacy and better protect against Canadian pest insects, or improve vaccine production.

昆虫杆状病毒感染农业和森林的许多昆虫害虫和讨厌的害虫如蚊子和黑蝇，使它们作为生物防治剂发挥主要作用。杆状病毒也被用作有效的表达载体（例如Cervarix疫苗）。它的大dsDNA基因组编码约150个在病毒复制周期中重要的基因。这些杆状病毒基因中的一些的功能是已知的，一些用于初始感染、病毒体蛋白的产生、从细胞中排出、细胞和宿主调节以及病毒传播。 对其他基因的了解要少得多。其中包括me 53基因，一个主要的早期基因，编码53 kDa的蛋白质。Me 53在感染后不久和之后转录。我们发现它是一种重要的病毒蛋白，因为在它缺失的情况下，病毒复制受到严重影响。由于它具有与转录因子一致的蛋白质结构域，我们认为它必须在细胞核中起作用以调节转录。我们发现，ME 53不仅位于细胞核中（如预期的转录因子），而且令人惊讶地位于细胞周边的病灶中，在那里它与病毒包膜蛋白GP 64共定位。由此我们推测，ME 53可能有助于病毒体在这些ME 53/GP 64焦点处穿过膜出芽。但我们仍然被这个问题所困扰，ME 53的作用是什么？我们提出了两个假设，一个是关于它在转录调控中的作用，另一个是关于病毒的逃逸。我们的目标之一是跟踪ME 53缺失病毒（deltaME 53）与野生型病毒相比在病毒复制周期中的差异。我们将通过电子显微镜进行超微结构比较（例如，观察病毒发生基质和病毒体在细胞核和细胞膜中的组装/转运）。我们还将比较病毒（和细胞）转录谱。为了区分ME 53的核特异性功能与细胞膜特异性功能，第二个目标是绘制核易位（NTD）和膜易位（MTD）所需的ME 53结构域。通过突变这些易位结构域，我们希望评估ME 53在不同细胞内位点的作用。例如，突变NTD将防止核易位，并且观察到的任何差异（例如，对于NTD突变体缺乏某些病毒或甚至宿主基因的调节）必须是由于其核功能，并且对于MTD突变体反之亦然。虽然ME 53与GP 64和衣壳蛋白VP 39相关，但我们怀疑它也与其他病毒（或宿主）蛋白相互作用，例如帮助ME 53易位到细胞核或质膜。为此，我们将使用遗传、酵母双杂交筛选和生物化学“下拉”实验来寻找ME 53蛋白伴侣（ME 53相互作用组），以从细胞制备物中去除ME 53，但仍附着有相关蛋白。通过非常精确的分光光度测量，将从这些“唐斯”中识别出结合蛋白。这之后将进行进一步的诱变，以试图鉴定ME 53中对结合不同配偶体重要的结构域（并使配偶体本身突变）。然后将评估无法与某些伴侣结合的ME 53突变对病毒复制的影响。在结合结构域突变体ME 53和正常ME 53之间注意到的任何差异必须与该特定蛋白的重要性有关。从这项工作中，我们应该更好地了解在杆状病毒的生活周期中的ME 53的功能。虽然这些发现推进了基础科学，但我们无法预测这些知识是否会有任何实用或商业价值。由于这些病毒被用作生物控制剂，也许这些知识可以用来提高它们的效力，更好地防止加拿大害虫，或提高疫苗生产。