Synthetic herpesvirus genomes with an expanded genetic code

合成生物学家重新设计生物系统，以加速基础和应用研究。基于我实验室疱疹病毒研究的基础和达尔豪西iGEM合成生物学团队的领导，我们将使用合成生物学技术创建一个新的人类疱疹病毒8（HHV 8）诱变平台。目前用于HHV 8基因组诱变的方法是低效的，并且由于紧凑的病毒基因组结构而复杂化，其中许多基因紧密相关或重叠，并且从两条DNA链转录。在与J.克雷格文特尔研究所和约翰霍普金斯大学的合作中，我们正在利用酵母中的有效同源重组组装一个新的合成HHV 8基因组。这种方法将使疱疹病毒基因组在多个位点的有效，同时重新编码，以及用合成DNA取代基因组的大片段。这种在酵母中组装和突变病毒基因组的新平台将加速重要基因座的定位和基因功能的阐明。在病毒感染的背景下，只有少数HHV 8蛋白质被彻底表征。为了克服这一点，我们将扩大HHV 8的遗传密码，使有效的位点特异性掺入非典型氨基酸（ncAA）与有用的特性到HHV 8蛋白。我们的第一个目标将是在酵母中重新编码HHV 8基因组，用替代的TAA或TGA终止密码子取代所有29个映射的琥珀（TAG）终止密码子。这将释放TAG密码子用于将ncAA掺入病毒蛋白中。我们将确认携带该重新编码基因组的HHV 8病毒显示正常的基因表达和复制动力学。使用这个修改后的基因组模板，我们将创建一个TAG密码子直接插入每个注释的翻译起始位点下游的基因组集合。因此，在缺乏ncAA的正常细胞中，每种重组病毒将基本上是单基因敲除;添加与正交氨酰-tRNA合成酶/tRNA对匹配的同源ncAA将允许ncAA掺入靶蛋白中。该集合将使我们能够绘制必需基因座，并创建这些基因座的新的含ncAA的蛋白质产物，这将有助于蛋白质的结构和功能表征。具体而言，该系统将使我们能够将ncAA与内在荧光结合，这将使我们能够监测病毒蛋白质和较大结构（如衣壳）的亚细胞定位，从而促进病毒组装和外出的研究。 利用合成生物学的原理，我们的NSERC研究计划将创建以前不可用的资源，以广泛共享病毒基因组操作和病毒蛋白质功能的研究。

Synthetic biologists re-design biological systems to accelerate fundamental and applied research. Building upon a foundation in herpesvirus research in my laboratory and leadership of the Dalhousie iGEM synthetic biology team, we will use synthetic biology techniques to create a new platform for mutagenesis of human herpesvirus-8 (HHV8). Current approaches for HHV8 genome mutagenesis are inefficient, and complicated by compact viral genome architecture, with many genes closely-associated or overlapping, and transcribed from both DNA strands. In collaboration with the J. Craig Venter Institute and Johns Hopkins University we are assembling a new synthetic HHV8 genome by capitalizing on efficient homologous recombination in yeast. This approach will enable efficient, simultaneous re-coding of herpesvirus genomes at multiple loci, as well as substitution of large segments of the genome with synthetic DNA. This new platform for assembling and mutating virus genomes in yeast will accelerate mapping of essential loci and elucidation of gene function. Only a handful of HHV8 proteins have been thoroughly characterized in the context of viral infection. To overcome this, we will expand the HHV8 genetic code to enable efficient site-specific incorporation of non-canonical amino acids (ncAAs) with useful properties into HHV8 proteins. Our first objective will be to re-code the HHV8 genome in yeast, substituting all 29 mapped amber (TAG) stop codons with alternative TAA or TGA stop codons. This will liberate TAG codons for incorporation of ncAAs into viral proteins. We will confirm that HHV8 viruses bearing this re-coded genome display normal gene expression and replication kinetics. Using this revised genome template, we will create a collection of genomes with TAG codons inserted directly downstream of each annotated translation start site. Thus, in normal cells lacking ncAAs, each recombinant virus will essentially be a single-gene knockout; addition of a cognate ncAA matched to the orthogonal aminoacyl-tRNA synthetase/tRNA pair will allow ncAA incorporation into the targeted protein. This collection will allow us to map essential loci and create new ncAA-containing protein products of these loci that will aid structural and functional protein characterization. Specifically, this system will allow us to incorporate ncAAs with intrinsic fluorescence that will allow us to monitor subcellular localization of viral proteins and larger structures like capsids, facilitating studies of viral assembly and egress. Using the principles of synthetic biology, our NSERC research program will create previously unavailable resources to be widely shared for viral genome manipulation and investigation of viral protein function.