Roddy Kellen, Grzesik Peter, Smith Barbara, Ko Nathan, Vashee Sanjay, Desai Prashant J
Department of Oncology, Johns Hopkins University School of Medicine, Baltimore.
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore.
bioRxiv. 2024 Nov 10:2024.11.10.622843. doi: 10.1101/2024.11.10.622843.
Previously, we had developed synthetic genomics methods to assemble an infectious clone of herpes simplex virus type-1 (HSV-1). To do this, the genome was assembled from 11 separate cloned fragments in yeast using transformation associated recombination. The eleven fragments or "parts" spanned the 152 kb genome and recombination was achieved because of the overlapping homologous sequences between each fragment. To demonstrate the robustness of this genome assembly method for reverse genetics, we engineered different mutations that were located in distant loci on the genome and built a collection of HSV-1 genomes that contained single and different combination of mutations in 5 conserved HSV-1 genes. The five genes: UL7, UL11, UL16, UL21 and UL51 encode virion structural proteins and have varied functions in the infected cell. Each is dispensable for virus replication in cell culture, however, combinatorial analysis of deletions in the five genes revealed "synthetic-lethality" of some of the genetic mutations. Thus, it was discovered that any virus that carried a UL21 mutation in addition to the other gene was unable to replicate in Vero cells. Replication was restored in a complementing cell line that provided pUL21 in trans. One particular combination (UL16-UL21) was of interest because the proteins encoded by these genes are known to physically interact and are constituents of the tegument structure. Furthermore, their roles in HSV-1 infected cells are unclear. Both are dispensable for HSV-1 replication, however, in HSV-2 their mutation results in nuclear retention of assembled capsids. We thus characterized these viruses that carry the single and double mutant. What we discovered is that in cells where both pUL16 and pUL21 are absent, cytoplasmic capsids were evident but did not mature into enveloped particles. The capsid particles isolated from these cells showed significantly lower levels of incorporation of both VP16 and pUL37 when compared to the wild-type capsids. These data now show that of the tegument proteins, like the essential pUL36, pUL37 and VP16; the complex of pUL16 and pUL21 should be considered as important mediators of cytoplasmic maturation of the particle.
此前，我们开发了合成基因组学方法来组装单纯疱疹病毒1型（HSV-1）的感染性克隆。为此，利用转化相关重组技术在酵母中从11个单独的克隆片段组装基因组。这11个片段或“部分”跨越了152 kb的基因组，由于每个片段之间存在重叠的同源序列，从而实现了重组。为了证明这种基因组组装方法在反向遗传学中的稳健性，我们设计了位于基因组遥远位点的不同突变，并构建了一系列HSV-1基因组，这些基因组在5个保守的HSV-1基因中包含单个和不同组合的突变。这5个基因：UL7、UL11、UL16、UL21和UL51编码病毒体结构蛋白，在受感染细胞中具有多种功能。每个基因对于病毒在细胞培养中的复制都是可有可无的，然而，对这5个基因缺失的组合分析揭示了一些基因突变的“合成致死性”。因此，发现任何除了其他基因还携带UL21突变的病毒都无法在Vero细胞中复制。在一个反式提供pUL21的互补细胞系中恢复了复制。一种特殊的组合（UL16-UL21）很有意思，因为这些基因编码的蛋白质已知会发生物理相互作用，并且是被膜结构的组成部分。此外，它们在HSV-1感染细胞中的作用尚不清楚。两者对于HSV-1复制都是可有可无的，然而，在HSV-2中它们的突变导致组装好的衣壳滞留在细胞核中。因此，我们对携带单突变和双突变的这些病毒进行了表征。我们发现，在同时缺失pUL16和pUL21的细胞中，细胞质衣壳很明显，但没有成熟为包膜颗粒。与野生型衣壳相比，从这些细胞中分离出的衣壳颗粒显示VP16和pUL37的掺入水平显著降低。这些数据现在表明，在被膜蛋白中，与必需的pUL36、pUL37和VP16一样；pUL16和pUL21的复合物应被视为颗粒细胞质成熟的重要介质。
