The role of UL128 and RL13 in cell-associated spread of human cytomegalovirus

人巨细胞病毒（HCMV）是一种疱疹病毒，其在全世界40- 100%的人群中流行，并且在免疫防御降低的情况下导致显著的发病率。原则上，HCMV可以通过将感染性病毒体释放到细胞外空间而无细胞传播，也可以通过病毒后代的直接细胞间传播而细胞相关传播。显然，在宿主内的传播主要是由于细胞相关模式。在血液中发现几乎完全在白细胞部分中的粘附性，并且新鲜分离的HCMV几乎总是以严格的细胞相关方式传播。本项目的总体目标是在研究HCMV细胞相关传播的分子因素方面取得实质性进展，因为我们相信，这种不明确的传播模式是在临床相关情况下更好地控制病毒的关键。病毒基因RL 13和UL 128被鉴定为保持HCMV细胞相关的因子，但它们的作用模式在很大程度上是未知的，因为它们在从临床标本中分离HCMV后迅速被破坏。RL 13和UL 128的缺失通过促进无细胞扩散提供了强选择性优势。这就是为什么完整的病毒基因组还没有成功地克隆在细菌载体（BAC），这是一个先决条件，基因工程的详细分子研究。我们的目标是克服这些限制，并获得实质性的见解，详细的贡献，病毒和细胞因子参与细胞相关的传播的HCMV。1。 从技术上讲，我们将最近的HCMV分离株克隆为BAC，这将首次允许对此类分离株进行靶向遗传修饰。我们已经开发了一种新的方法，用于从分离感染的成纤维细胞培养物中释放无细胞感染性，从而打开了这种选择。我们将产生一个BAC克隆的HCMV变异体的集合，代表各种多态性糖蛋白的所有基因型，我们将构建适合于抗病毒筛选方法或病毒颗粒的实时成像的报告病毒。这些收集的病毒将与科学界分享2。 关于细胞相关传播的分子机制，我们将重点关注病毒基因UL 128和RL 13的作用。我们将研究它们的表达和病毒的细胞结合之间的剂量反应关系，并分析这两种蛋白质在感染细胞中是否相互作用以及它们如何降低无细胞感染性。我们将尝试确定细胞类型或培养条件，使细胞释放-总的来说，该项目的预期进展旨在为未来开发特异性靶向HCMV细胞相关传播的抗病毒剂提供基础。

Human cytomegalovirus (HCMV) is a herpesvirus that is prevalent in 40-100 % of the population worldwide and causes significant morbidity under conditions of reduced immune defenses. In principle, HCMV can spread both cell-free by releasing infectious virions into the extracellular space and cell-associated by direct cell-to-cell transmission of viral progeny. Apparently, dissemination within that host is greatly due to the cell-associated mode. Infectivity in the bloodstream is found almost exclusively in the leukocyte fraction, and freshly isolated HCMV almost always spreads in a strictly cell-associated manner.The general objective of this project is to make substantial progress in the investigation of the molecular factors underlying cell-associated spread of HCMV, as we are convinced that this ill-defined transmission mode is a key for better control of the virus in clinically relevant situations. The viral genes RL13 and UL128 were identified as factors that keep HCMV cell-associated, but their mode of action is largely unknown, since they are disrupted quickly after isolation of HCMV from clinical specimens. Loss of RL13 and UL128 provides a strong selective advantage by promoting cell-free spread. This why intact viral genomes have not successfully been cloned in bacterial vectors (BACs), which is a prerequisite for genetic engineering for detailed molecular studies.We aim to overcome such limitations and to gain substantive insights regarding the detailed contribution of viral and cellular factors involved in cell-associated spread of HCMV.1. Technically, we address cloning of recent HCMV isolates as BACs, which would allow for the first time the targeted genetic modification of such isolates. We have developed a novel method for releasing cell-free infectivity from isolate-infected fibroblast cultures that opens this option. We will generate a collection of BAC-cloned HCMV variants representing all genotypes of the various polymorphic glycoproteins, and we will construct reporter viruses suitable for antiviral screening approaches or live imaging of virus particles. These collections of viruses will be shared with the scientific community2. Concerning the molecular mechanisms underlying cell-associated spread, we will focus on the role of the viral genes UL128 and RL13. We will investigate the dose-response relationship between their expression and cell-association of the virus and analyze whether the two proteins interact in infected cells and how they reduce cell-free infectivity.In an attempt to develop culture models reflecting virus shedding compartments, we will try to identify cell types or culture conditions that allow the release of cell-free virus from isolates e.g. by downmodulation of UL128 or RL13.Taken together, the intended advances in this project are meant to provide a basis for the future development of antiviral agents that specifically target the cell-associated spread of HCMV.