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

人巨细胞病毒(HCMV)是一种在全球40-100%的人群中流行的疱疹病毒，在免疫防御能力降低的情况下会导致显著的发病率。原理上，巨细胞病毒既可以通过将感染性病毒粒子释放到细胞外空间而进行无细胞传播，也可以通过病毒后代的直接细胞间传播而与细胞相关。显然，在宿主内的传播在很大程度上是由于细胞相关模式。血液中的传染性几乎只存在于白细胞中，新分离的HCMV几乎总是以严格的细胞相关方式传播。这个项目的总体目标是在研究细胞相关传播的分子因素方面取得实质性进展，因为我们相信这种定义不清的传播方式是在临床相关情况下更好地控制病毒的关键。病毒基因RL13和UL128被鉴定为保持与HCMV细胞相关的因子，但它们的作用模式很大程度上尚不清楚，因为它们在从临床标本中分离后很快被破坏。RL13和UL128的缺失通过促进无细胞扩散提供了强大的选择优势。这就是为什么完整的病毒基因组没有成功地克隆到细菌载体(BAC)中，这是进行详细分子研究的基因工程的先决条件。我们的目标是克服这些限制，并获得关于病毒和细胞因素在细胞相关传播中的详细贡献的实质性见解。从技术上讲，我们致力于克隆最近的HCMV分离物作为BAC，这将首次允许对此类分离物进行有针对性的基因改造。我们已经开发了一种从分离感染的成纤维细胞培养中释放无细胞传染性的新方法，从而开启了这一选择。我们将产生一组BAC克隆的HCMV变异体，代表各种多态糖蛋白的所有基因类型，并构建适合于抗病毒筛选方法或病毒颗粒活体成像的报告病毒。这些病毒收集将与科学界共享2。关于细胞相关传播的分子机制，我们将重点介绍病毒基因UL128和RL13的作用。我们将研究它们的表达与病毒的细胞结合之间的剂量-反应关系，并分析这两种蛋白是否在感染细胞中相互作用以及它们如何降低无细胞感染力。为了试图建立反映病毒脱落间隔的培养模型，我们将尝试确定允许从分离株释放无细胞病毒的细胞类型或培养条件，例如通过下调UL128或RL13。综上所述，本项目的预期进展旨在为未来专门针对HCMV细胞相关传播的抗病毒药物的开发提供基础。

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.