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

UL128和RL13在人巨细胞病毒细胞相关传播中的作用

• 批准号: 470266494
• 负责人: Professor Dr. Christian Sinzger
• 金额: --
• 依托单位: Institut für Virologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/470266494?language=en
• 关键词: role; UL128; RL13; cell; associated

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.

人类巨细胞病毒 (HCMV) 是一种疱疹病毒，在全球 40-100% 的人口中流行，在免疫防御降低的情况下会导致显着的发病率。原则上，HCMV 可以通过将感染性病毒颗粒释放到细胞外空间进行无细胞传播，也可以通过病毒子代的直接细胞间传播进行细胞结合传播。显然，在该宿主内的传播很大程度上归因于细胞相关模式。血流中的感染性几乎全部存在于白细胞部分中，而新鲜分离的 HCMV 几乎总是以严格的细胞相关方式传播。该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 RL13 和 UL128 被确定为保持 HCMV 细胞相关性的因子，但其作用方式很大程度上未知，因为从临床标本中分离出 HCMV 后，它们很快就会被破坏。 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尚未成功克隆到细菌载体 (BAC) 中，而细菌载体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，并获得有关 HCMV.1 细胞相关传播中病毒和细胞因子的详细贡献的实质性见解。 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有针对性的遗传修饰。我们开发了一种从分离物感染的成纤维细胞培养物中释放无细胞感染性的新方法，打开了这种选择。我们将生成代表各种多态性糖蛋白的所有基因型的 BAC 克隆 HCMV 变体的集合，并且我们将构建适合抗病毒筛选方法或病毒颗粒实时成像的报告病毒。这些病毒集合将与科学界共享2。 关于细胞相关传播的分子机制，我们将重点关注病毒基因 UL128 和 RL13 的作用。我们将研究它们的表达与病毒的细胞关联之间的剂量反应关系，并分析这两种蛋白是否在受感染的细胞中相互作用以及它们如何降低无细胞感染性。通过下调 UL128 或 RL13。总之，该项目的预期进展旨在为未来开发专门针对 HCMV 细胞相关传播的抗病毒药物奠定基础。