Die Infektion endothelialer Zellen mit dem humanen Herpesvirus 8 (KSHV/HHV8): Mechanismen der Persistenz, Replikation und abnormaler Differenzierung

卡波西氏肉瘤相关疱疹病毒（KSHV/HHV-8）是卡波西氏肉瘤（KS）发病机制中的一个重要因素，KS是一种源自内皮细胞的肿瘤。KSHV感染内皮细胞，并可能持续存在，诱导其非典型分化为KS梭形细胞，这是该肿瘤的标志。潜伏持续和裂解复制过程中表达的病毒蛋白可能有助于KS梭形细胞的发育。然而，在表征单个病毒蛋白的功能时，所有先前的研究都是在分离的情况下研究它们的，而不是在病毒基因组的其余部分的背景下，而且大多是在过表达之后。其他疱疹病毒的经验表明，这可能导致误导性的解释。迄今为止，缺乏有效的体外培养系统阻碍了KSHV突变体的实验。鉴于最近KSHV细胞培养系统的改进，我们提出在“细菌人工染色体”（BAC）中构建重组KSHV分子克隆，并将其用于突变转座子文库的生成以及单个靶向基因的突变体。该提案的目的是鉴定在内皮细胞中失去复制和/或持续存在能力的KSHV突变体，并在原代内皮细胞中诱导梭形细胞形成。这将允许鉴定对内皮细胞中kshv介导的发病机制至关重要的病毒基因。该结果将有助于我们了解疱疹病毒如何在内皮细胞中感染和持续存在。

Kaposi's Sarcoma associated herpesvirus (KSHV/HHV-8), is an essential factor in the pathogenesis of Kaposi's sarcoma (KS), a tumour derived from endothelial cells. KSHV infects, and probably persists in, endothelial cells and induces their atypical differentiation into KS spindle cells, the hallmark of this tumour. Viral proteins expressed during latent persistence and lytic replication may contribute to the development KS spindle cells. However, in characterising the function of individual viral proteins, all previous studies have studied them in isolation, outside the context of the remainder of the virus genome, and mostly after overexpression. The experience with other herpesviruses suggests that this may lead to misleading interpretations. The lack of an efficient in vitro culture system has so far precluded experiments with KSHV mutants. Given the recent improvements in cell culture systems for KSHV we propose to construct a recombinant molecular clone of KSHV in a 'bacterial artificial chromosome' (BAC) and to use it for the generation of a mutant transposon library as well as mutants of individually targeted genes. The aim of the proposal is to identify KSHV mutants that have lost the ability to replicate and/or persist in endothelial cells and to induce spindle cell formation in primary endothelial cells. This will allow the identification of viral genes that are essential for KSHV-mediated pathogenesis in endothelial cells. The results will contribute to our understanding of how herpesviruses in general infect and persist in endothelial cells.