Gammaherpesviral tegument proteins - crucial effectors of viral infection

所有的伽玛疱疹病毒都编码与细胞型PFAS(FGARAT)同源的被膜蛋白，FGARAT是一种在所有生命形式中保守的嘌呤核苷酸合成途径酶。然而，这些病毒已经进化出这些酶的同源物，朝着干扰ND10介导的固有免疫的不同效应器功能方向发展。由于病毒和真核生物缺乏高分辨率的结构信息，我们将对病毒FGARAT(KSHV ORF75、RRV ORF75和HVS ORF3)与其细胞同行人PFAS的相互作用进行基于结构的分析。到目前为止，用于结构分析的病毒ORF75(KSHV，RRV)和细胞PFAS(人)的蛋白表达正在进行中。KSHV ORF75蛋白是复制所必需的，因此是开发新型抗病毒抑制剂的靶点。分子和生化分析将包括基于MS的技术和CRISPR/Cas9介导的基因敲除以及实验室建立的合成激活介体(SAM)技术。病毒FGARATs对细胞核和细胞质蛋白质组的更广泛影响将由SILAC在有条件地表达病毒效应物的细胞中进行定量分析。基于MS的交互伙伴识别已经显示Smchd1、细胞PFAS和USP15是重要的目标。值得注意的是，细胞肿瘤抑制基因Smchd1正被KSHV ORF75重新定位到ND10。Smchd1是X染色体紧凑和失活的关键介质，它参与DNA损伤反应，表明病毒干扰Smchd1与癌症之间存在联系。USP15是已知的转化生长因子β信号和肿瘤发生的启动子。根据目前的结果，我们建议病毒FGARAT解决几条细胞防线，项目计划将解决以下假设：-RhadinoVirus vFGARAT被膜蛋白针对不同的ND10相关染色质重塑因子，以获得有利于病毒基因在从头感染和重新激活期间表达的染色质状态。-KSHV vFGARAT通过与在人类癌症和DNA修复中起作用的细胞靶点的相互作用，促进病毒癌症的发展。揭示新发现的病毒效应器与宿主细胞蛋白相互作用的机制将扩大对病毒和细胞复制和中央生物合成途径以及病毒相关癌症的易感性的了解。

All Gammaherpesviruses encode tegument proteins with homology to cellular PFAS (FGARAT), a purine nucleotide synthesis pathway enzyme conserved in all life forms. The viruses, however, have evolved these enzyme homologs toward diverse effector functions that interfere with ND10 mediated intrinsic immunity. Since high-resolution structural information is lacking from both viruses and eukaryotes, we will perform a structure-based analysis of viral FGARAT interactions (KSHV ORF75, RRV ORF75, and HVS ORF3) with their cellular counterpart, the human PFAS. Hitherto, protein expression of viral ORF75 (KSHV, RRV) and cellular PFAS (human) for structural analysis is in progress. The KSHV ORF75 protein is essential for replication and thus a target for the development of a new class of antiviral inhibitors. The molecular and biochemical analysis will include MS-based technologies and CRISPR/Cas9 mediated knockout as well as synthetic activation mediator (SAM) technologies established in the laboratory. The broader effects of viral FGARATs on the nuclear and cytoplasmic proteome will be quantitatively analyzed by SILAC in cells conditionally expressing the viral effectors. MS-based identification of interaction partners has already revealed SMCHD1, cellular PFAS, and USP15 as important targets. Notably, the cellular tumor suppressor SMCHD1 is being relocated by KSHV ORF75 to ND10. SMCHD1 is a crucial mediator of X-chromosome compaction and inactivation, and it is involved in DNA-Damage response, suggesting a link between viral interference with SMCHD1 and cancer. USP15 is a known promoter of TGFbeta signaling and oncogenesis. From the current results, we propose that viral FGARATs tackle several lines of cellular defense, and the project plan will address the following hypotheses:- Rhadinoviral vFGARAT tegument proteins target distinct ND10 associated chromatin remodeling factors to achieve a chromatin status favorable for viral gene expression during de-novo infection and during reactivation.- KSHV vFGARAT contributes to the development of viral cancers through interactions with cellular targets that have roles in human cancer and DNA repair.Uncovering the mechanisms of newly identified viral effector interactions with host cell proteins will extend knowledge on viral and cellular replication and central biosynthesis pathways, and on predisposition to virus-associated cancer.