Gammaherpesviral tegument proteins - crucial effectors of viral infection

所有γ疱疹病毒编码的被膜蛋白与细胞PFAS （FGARAT）同源，PFAS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同源物，具有不同的效应功能，干扰ND10介导的内在免疫。由于病毒和真核生物都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）与其细胞对应物人类PFAS的相互作用进行基于结构的分析。迄今为止，病毒ORF75 （KSHV， RRV）和细胞PFAS（人）的蛋白表达正在进行结构分析。KSHV ORF75蛋白对病毒复制至关重要，因此是开发一类新型抗病毒抑制剂的靶标。分子和生化分析将包括基于ms的技术和CRISPR/Cas9介导的敲除以及在实验室建立的合成激活介质（SAM）技术。病毒fgarat对细胞核和细胞质蛋白质组的广泛影响将在有条件表达病毒效应物的细胞中通过SILAC定量分析。基于质谱的相互作用伙伴鉴定已经发现SMCHD1、细胞PFAS和USP15是重要的靶点。值得注意的是，细胞肿瘤抑制因子SMCHD1被KSHV ORF75重新定位到ND10。SMCHD1是x染色体压实和失活的重要介质，它参与dna损伤反应，提示病毒干扰SMCHD1与癌症之间存在联系。USP15是已知的tgf - β信号传导和肿瘤发生的启动子。根据目前的结果，我们提出病毒fgarat解决了几条细胞防御线，项目计划将解决以下假设：- Rhadinoviral vFGARAT被膜蛋白靶向不同的ND10相关染色质重塑因子，以在去novo感染和再激活期间实现有利于病毒基因表达的染色质状态。- KSHV vFGARAT通过与在人类癌症和DNA修复中起作用的细胞靶点相互作用，促进病毒性癌症的发展。揭示新发现的病毒效应物与宿主细胞蛋白相互作用的机制将扩展对病毒和细胞复制、中心生物合成途径以及病毒相关癌症易感性的认识。

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.