The role of TREX1 for innate sensing human endogenous retroviruses

HIV-1逆转录(RT)产物主要通过环状GMP-AMP合成酶cGAS-STING途径在大多数细胞类型中被检测到。宿主细胞胞质外切酶TREX1被描述为在哺乳动物细胞胞浆中代谢外源逆转录病毒RT产物，在TREX1基因敲除小鼠中的研究表明，TREX1在消除ERV RT产物和DNA损伤引起的细胞质DNA产物方面具有重要作用。根据文献中报道的这些结果，该项目的一个中心假设是，TREX1通过降低逆转录病毒和细胞质DNA的数量来避免先天性免疫感知，在控制先天性(自身)免疫反应中发挥核心作用。在第一个资助期的工作中，建立了具有或不具有TREX1表达的先天免疫感知功能细胞系统，用于分析TREX1在感染外源性逆转录病毒或诱导内源性逆转录病毒表达或DNA损伤时的功能。同时，建立了TREX1体外酶活性及其在细胞中的免疫学影响的定量检测方法。最后，对TREX1在人类细胞中的表达进行了分析，发现了一种出人意料的复杂的细胞类型和各种TREX1亚型的刺激依赖性表达模式，这些亚型的底物特异性不同，并揭示了TREX1可以受到蛋白水解性切割。第二个资助期的工作将以这些工具和研究结果为基础，全面了解人类细胞中不同的TREX1亚型和加工产物在逆转录病毒感染和DNA损伤方面的作用。由于TREX1是一种定位于内质网和细胞核的跨膜蛋白，另一个核心问题是外切酶如何以及在哪里获得逆转录病毒RT产物。为了解决这些问题，我们将进行(I)TREX1亚型和切割产物的全面功能鉴定和(Ii)剖析细胞质DNA代谢的免疫学后果。这些分析将系统地比较外源性感染HIV-1与诱导ERV表达或DNA损伤。总之，我们的目标是在TREX1和逆转录病毒复制之间的相互作用中提供新的时空和机械洞察力。

HIV-1 reverse transcription (RT) products are predominantly sensed in most cell types by the cyclic GMP-AMP synthase cGAS-STING pathway. The host cell cytoplasmic exonuclease TREX1 was described to metabolize exogenous retroviral RT products in the cytoplasm of mammalian cells and studies in trex1 knock-out mice suggested that TREX1 has an important role in the elimination of ERV RT products as well as of cytoplasmic DNA products resulting from DNA damage. Based on these results reported in the literature, a central hypothesis of this project was that TREX1 plays a central role in controlling innate (auto) immune responses by lowering the amounts of retroviral and cellular cytoplasmic DNA to avoid innate immune sensing. Work in the first funding period established innate immune sensing competent and –incompetent cell systems with or without TREX1 expression for the analysis of TREX1 function in response to infection with exogenous retrovirus or upon induction of expression of endogenous retroviruses or DNA damage. In parallel, assays were established for the quantification of enzymatic activities of TREX1 in vitro and their immunological consequences in cells. Finally, the analysis of TREX1 expression in human cells yielded an unexpectedly complex pattern of cell-type and stimulation-dependent expression of various TREX1 isoforms that differ in their substrate specificity and revealed that TREX1 can be subject to proteolytic cleavage. Work in the second funding period will build on these tools and findings to achieve a comprehensive understanding of the role of diverse TREX1 isoforms and processing products in human cells in the context of retrovirus infection and DNA damage. Since TREX1 is a transmembrane protein localized at the endoplasmic reticulum as well as in the nucleus, another central question is how and where the exonuclease has access to retroviral RT products. To address these questions, we will conduct (i) a comprehensive functional characterization of TREX1 isoforms and cleavage products and (ii) dissect the immunological consequences of the metabolism of cytoplasmic DNA. These analyses will systematically compare exogenous infections with HIV-1 with the induction of ERV expression or DNA damage. Together, we aim at providing novel tempo-spatial and mechanistic insight in the interplay between TREX1 and retroviral replication.