The role of TREX1 for innate sensing human endogenous retroviruses

HIV-1逆转录（RT）产物在大多数细胞类型中主要通过环GMP-AMP合成酶cGAS-STING途径被感知。宿主细胞质外切酶TREX1被描述为代谢哺乳动物细胞质中外源性逆转录病毒RT产物，在TREX1敲除小鼠中的研究表明，TREX1在消除ERV RT产物以及DNA损伤引起的细胞质DNA产物中起重要作用。基于这些文献报道的结果，本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的数量来避免先天免疫感应，在控制先天（自身）免疫反应中起核心作用。第一个资助期的工作建立了具有或不具有TREX1表达的先天免疫感知能力和非能力细胞系统，用于分析TREX1在响应外源性逆转录病毒感染或诱导内源性逆转录病毒表达或DNA损伤时的功能。同时，建立了体外定量TREX1酶活性及其在细胞中的免疫学影响的测定方法。最后，对TREX1在人类细胞中的表达的分析得出了一个出乎意料的复杂的细胞类型和刺激依赖的各种TREX1亚型的表达模式，这些亚型的底物特异性不同，并揭示了TREX1可以受到蛋白水解裂解的影响。第二个资助期的工作将以这些工具和发现为基础，全面了解逆转录病毒感染和DNA损伤情况下人类细胞中各种TREX1亚型和加工产物的作用。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何处获得逆转录病毒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表征和（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，我们的目标是为TREX1和逆转录病毒复制之间的相互作用提供新的时空和机制见解。

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.