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.