Fig. 4. Recognition of HIV RT products by enzymatically active TREX1 suppresses IFN induction.

(a) Schematic of synthetic nucleic acids used in (b) as they are generated during reverse transcription. (b) DNA-containing RT products trigger a stronger IFN response in Trex1^−/− compared to WT cells. IFN-β expression was measured 6 h post transfection of indicated nucleic acids. (c) TREX1 metabolizes reverse-transcribed HIV DNA and transfected ssDNA and dsDNA in both mouse and human fibroblasts. WT and KO MEFs or WT and TREX1 mutant (D18N) human fibroblasts were either infected with HIV or transfected with ssDNA and dsDNA gag sequence oligonucleotides (100 bp), and cytosolic DNA was measured by qPCR using gag primers 10 hpi or 3 h post transfection. *, P <0.01, Student's t-test. Error bars indicate S.D. of three independent experiments. (d) HIV DNA, but not RNA, is pulled down with FLAG antibody from cytosolic lysates of HeLa-CD4 cells expressing FLAG-TREX1 that were infected with HIV_IIIB for 10 h. Lysates were immunoprecipitated (IP) with FLAG or IgG antibodies. DNA and RNA extracted from the IP were quantified by qPCR or qRT-PCR, respectively. *, P <0.05, Student's t-test. Error bars indicate S.D. of triplicate replicates of two independent experiments. (e,f) TREX1 enzymatic activity is required to support HIV infection. (e) WT and Trex1^−/− (KO) MEFs expressing GFP alone or wild type or enzymatically inactive mutant (D18N) GFP-tagged TREX1 were infected with HIV-Luc, and luciferase activity was measured 48 hpi. TREX1 expression was comparable for each construct by immunoblot. (f) GFP-TREX1, but not the D18N mutant, eliminates excess HIV DNA in Trex1^−/− MEFs. *, P <0.01, Student's t-test. Error bars indicate S.D. of triplicate replicates of two independent experiments.