Repair Exonuclease Research Articles

Synthesis and Excision Repair of Site-Specific 3'-End DNA-Histone Cross-Links Derived from Abasic Sites.

Histones catalyze the DNA strand incision at apurinic/apyrimidinic (AP) sites accompanied by formation of reversible but long-lived DNA-protein cross-links (DPCs) at 3'-DNA termini within single-strand breaks. These DPCs need to be removed because 3'-hydroxyl is required for gap-filling DNA repair synthesis but are challenging to study because of their reversible nature. Here we report a chemical approach to synthesize stable and site-specific 3'-histone-DPCs and their repair by three nucleases, human AP endonuclease 1, tyrosyl-DNA phosphodiesterase 1, and three-prime repair exonuclease 1. Our method employs oxime ligation to install an alkyne to 3'-DNA terminus, genetic incorporation of an azidohomoalanine to histone H4 at a defined position, and click reaction to conjugate DNA to H4 site-specifically. Using these model DPC substrates, we demonstrated that the DPC repair efficiency is highly affected by the local protein environment, and prior DPC proteolysis facilitates the repair.

Meloxicam inhibits STING phosphorylation and alleviates intracellular DNA-mediated autoimmune responses

BackgroundCyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is critical for cytosolic DNA-sensing and the subsequent immune responses. The inappropriate activation of this pathway leads to DNA-induced autoimmune response. Understanding the precise regulation of cGAS-STING pathway is important for developing therapeutics to treat several autoimmune diseases caused by self-DNA.ResultsWe report that Meloxicam (MXC) inhibits intracellular DNA-, but not RNA-induced immune responses. We find that MXC inhibits the phosphorylation of STING by examining in different cells with various DNA stimulations. We further find that MXC significantly dampens the expression levels of interferon-stimulated genes (ISGs) by using DNA 3’ repair exonuclease 1 (TREX1)-deficient cell, an experimental model for self-DNA-induced autoimmune disease. Importantly, we demonstrate that MXC could promote the survival in Trex1–/– mouse model for Aicardi-Goutières syndrome (AGS).ConclusionsOur study identified a non-steroidal anti-inflammatory drug, MXC, that exhibits potential effect in treating the autoimmunity caused by self-DNA.

Abstract 1636: Generation of novel potent human TREX1 inhibitors facilitated by crystallography

Abstract Background: STING is an innate immune sensor critical for the development of immunity. Tumor cells can inactivate the STING pathway to avoid immune recognition, emphasizing its importance in generating tumor-specific immunity. Broad activation of STING in advanced cancers may be required to initiate CD8+ T cell priming against unique antigenic repertoires among distinct metastases and to reverse an immune-suppressive TME. Due its ubiquitous expression, systemic delivery of untargeted STING agonists may not achieve a therapeutic index. In contrast, expression of the cytosolic DNAse 3’ repair exonuclease (TREX1) is upregulated in tumor cells in response to genomic instability, inflammatory stimuli, and DNA replication, providing an opportunity for selective activation of the STING pathway. In addition to modulating cGAS/STING signaling, interaction with DNA replication enzymes that generate immunogenic DNA waste highlight a facet of TREX1 biology that may inform clinical development of targeted inhibitors. Methods: Using a structure-based drug design strategy, we designed and optimized small-molecule inhibitors of TREX1 with drug-like physicochemical properties that were profiled in biochemical and cell-based assays. X-ray crystallography studies, thermal shift, and biochemical assays were employed to determine mechanism of action. We evaluated the in vivo profile of select compounds. Results: Inhibitors of TREX1 with < 100 µM potency were optimized into a series with nanomolar potency against purified, recombinant murine and human TREX1 protein in biochemical assays. Inhibitors had similar IC50 values against TREX1 nuclease in an intact cell-based assay. Finally, TSA results demonstrated that compound interaction required magnesium. To our knowledge, we produced the first high-resolution co-crystal structures of inhibitor-bound human TREX1. We used this to dissect mouse- and human-specific interactions, confirming MOA predicted by earlier modeling efforts. Lead compounds demonstrated good bioavailability and achieved exposures necessary for target engagement in mouse models and resulted in tumor growth inhibition when combined with sub-therapeutic doses of doxorubicin. Conclusions: We present the identification and characterization of a potential first-in-class TREX1 inhibitor with nanomolar potency against human and mouse TREX1. Treatment with TREX1 inhibitors conferred profound anti-tumor activity when combined with DNA-damaging agents. Here we demonstrate that targeting TREX1 can specifically and locally engage the STING pathway in the tumor microenvironment, enhance tumor-specific immunity, and provide therapeutic benefit. Citation Format: Valerie Chen, Brian Francica, David Hsieh, Dave Freund, Anja Holtz, Saheli Samanta, Jamie Cope, Ryan Clark, Peppi Prasit, Anne Moon, Henry Johnson, Thomas Dubensky, Dara Burdette. Generation of novel potent human TREX1 inhibitors facilitated by crystallography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1636.

TREX1 cytosolic DNA degradation correlates with autoimmune disease and cancer immunity.

Three Prime Repair Exonuclease 1 (TREX1) is a major 3'-5' exonuclease in mammalian cells that primarily degrades DNA substrates in the cytoplasm. TREX1 deficiency results in an aberrant accumulation of self-DNA in the cytosol, predominantly activating the innate immune response via the cGAS-STING-IFN pathways. Characterized by interferon production, autoimmune diseases, such as Aicardi-Goutieres syndrome (AGS), systemic lupus erythematosus (SLE), familial chilblain lupus (FCL), and retinal vasculopathy and leukodystrophy (RVCL), are associated with TREX1 loss of function. Besides, abnormal expression of TREX1 also appears in different types of tumor cells. In this review, we described the structure and mechanisms of TREX1 in degrading self-DNA and discussed the potential sources of these aberrant nucleic acids. We also summarized the major research advances in TREX1-related diseases, including autoimmune diseases and cancer. These contents will serve as a foundation for further study on the function of TREX1 and provide advances in the diagnosis, treatment, and prognosis of related disorders.

Type-1 interferon-dependent and -independent mechanisms in cyclic GMP–AMP synthase–stimulator of interferon genes-driven auto-inflammation

The cyclic cyclic gaunosine monophosphate adenosine monophosphate (GMP-AMP) synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic dsDNA and initiates immune responses against pathogens. It is also implicated in several auto-inflammatory diseases known as monogenic interferonopathies, specifically Three prime repair exonuclease 1 (Trex1) loss-of-function (LOF), Dnase2 LOF, and stimulator of interferon genes-associated-vasculopathy-with-onset-in-infancy (SAVI). Although monogenic interferonopathies have diverse clinical presentations, they are distinguished by the elevation of type-1 interferons (T1IFNs). However, animal models have demonstrated that T1IFNs contribute to only some disease outcomes and that cGAS-STING activation also promotes T1IFN-independent pathology. For example, while T1IFNs drive the immunopathology associated with Trex1 LOF, disease in Dnase2 LOF is partially independent of T1IFNs, while disease in SAVI appears to occur entirely independent of T1IFNs. Additionally, while the cGAS-STING pathway is well characterized in hematopoietic cells, these animal models point to important roles for STING activity in nonhematopoietic cells in disease. Together, these models illustrate the complex role that cGAS-STING-driven responses play in the pathogenesis of inflammatory diseases across tissues.

NMR applications to find and progress TREX1 binders

Inhibition of the exonuclease activity of Three-prime Repair EXonuclease 1 (TREX1) has emerged as a potential novel immunotherapeutic strategy. Herein we describe our screening approach to find initial low-affinity fragment hits for TREX1 from our 19F library and the use of both ligand- and target-based NMR methods for hit confirmation and validation. In a further step to support the early hit expansion stage we describe our setup of a 19F competition assay for KI determination of analogs.

A case of retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations: Long‐term treatment with repeated courses of immunotherapy

AbstractWe present here the case of a 48‐year‐old man who first developed retinopathy, and motor deficit and aphasia occurred 8 years later. A tumor‐like lesion was seen by brain MRI. He was diagnosed with retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL‐S) because a three prime repair exonuclease 1 (TREX1) gene abnormality was identified. After intravenous methylprednisolone pulse (IVMP) and intravenous immunoglobulin (IVIg) therapy, cerebrospinal fluid (CSF) test results and clinical manifestations improved. However, a new lesion appeared 3 years later. Immunotherapy became ineffective, and repeated relapses occurred. We evaluated the results of laboratory tests and brain MRI for 6 years after the first treatment. Coagulation tests, including measurements of vWF activity, remained high, so immunotherapy was thought to have a limited effect.

Generation of induced pluripotent stem cell lines from two patients with Aicardi-Goutières syndrome type 1 due to biallelic TREX1 mutations

Mutations in TREX1, encoding three prime repair exonuclease 1, cause Aicardi-Goutières syndrome (AGS) 1, an autoinflammatory disease characterized by neurodegeneration and constitutive activation of the antiviral cytokine type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts from two AGS patients with biallelic TREX1 mutations. These cell lines offer a unique resource to investigate disease processes in a cell-type specific manner.

Involvement of retroelements in the autoimmune response in humans

Retroelements are mobile genomic components requiring an RNA intermediate which is reverse-transcribed into complementary DNA for transposition. Human genome contains a vast amount of retroelements including retrotransposons and endogenous retroviruses. These elements are categorized according to presence or absence of long terminal repeats, LTRs or non-LTRs, as well as autonomous and non-autonomous according to involvement of reverse transcriptase. The retroelements have been accumulated in mammalian genomes over all evolutionary times through vertical transmission, and many of them were inactivated through accumulation of mutations. However, the retroelements entered into genome within the last 200,000 years are mostly functional. Some of the active retroelements are associated with varying autoimmune diseases because anti-retroelement antibodies might cross-react with other proteins in the human body. For instance, autoimmunity and inflammation could be stimulated by increased expression of long interspersed element 1 (LINE-1 or L1) or decreased L1 degradation. Different regulation of L1 expression might be related to the genetic and sex-related variations or environmental factors. Activation of retroelements is also controlled by epigenetic silencing mechanisms such as histone modification. Elevated levels of L1 retroelements could trigger the production of type I interferon, a crucial innate defense mechanism in mammals against viruses, and systemic autoimmune response is induced. Loss-of-function in some deoxyribonucleases (DNases) such as three prime repair exonuclease 1 that degrades reverse-transcribed DNA is also related to autoimmune diseases. Additionally, human endogenous retroviruses also play a role in autoimmune diseases. Involvement of retroelements in autoimmune disorders is exemplified with three diseases, i.e. systemic lupus erythematosus, Aicardi–Goutières syndrome, and multiple sclerosis.

Analysis of clinical characteristics of children with Aicardi-Goutieres syndrome in China

BackgroundAicardi-Goutieres syndrome (AGS) is an inflammatory disorder belonging to the type I interferonopathy group. The clinical diagnosis of AGS is difficult, which can lead to a high mortality rate. Overall, there is a lack of large-sample research data on AGS in China. We aim to summarize the clinical characteristics of Chinese patients with AGS and provide clues for clinical diagnostic.MethodsThe genetic and clinical features of Chinese patients with AGS were collected. Real-time polymerase chain reaction was used to detect expression of interferon-stimulated genes (ISGs).ResultsA total of 23 cases were included, consisting of 7 cases of AGS1 with three prime repair exonuclease 1 mutations, 3 of AGS2 with ribonuclease H2 subunit B (RNASEH2B) mutations, 3 of ASG3 with RNASEH2C, 1 of AGS4 with RNASEH2A mutations, 2 of AGS6 with adenosine deaminase acting on RNA 1 mutations, and 7 of AGS7 with interferon induced with helicase C domain 1 mutations. Onset before the age of 3 years occurred in 82.6%. Neurologic involvement was most common (100%), including signs of intracranial calcification which mainly distributed in the bilateral basal ganglia, leukodystrophy, dystonia, epilepsy, brain atrophy and dysphagia. Intellectual disability, language disability and motor skill impairment were also observed. Skin manifestations (60.87%) were dominated by a chilblain-like rash. Features such as microcephaly (47.62%), short stature (52.38%), liver dysfunction (42.11%), thyroid dysfunction (46.15%), positive autoimmune antibodies (66.67%), and elevated erythrocyte sedimentation rate (53.85%) were also found. The phenotypes of 2 cases fulfilled the diagnostic criteria for systemic lupus erythaematosus (SLE). One death was recorded. ISGs expression were elevated.ConclusionsAGS is a systemic disease that causes sequelae and mortality. A diagnosis of AGS should be considered for patients who have an early onset of chilblain-like rash, intracranial calcification, leukodystrophy, dystonia, developmental delay, positive autoimmune antibodies, and elevated ISGs, and for those diagnosed with SLE with atypical presentation who are nonresponsive to conventional treatments. Comprehensive assessment of vital organ function and symptomatic treatment are important.

Genome Replication Is Associated With Release of Immunogenic DNA Waste

Innate DNA sensors detect foreign and endogenous DNA to induce responses to infection and cellular stress or damage. Inappropriate activation by self-DNA triggers severe autoinflammatory conditions, including Aicardi-Goutières syndrome (AGS) that can be caused by defects of the cytosolic DNase 3’repair exonuclease 1 (TREX1). TREX1 loss-of-function alleles are also associated with systemic lupus erythematosus (SLE). Chronic activation of innate antiviral immunity in TREX1-deficient cells depends on the DNA sensor cGAS, implying that accumulating TREX1 DNA substrates cause the inflammatory pathology. Retrotransposon-derived cDNAs were shown to activate cGAS in TREX1-deficient neuronal cells. We addressed other endogenous sources of cGAS ligands in cells lacking TREX1. We find that induced loss of TREX1 in primary cells induces a rapid IFN response that requires ongoing proliferation. The inflammatory phenotype of Trex1-/- mice was partially rescued by additional knock out of exonuclease 1, a multifunctional enzyme providing 5’ flap endonuclease activity for Okazaki fragment processing and postreplicative ribonucleotide excision repair. Our data imply genome replication as a source of DNA waste with pathogenic potential that is efficiently degraded by TREX1.

Human TREX1 Repairs 3'-End DNA Lesions in Vitro.

Human three-prime repair exonuclease 1 (TREX1) is the major 3' to 5' exonuclease that functions to deplete the cytosolic DNA to prevent the autoimmune response. TREX1 is upregulated and translocates from cytoplasm to the nucleus in response to genotoxic stress, but the function of nuclear TREX1 is not well understood. Herein, we wish to report our in vitro finding that TREX1 efficiently excises 3'-phospho-α,β-unsaturated aldehyde and 3'-deoxyribose phosphate that are commonly produced as base excision repair intermediates and also from the nonenzymatic strand incision at abasic sites.

CDK inhibitor Palbociclib targets STING to alleviate autoinflammation.

Aberrant activation of stimulator of interferon genes (STING) is tightly associated with multiple types of disease, including cancer, infection, and autoimmune diseases. However, the development of STING modulators for the therapy of STING-related diseases is still an unmet clinical need. We employed a high-throughput screening approach based on the interaction of small-molecule chemical compounds with recombinant STING protein to identify functional STING modulators. Intriguingly, the cyclin-dependent protein kinase (CDK) inhibitor Palbociclib was found to directly bind STING and inhibit its activation in both mouse and human cells. Mechanistically, Palbociclib targets Y167 of STING to block its dimerization, its binding with cyclic dinucleotides, and its trafficking. Importantly, Palbociclib alleviates autoimmune disease features induced by dextran sulphate sodium or genetic ablation of three prime repair exonuclease 1 (Trex1) in mice in a STING-dependent manner. Our work identifies Palbociclib as a novel pharmacological inhibitor of STING that abrogates its homodimerization and provides a basis for the fast repurposing of this Food and Drug Administration-approved drug for the therapy of autoinflammatory diseases.

Human TDP1, APE1and TREX1 repair 3'-DNA-peptide/protein cross-links arising from abasic sites in vitro.

Histones and many other proteins react with abundant endogenous DNA lesions, apurinic/apyrimidinic (abasic, AP) sites and/or 3′-phospho-α,β-unsaturated aldehyde (3′-PUA), to form unstable but long-lived Schiff base DNA–protein cross-links at 3′-DNA termini (3′-PUA–protein DPCs). Poly (ADP-ribose) polymerase 1 (PARP1) cross-links to the AP site in a similar manner but the Schiff base is reduced by PARP1’s intrinsic redox capacity, yielding a stable 3′-PUA–PARP1 DPC. Eradicating these DPCs is critical for maintaining the genome integrity because 3′-hydroxyl is required for DNA synthesis and ligation. But how they are repaired is not well understood. Herein, we chemically synthesized 3′-PUA-aminooxylysine-peptide adducts that closely resemble the proteolytic 3′-PUA–protein DPCs, and found that they can be repaired by human tyrosyl-DNA phosphodiesterase 1 (TDP1), AP endonuclease 1 (APE1) and three-prime repair exonuclease 1 (TREX1). We characterized these novel repair pathways by measuring the kinetic constants and determining the effect of cross-linked peptide length, flanking DNA structure, and the opposite nucleobase. We further found that these nucleases can directly repair 3′-PUA–histone DPCs, but not 3′-PUA–PARP1 DPCs unless proteolysis occurs initially. Collectively, we demonstrated that in vitro 3′-PUA–protein DPCs can be repaired by TDP1, APE1, and TREX1 following proteolysis, but the proteolysis is not absolutely required for smaller DPCs.

Proceedings to the 7th Annual Conference of the Particle Therapy Cooperative Group North America (PTCOG-NA)

Proceedings to the 7th Annual Conference of the Particle Therapy Cooperative Group North America (PTCOG-NA)

Targeting Bcl6 in the TREX1 D18N murine model ameliorates autoimmunity by modulating T-follicular helper cells and germinal center B cells.

The Three Prime Repair EXonuclease I (TREX1) is critical for degrading post‐apoptosis DNA. Mice expressing catalytically inactive TREX1 (TREX1 D18N) develop lupus‐like autoimmunity due to chronic sensing of undegraded TREX1 DNA substrates, production of the inflammatory cytokines, and the inappropriate activation of innate and adaptive immunity. This study aimed to investigate Thelper (Th) dysregulation in the TREX1 D18N model system as a potential mechanism for lupus‐like autoimmunity. Comparison of immune cells in secondary lymphoid organs, spleen and peripheral lymph nodes (LNs) between TREX1 D18N mice and the TREX1 null mice revealed that the TREX1 D18N mice exhibit a Th1 bias. Additionally, the T‐follicular helper cells (Tfh) and the germinal celter (GC) B cells were also elevated in the TREX1 D18N mice. Targeting Bcl6, a lineage‐defining transcription factor for Tfh and GC B cells, with a commercially available Bcl6 inhibitor, FX1, attenuated Tfh, GC, and Th1 responses, and rescued TREX1 D18N mice from autoimmunity. The study presents Tfh and GC B‐cell responses as potential targets in autoimmunity and that Bcl6 inhibitors may offer therapeutic approach in TREX1‐associated or other lupus‐like diseases.

187 Photosensitivity and cGAS-dependent type I IFN activation in lupus patients with TREX1 deficiency

The exonuclease three prime repair exonuclease 1 (TREX1) safeguards the cell against DNA accumulation in the cytosol and thereby prevents innate immune activation and autoimmunity. TREX1 mutations lead to chronic DNA damage and a cell-intrinsic type I interferon (IFN) response. Associated disease phenotypes include Aicardi-Goutières syndrome, familial chilblain lupus and systemic lupus erythematosus. Given the role of ultraviolet (UV) light in lupus pathogenesis, we assessed sensitivity to UV light in lupus patients with TREX1 mutation by phototesting which revealed an enhanced photosensitivity compared to healthy controls.

Nucleic Acid Sensing in the Tumor Vasculature.

Simple SummaryOur cells can recognize DNA or RNA from pathogens, such as viruses. The proteins that recognize these nucleic acids are known as nucleic acid sensors. Upon activation, they trigger immune responses that result in the elimination of the infected cells. Recent research has shown how we can mimic this process in cancer and recruit immune cells against the tumor. Among the different cell types within a tumor, endothelial cells that line the blood vessels play a main role as conduits for nutrients and oxygen and highways for the immune cells. In this review, we discuss two different nucleic acid sensors—the three-prime repair exonuclease 1 (TREX1) and the retinoic acid-inducible gene 1 (RIG-I)—and how they play a role in endothelial cells. We present some approaches to target these pathways within the cancer blood vessels to disrupt the blood supply and attract immune responses to cancers.Endothelial cells form a powerful interface between tissues and immune cells. In fact, one of the underappreciated roles of endothelial cells is to orchestrate immune attention to specific sites. Tumor endothelial cells have a unique ability to dampen immune responses and thereby maintain an immunosuppressive microenvironment. Recent approaches to trigger immune responses in cancers have focused on activating nucleic acid sensors, such as cGAS-STING, in combination with immunotherapies. In this review, we present a case for targeting nucleic acid-sensing pathways within the tumor vasculature to invigorate tumor-immune responses. We introduce two specific nucleic acid sensors—the DNA sensor TREX1 and the RNA sensor RIG-I—and discuss their functional roles in the vasculature. Finally, we present perspectives on how these nucleic acid sensors in the tumor endothelium can be targeted in an antiangiogenic and immune activation context. We believe understanding the role of nucleic acid-sensing in the tumor vasculature can enhance our ability to design more effective therapies targeting the tumor microenvironment by co-opting both vascular and immune cell types.

Serial magnetic resonance imaging changes of pseudotumor lesions in retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations: a case report

BackgroundRetinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is an adult-onset rare monogenic microvasculopathy. Its typical neuroimaging features are punctate white matter lesions or pseudotumor alterations. RVCL-S is often under-recognized and misdiagnosed because of its rarity and similar imaging manifestations to multiple sclerosis or brain malignant mass.Case presentationHere we report a case of a 36-year-old Chinese man who developed multiple tumefactive brain lesions spanning over two years leading to motor aphasia, cognitive decline, and limb weakness. He also presented with slight vision loss, and fundus fluorescein angiography indicated retinal vasculopathy. He underwent brain biopsies twice and showed no evidence of malignancy. Given the family history that his father died of a brain mass of unclear etiology, RVCL-S was suspected, and genetic analysis confirmed the diagnosis with a heterozygous insertion mutation in the three-prime repair exonuclease 1 gene. He was given courses of corticosteroids and cyclophosphamide but received little response.ConclusionsThe present case is one of the few published reports of RVCL-S with two-year detailed imaging data. Serial magnetic resonance images showed the progression pattern of the lesions. Our experience emphasizes that a better understanding of RVCL-S and considering it as a differential diagnosis in patients with tumefactive brain lesions may help avoid unnecessary invasive examinations and make an earlier diagnosis.

Human Three Prime Repair Exonuclease 1 Promotes HIV-1 Integration by Preferentially Degrading Unprocessed Viral DNA.

Three prime repair exonuclease 1 (TREX1) is the most abundant 3'→5' exonuclease in mammalian cells. It has been suggested that TREX1 degrades HIV-1 DNA to enable the virus to evade the innate immune system. However, the exact role of TREX1 during early steps of HIV-1 infection is not clearly understood. In this study, we report that HIV-1 infection is associated with upregulation, perinuclear accumulation, and nuclear localization of TREX1. However, TREX1 overexpression did not affect reverse transcription or nuclear entry of the virus. Surprisingly, HIV-1 DNA integration was increased in TREX1-overexpressing cells, suggesting a role of the exonuclease in the post-nuclear entry step of infection. Accordingly, preintegration complexes (PICs) extracted from TREX1-overexpressing cells retained higher levels of DNA integration activity. TREX1 depletion resulted in reduced levels of proviral integration, and PICs formed in TREX1-depleted cells retained lower DNA integration activity. Addition of purified TREX1 to PICs also enhanced DNA integration activity, suggesting that TREX1 promotes HIV-1 integration by stimulating PIC activity. To understand the mechanism, we measured TREX1 exonuclease activity on substrates containing viral DNA ends. These studies revealed that TREX1 preferentially degrades the unprocessed viral DNA, but the integration-competent 3'-processed viral DNA remains resistant to degradation. Finally, we observed that TREX1 addition stimulates the activity of HIV-1 intasomes assembled with the unprocessed viral DNA but not that of intasomes containing the 3'-processed viral DNA. These biochemical analyses provide a mechanism by which TREX1 directly promotes HIV-1 integration. Collectively, our study demonstrates that HIV-1 infection upregulates TREX1 to facilitate viral DNA integration. IMPORTANCE Productive HIV-1 infection is dependent on a number of cellular factors. Therefore, a clear understanding of how the virus exploits the cellular machinery will identify new targets for inhibiting HIV-1 infection. The three prime repair exonuclease 1 (TREX1) is the most active cellular exonuclease in mammalian cells. It has been reported that TREX1 prevents accumulation of HIV-1 DNA and enables the virus to evade the host innate immune response. Here, we show that HIV-1 infection results in the upregulation, perinuclear accumulation, and nuclear localization of TREX1. We also provide evidence that TREX1 promotes HIV-1 integration by preferentially degrading viral DNAs that are incompatible with chromosomal insertion. These observations identify a novel role of TREX1 in a post-nuclear entry step of HIV-1 infection.