Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties.

HIV-1 integration into the human genome is dependent on 3'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼2-fold) for the 3'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.

Optogenetically-induced multimerization of the dopamine transporter increases uptake and trafficking to the plasma membrane.

The dopamine transporter (DAT) is essential for the reuptake of the released neurotransmitter dopamine (DA) in the brain. Psychostimulants, methamphetamine and cocaine, have been reported to induce the formation of DAT multimeric complexes in vivo and in vitro. The interpretation of DAT multimer function has been primarily in the context of compounds that induce structural and functional modifications of the DAT, complicating the understanding of the significance of DAT multimers. To examine multimerization in the absence of DAT ligands as well as in their presence, we developed a novel, optogenetic fusion chimera of cryptochrome 2 and DAT with an mCherry fluorescent reporter (Cry2-DAT). Using blue light to induce Cry2-DAT multimeric protein complex formation, we were able to simultaneously test the functional contributions of DAT multimerization in the absence or presence of substrates or inhibitors with high spatiotemporal precision. We found that blue light-stimulated Cry2-DAT multimers significantly increased IDT307 uptake and MFZ 9-18 binding in the absence of ligands as well as after methamphetamine and nomifensine treatment. Blue light-induced Cry2-DAT multimerization increased colocalization with recycling endosomal marker Rab11 and had decreased presence in Rab5-positive early endosomes and Rab7-positive late endosomes. Our data suggest that the increased uptake and binding results from induced and rapid trafficking of DAT multimers to the plasma membrane. Our data suggest that DAT multimers may function to help maintain DA homeostasis.

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.

Three Prime Repair Exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural and conformational properties.

HIV-1 integration into the human genome is dependent on 3'-processing of the reverse transcribed viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower than the unprocessed HIV-1 DNA by TREX1. The efficiency of degradation (kcat/KM) of the 3'-processed DNA was also significantly lower than the unprocessed DNA. Furthermore, the binding affinity (Kd) of TREX1 was markedly lower to the 3'-processed DNA compared to the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the biochemical mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.

HIV-1 mutants that escape the cytotoxic T-lymphocytes are defective in viral DNA integration.

HIV-1 replication is durably controlled without antiretroviral therapy (ART) in certain infected individuals called elite controllers (ECs). These individuals express specific human leukocyte antigens (HLA) that tag HIV-infected cells for elimination by presenting viral epitopes to CD8+ cytotoxic T-lymphocytes (CTL). In HIV-infected individuals expressing HLA-B27, CTLs primarily target the viral capsid protein (CA)-derived KK10 epitope. While selection of CA mutation R264K helps HIV-1 escape this potent CTL response, the accompanying fitness cost severely diminishes virus infectivity. Interestingly, selection of a compensatory CA mutation S173A restores HIV-1 replication. However, the molecular mechanism(s) underlying HIV-1 escape from this ART-free virus control by CTLs is not fully understood. Here, we report that the R264K mutation-associated infectivity defect arises primarily from impaired HIV-1 DNA integration, which is restored by the S173A mutation. Unexpectedly, the integration defect of the R264K variant was also restored upon depletion of the host cyclophilin A. These findings reveal a nuclear crosstalk between CA and HIV-1 integration as well as identify a previously unknown role of cyclophilin A in viral DNA integration. Finally, our study identifies a novel immune escape mechanism of an HIV-1 variant escaping a CA-directed CTL response.