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

Abstract

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 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 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 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 to chromosomal insertion. These observations identify a novel role of TREX1 in a post-nuclear entry step of HIV-1 infection.

HIV-1 preintegration complex preferentially integrates the viral DNA into nucleosomes containing trimethylated histone 3-lysine 36 modification

“…9A). The genomic DNA was extracted from the WT and H1 0 overexpressing cells and the proviral integration (PVI) was measured by nested Alu-gag PCR method 73, 74, 102 . Our results revealed that viral DNA integration was significantly reduced in the H1 0 overexpressing cells relative to the control cells (Fig.…”

“…To better understand the mechanism of HIV-1 integration into chromatin, we adopted a method that measured PIC-associated viral DNA integration (PIC-VDI) using 300 ng of target substrates from isolated chromatin DNA, de-chromatinized genomic DNA, biochemically reconstituted nucleosome core particles (NCP) with or without linker DNA and the analogous DNA sequences. HIV-1 PICs were extracted from acutely infected SupT1 cells 71–74 and are competent for viral DNA integration in vitro (SI-Fig. 1A-B).…”

“…1C). To probe the preference of PIC-VDI into these substrates, PICs were incubated with either the chromatin or gDNA preparations containing equivalent amount of DNA and PIC-VDI was measured by alu - based nested qPCR 72–74 . Results from these measurements revealed that PIC-VDI levels were significantly higher with the chromatin substrate compared to the isolated gDNA (Fig.…”

“…Therefore, we employed a biochemical approach involving the extraction of PICs from acutely infected cells and quantified viral DNA integration activity in vitro (SI-Fig. 1A-B) 72–74 . We coupled PIC-VDI with biochemical assembled nucleosomes to define target substrate preference for HIV-1 DNA integration.…”

“…HIV-1 PICs were isolated from acutely HIV-1 infected T cells as described by Balasubramaniam and colleagues 71–74 . Briefly, 8 X 10 7 of SupT1 cells were spinoculated (480 X g ) with DNase I-treated wild type virions for 2 hours at 25°C, and then cultured for 5 hours at 37°C.…”