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HIV-1 and HIV-2 Exhibit Divergent Interactions with HLTF and UNG2

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HIV-1 and HIV-2 Exhibit Divergent Interactions with HLTF and UNG2 HIV-1 and HIV-2 exhibit divergent interactions with PNAS PLUS HLTF and UNG2 DNA repair proteins Kasia Hreckaa,1, Caili Haoa,1, Ming-Chieh Shuna, Sarabpreet Kaura, Selene K. Swansonb, Laurence Florensb, Michael P. Washburnb,c, and Jacek Skowronskia,2 aDepartment of Molecular Biology and Microbiology, Case Western Reserve School of Medicine, Cleveland, OH 44106; bStowers Institute for Medical Research, Kansas City, MO 64110; and cDepartment of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160 Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved May 20, 2016 (received for review March 29, 2016) HIV replication in nondividing host cells occurs in the presence antiviral proteins for degradation by the proteasome (13). In of high concentrations of noncanonical dUTP, apolipoprotein B particular, Vif loads antiviral APOBEC3 family proteins onto a mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) CRL5 E3 ubiquitin ligase for polyubiquitination and subsequent cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP proteasome-dependent degradation (14). Vpx, encoded by the triphosphohydrolase) dNTPase, which maintains low concentra- HIV-2/simian immunodeficiency virus (SIV) SIVsm lineage of tions of canonical dNTPs in these cells. These conditions favor primate lentiviruses, and closely related Vpr proteins from a the introduction of marks of DNA damage into viral cDNA, and subset of SIV viruses isolated from various primate species, thereby prime it for processing by DNA repair enzymes. Accessory counteract SAMHD1-mediated restriction (6, 7, 11). Specifically, protein Vpr, found in all primate lentiviruses, and its HIV-2/simian Vpx binds to the DCAF1 substrate receptor subunit of the immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4DCAF1 E3 ubiquitin ligase and loads SAMHD1 onto this CRL4DCAF1 E3 ubiquitin ligase to alleviate some of these conditions, enzyme, thereby targeting it for degradation (15). Although HIV-1 but the extent of their interactions with DNA repair proteins has does not counteract SAMHD1 directly, it was proposed to bypass not been thoroughly characterized. Here, we identify HLTF, a post- the SAMHD1-imposed restriction as a result of a more efficient replication DNA repair helicase, as a common target of HIV-1/ reverse transcriptase that can synthesize viral cDNA in a low-dNTP SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms environment (16). The failure of the aforementioned viral coun- DCAF1 CRL4 E3 to direct HLTF for proteasome-dependent degrada- termeasures flags HIV cDNA for processing by DNA repair tion independent from previously reported Vpr interactions with enzymes, which, if not successfully completed in a low-dNTP base excision repair enzyme uracil DNA glycosylase (UNG2) and environment set up by SAMHD1, could lead to the initiation of crossover junction endonuclease MUS81, which Vpr also directs an innate response to viral nucleic acids, increased HIV-1 mu- for degradation via CRL4DCAF1 E3. Thus, separate functions of – DCAF1 tation rate, and/or inhibition of HIV-1 infection (17 19). HIV-1 Vpr usurp CRL4 E3 to remove key enzymes in three Vpr, a paralogue of Vpx found in all primate lentiviruses, coor- DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable dinates interactions with postreplication DNA repair machinery, to efficiently program HLTF or UNG2 for degradation. Our find- whose role for the replication cycle of primate lentiviruses is not MICROBIOLOGY ings reveal complex interactions between HIV-1 and the DNA re- well understood. Early studies revealed that HIV-1 Vpr modulates pair machinery, suggesting that DNA repair plays important roles mutation rates in plasmid shuttle vectors in model systems (20, in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these Significance viruses use different strategies to guard their genomes and facil- itate their replication in the host. In nondividing host cells, HIV is targeted by intrinsic antiviral HIV | Vpr | postreplication DNA repair | SAMHD1 | restriction defense mechanisms that introduce marks of damage into viral cDNA, thereby tagging it for processing by cellular DNA repair machinery. Surprisingly, our findings reveal that the two main ondividing memory T cells and myeloid cells are the main types of HIV exhibit very different interactions with enzymes Ntargets of primate lentiviruses during the initial weeks of the – involved in DNA repair. HIV-1, but not HIV-2, efficiently removes acute, in vivo infection (1 4). Infection of these cells is inhibited select DNA repair enzymes, whereas HIV-2 increases dNTP supply by intrinsic and innate antiviral mechanisms, several of which con- in infected cells by removing SAMHD1 (a cell cycle-regulated dNTP verge on reverse transcription of the viral RNA genome. One such triphosphohydrolase) dNTPase. Our findings imply that increasing restriction is imposed by SAMHD1, a cell cycle-regulated dNTP dNTP supply during viral cDNA synthesis or repair, or blocking triphosphohydrolase that, in G1-phase leukocytes, maintains the cDNA processing by DNA repair enzymes, are alternative strate- concentrations of canonical dNTPs below the threshold required for gies used by HIV-2 and HIV-1 to guard their DNA genomes and efficient reverse transcription (5–8). Another is caused by a rela- facilitate their replication/persistence in the host. tively high concentration of noncanonical deoxyuridine triphosphate compared with canonical TTP. dUTP is a substrate for HIV reverse Author contributions: K.H., C.H., M.-C.S., S.K., S.K.S., L.F., M.P.W., and J.S. designed re- transcriptase, which leads to uracil incorporation into viral cDNA. search; K.H., C.H., M.-C.S., S.K., and S.K.S. performed research; L.F. and M.P.W. contrib- uted new reagents/analytic tools; K.H., C.H., M.-C.S., S.K., S.K.S., L.F., M.P.W., and J.S. HIV reverse transcripts are heavily uracilated in macrophages analyzed data; and J.S. wrote the paper. (9, 10). Moreover, viral cDNA is a substrate for apolipoprotein B The author declares no conflict of interest. mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3)- This article is a PNAS Direct Submission. family editing enzymes, which convert cytidine to uridine in the Data deposition: The complete MudPIT mass spectrometry dataset (raw files, peak files, minus strand of HIV reverse-transcription intermediates (6, 7, 11, search files, as well as DTASelect result files) can be obtained from the MassIVE database 12). The latter two mechanisms flag viral cDNA for processing by via ftp://[email protected] with password KHJS60144. cellular DNA repair enzymes. 1K.H. and C.H. contributed equally to this work. The restriction mechanisms that target lentivirus genome rep- 2To whom correspondence should be addressed. Email: [email protected]. lication are counteracted by accessory virulence proteins Vif, Vpx, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and Vpr, which usurp specific cellular E3 ubiquitin ligases to direct 1073/pnas.1605023113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1605023113 PNAS | Published online June 22, 2016 | E3921–E3930 21). Vpr, like Vpx, binds the DCAF1 subunit of the CRL4DCAF1 (41, 42). The presence of multiple proteins involved with DNA E3 complex and hijacks this enzyme (22). This interaction is repair was not surprising, as HIV-1 Vpr and DCAF1 were reported associated with the induction of DNA repair foci and activation to colocalize with DNA repair foci in chromatin (23). The presence of the serine/threonine kinase ATR-controlled DNA damage of UNG2, a known, specific substrate of the CRL4DCAF1-H1.Vpr checkpoint; the latter usually reflects the presence of, or failure E3, in MudPIT datasets indicates that our experimental approach to repair, damaged DNA at replication forks (23–25). Al- detects cellular proteins that Vpr recruits to the CRL4DCAF1 E3 though the exact mechanism by which Vpr mediates the in- ubiquitin ligase and thereby directs for proteasome-dependent duction of replication stress is unclear, it was recently linked degradation. to the Vpr-mediated recruitment of SLX4-SLX1/MUS81-EME1 structure-specific endonucleases to DCAF1, resulting in activation HIV-1 Vpr Down-Regulates HLTF, a Postreplication DNA Repair Helicase. of the MUS81-EME1 endonuclease and, surprisingly, DCAF1- To assess whether any of the 21 identified DNA repair proteins is a and proteasome-dependent MUS81 degradation (26). Sepa- potential substrate of CRL4DCAF1-H1.Vpr E3, we first tested their rately, HIV-1 and HIV-2 Vpr were reported to bind a base levels in CEM.SS-iH1.Vpr and/or U2OS-iH1.Vpr, the latter also excision repair enzyme, uracil DNA glycosylase (UNG2), harboring a doxycycline-inducible HIV-1 NL4-3 Vpr transgene (Fig. which can restrict HIV-1 infection in cells with high concen- S1). Of note, U2OS cells retain many of the cell cycle regulation trations of dUTP (19, 27–29). HIV-1 Vpr was shown to tar- characteristics of normal cells and are commonly used for cell cycle/ get UNG2 to the ubiquitin proteasome pathway via the DNA repair/replication studies. Interestingly, the levels of endoge- CRL4DCAF1 E3 complex and thereby disrupt UNG2-initiated base nous HLTF were much lower in CEM.SS-iH1.Vpr and U2OS-iH1. excision repair in HIV-1–infected cells (30–32). Vpr cells that had been arrested by Vpr at the DNA damage The fact that cellular restriction factors flag HIV cDNA for checkpoint in the G2 phase of the cell cycle compared with control processing by DNA repair enzymes, taken together with the asynchronously dividing cells that did not express Vpr (Fig. S1). considerable complexity of cellular repair machineries, raises the Significantly, HLTF was not depleted in control cells arrested possibility that Vpr coordinately engages DNA repair pathways.
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