HIV-1 and HIV-2 exhibit divergent interactions with PNAS PLUS HLTF and UNG2 DNA repair

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 (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 for polyubiquitination and subsequent cytidine deaminases, and SAMHD1 (a -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 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, 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. in late S/G2 phase by etoposide or in early M phase by nocodazole Unveiling additional targets in these pathways would lead to a treatments. These observations are consistent with the possibility more integrated model for the role of Vpr and DNA repair in that HLTF, a DNA repair protein expressed in natural target cells the HIV life cycle. Here, we performed a proteomic screen for of HIV-1 infection (Fig. S2),isaspecifictargetofHIV-1Vpr. Vpr-interacting DNA repair proteins, aiming in particular to identify novel substrates of the reprogrammed by HIV-1 Vpr HIV-1 Vpr Down-Regulates HLTF Independently of Cell Cycle Position. CRL4DCAF1 E3 ubiquitin ligase (CRL4DCAF1-H1.Vpr). We show Vpr activates the ATR-controlled DNA damage checkpoint, that human HLTF DNA repair helicase is such a target of HIV-1 thereby arresting cells in G2 phase (24). The possibility existed that Vpr. Our findings reveal that HIV-1 Vpr impacts three distinct types HLTF down-regulation is an indirect consequence of Vpr-induced of DNA repair transactions and illustrate the complexity of HIV-1 cell cycle perturbations. Hence, to demonstrate that HLTF de- Vpr interactions with cellular postreplication DNA repair machin- pletion by Vpr is independent of cell cycle phase and ATR acti- ery. We also show that HLTF and UNG2 are common targets of vation, additional experiments were performed. Vpr proteins from all main groups of HIV-1, but, somewhat un- First, we asked whether Vpr can deplete HLTF in U2OS-iH1. expectedly, not of those found in HIV-2 A or B. Thus, our findings Vpr cells outside of the G2 phase. U2OS-iH1.Vpr were syn- support the possibility that the two main types of HIV use very chronized in late G1/early S phase by double-thymidine block, different strategies to stabilize their DNA genomes. HIV-2 increases and Vpr expression was induced at 8 h into the second thymidine dNTP supply in infected cells by removing SAMHD1, whereas treatment (Fig. 1A). Cells were harvested at 0, 6, 12, and 24 h HIV-1 inactivates DNA repair enzymes, possibly to compensate for after induction for flow cytometry analysis of cell cycle position its inability to drive up dNTP concentrations in primary target cells. and for immunoblot analysis of HLTF levels. As shown in Fig. 1B, U2OS-iH1.Vpr cells remained at the G1/S border through Results the duration of the experiment. Significantly, endogenous HLTF Proteomic Screen for DNA Repair Proteins Associated with HIV-1 Vpr. levels became severely depleted within 6 h of the induction of We searched for candidate DNA repair proteins that are targeted Vpr expression (Fig. 1C). by HIV-1 Vpr by purifying Vpr–protein complexes from T cells and To assess whether the Vpr effect on HLTF was linked to its characterizing their composition by multidimensional protein interaction with the CRL4DCAF1 E3 ubiquitin ligase, we next tested identification technology (MudPIT) (33). In brief, we constructed a the Vpr(H71R) variant that does not bind DCAF1 (32). Significantly, CEM.SS T-cell line harboring a doxycycline-inducible HIV-1 NL4-3 this mutant did not detectably modulate HLTF levels even at the Vpr allele tagged with a double HA-FLAG-epitope (CEM.SS-iH1. late 24-h time point. These findings link the ability of Vpr to deplete Vpr). Vpr expression was induced with doxycycline for 6 h, and Vpr, HLTF to its interaction with CRL4DCAF1 E3 Ub ligase. together with its associated proteins, was purified by successive Excess thymidine stresses replication forks (43), potentially immunoprecipitations via the HA and FLAG tags (6, 34). As ex- contributing to the observed Vpr-mediated HLTF depletion. To pected, the most abundant cellular polypeptides found to be asso- exclude this possibility, we characterized HLTF levels across the ciated with Vpr were DCAF1, DDB1, and DDA1, subunits of the cell cycle in asynchronously dividing U2OS-iH1.Vpr cells. The CRL4DCAF1 E3 complex, which Vpr binds via its DCAF1 subunit cells were cultured in the presence or absence of doxycycline for (Table S1)(34,35.AnalysisoftheMudPIT datasets using the 6 h, stained with a vital stain, Vybrant DyeCycle Green, to reveal Database for Annotation, Visualization and Integrated Discovery their DNA content, and then sorted into highly enriched G1, S, bioinformatics resource (36, 37) identified 21 Vpr-associated pro- and G2/M populations (Fig. 2A). Whole-cell extracts prepared teins that have been linked to DNA replication and/or repair. None from the sorted cells were analyzed by immunoblotting for of these proteins was detected in MudPIT datasets obtained for CyclinA2, HLTF, and UNG2, the previously identified specific purifications from control CEM.SS T cells that did not express Vpr. substrate of the CRL4DCAF1-H1.Vpr E3 ubiquitin ligase (Fig. 2B). Among the proteins identified (Table S1) were several that mediate CyclinA2 was detected in only the S and G2/M extracts, as postreplication DNA repair, including UNG2, MSH6, a mismatch expected, thus confirming the purity of the sorts. HLTF levels were repair protein, RFC clamp loader, and PCNA, replication factors similar in G1, S, and G2/M extracts from uninduced U2OS-iH1. that are central to DNA replication and repair (38–40), and HTLF, Vpr cells, indicating that HLTF is expressed in all cell cycle phases. one of two mammalian homologs of yeast RAD5 DNA helicase Significantly, HLTF levels were much lower in the induced U2OS- that controls the postreplication error-free DNA repair pathway iH1.Vpr cells, regardless of their cell cycle position. The levels of

E3922 | www.pnas.org/cgi/doi/10.1073/pnas.1605023113 Hrecka et al. PNAS PLUS A similar analysis was performed with the HIV-1 Vpr(W54R) variant, which is defective for UNG2 loading onto the CRL4DCAF1 complex but retains binding to DCAF1 (28, 30). This variant retained full ability to deplete HLTF and MUS81 levels (Fig. 3B). We conclude that Vpr exerts its effects on HLTF and MUS81 in a manner independent of UNG2, even though all three require the interaction with DCAF1 (26, 30). The possibility still remained that lower MUS81 levels in Vpr- expressing cells reflected a cellular response to HLTF depletion, rather than a direct effect of Vpr. Therefore, we asked whether HLTF and MUS81 levels are correlated in the absence of Vpr. Parental U2OS cells were subjected to RNAi targeting of HLTF or MUS81, and cell extracts were prepared 48 h later and analyzed by immunoblotting. Notably, HLTF knockdown led to elevated MUS81 levels, suggesting that the latter was to compensate for the loss of HLTF (Fig. 3C). In contrast, MUS81 depletion did not have a detectable effect on HLTF levels. This evidence supports the possibility that HLTF and MUS81 are specific and independent targets of the CRL4DCAF1-H1.Vpr E3 ubiquitin ligase.

HIV-1 Selectively Depletes Only the HLTF Homolog of Yeast RAD5 DNA Repair Protein. Mammalian cells possess two Rad5 homologs, HLTF and SHPRH, which serve distinct roles in postreplication DNA repair (41). Experiments were performed to assess whether Vpr selectively depletes HLTF or, instead, targets both Rad5 homologs. As shown in Fig. 3, SHPRH levels remained largely Fig. 1. HIV-1 Vpr depletes HLTF outside of G2 phase. (A) Experimental strategy. U2OS cells harboring the indicated HIV-1 NL4-3 Vpr transgenes were synchro- nized by double thymidine block and then kept arrested at the G1/S border for the duration of the experiment. HIV-1 Vpr expression was induced by doxycy- cline during the second thymidine treatment, and cells were collected at the indicated time points for FACS analysis of cell cycle position (B) and immunoblot analysis of the indicated proteins (C). Transcription factor II D (TFIID) was used as a loading control. The cell cycle profile of an asynchronously dividing cell pop- ulation that did not undergo double thymidine block is shown in B and labeled MICROBIOLOGY with “A”.

UNG2 were similarly depleted following induction of Vpr ex- pression. Finally, HLTF depletion by Vpr was not inhibited by culturing the cells in the presence of caffeine, indicating that the depletion was not a G2/M DNA damage checkpoint-mediated response (Fig. S3). Together, the aforementioned findings demonstrate that Vpr down-regulates HLTF levels independently of the cell cycle po- sition and of G2 DNA damage checkpoint, thus further sup- porting the possibility that HLTF is a substrate for the HIV-1 Vpr-modified CRL4DCAF1 E3 Ubiquitin ligase.

HIV-1 Vpr Targets HLTF Independently of MUS81. HIV-1 Vpr was re- cently reported to down-modulate the MUS81 subunit of a struc- ture-specific endonuclease in a DCAF1-dependent manner (26). Therefore, we characterized MUS81 levels in control and Vpr- expressing U2OS-iH1.Vpr sorted cell populations. Strikingly, the endogenous MUS81 levels were not detectably altered following a brief, 6-h-long pulse of Vpr expression. This contrasted with the robust depletion of HLTF in the same time frame (Fig. 2B). These findings implied that the effects of HIV-1 Vpr on HLTF and MUS81 are likely to be independent of each other. We further Fig. 2. HIV-1 Vpr depletes HLTF independently of cell cycle position. explored this by carefully comparing the time course of HLTF and (A) Purity of G1, S, and G2/M cell populations isolated by cell sorting. MUS81 depletion following induction of Vpr expression in U2OS- The DNA profiles of U2OS (mock) and U2OS-iH1.Vpr (HIV-1 Vpr) G1-, S-, and iH1.Vpr cells. As shown in Fig. 3A, depletion of HLTF levels was G2/M-phase cells isolated by sorting for DNA content are shown overlaid with that of the parental asynchronously growing unsorted population (la- almost complete at the 6-h time point. In contrast, MUS81 levels “ ” “ ” were down-regulated at a much slower rate, and 24–48 h were re- beled A ). (B) Cell extracts prepared from asynchronously dividing ( A ), G1, S, and G2/M populations were analyzed by immunoblotting with anti- quired for complete depletion, which coincides with the accumu- bodies reacting with HLTF, UNG2, MUS81, CyclinA2, FLAG-tagged Vpr, and lation of Vpr-expressing cells at the DNA damage checkpoint in TFIID loading control. Asterisk indicates a nonspecific background band late S/G2 phase. revealed by the α-UNG2 antibody.

Hrecka et al. PNAS | Published online June 22, 2016 | E3923 Fig. 3. Vpr selectively targets the HLTF homolog of RAD5, and this effect is independent from interactions with UNG2 and MUS81. (A and B) Time course of HLTF, SHPRH, MUS81, and UNG2 depletion by HIV-1 Vpr. U2OS-iH1.Vpr and control cells were treated with doxycycline, and cell extracts prepared at the indicated times were analyzed by immunoblotting. Lamin B and TFIID provided loading controls. (C) HLTF, MUS81, and UNG2 levels are not coregulated. U2OS cells were subjected to control nontargeting RNAi (scr) or RNAi targeting HLTF or MUS81, and cell extracts were immunoblotted for HLTF, MUS81, UNG2, and TFIID. unaltered with a small, at most approximately twofold, decrease HIV-1 Vpr Interaction with HLTF Defines Previously Unidentified at the 24- and 48-h time points. We conclude that Vpr selectively Functional Elements in both Proteins. HLTF comprises an N-ter- targets the HLTF homolog of yeast Rad5 helicase. minal DNA-binding HIRAN domain and a C-terminal RING domain possessing E3 Ubiquitin ligase activity (Fig. 5A). To map HIV-1 Vpr Targets HLTF for Proteasome-Dependent Degradation via the HLTF region that mediates the effect of Vpr, we analyzed the CRL4DCAF1 E3. We next tested whether Vpr directs HLTF for activity of HIV-1 NL4-3 Vpr toward a set of HLTF deletion mu- proteasome-dependent degradation. U2OS-iH1.Vpr cells were tants by using a transient coexpression assay in HEK 293T cells. treated with doxycycline in the absence or presence of MG132 This assay faithfully reproduced the Vpr-induced proteasome- proteasome inhibitor for 9 h. As shown in Fig. 4A, proteasome dependent HLTF down-modulation (Fig. S4). As shown in Fig. 5B, inhibition stabilized HLTF levels in Vpr expressing cells while an HLTF fragment lacking the N-proximal 151 residues, including having no detectable effect on HLTF in control cells. We con- the HIRAN domain [HLTF(152–1009)], as well as those lacking the clude that Vpr activates a proteasome-dependent mechanism to C-distal ∼700 residues comprising the RING domain and down-modulate HLTF levels. HELICc/DEXDc helicase components, were fully responsive to Next, we asked whether HIV-1 Vpr can recruit HLTF to the HIV-1 Vpr, thus tentatively mapping the Vpr target site to HLTF endogenously expressed CRL4DCAF1 E3 complex (Fig. 4 B and C). residues 152–299. This region appears to serve as a linker be- FLAG-tagged HLTF was expressed alone or coexpressed with tween the HIRAN domain and the N-proximal component of the HIV-1 NL4-3 Vpr or the Vpr(H71R) variant in HEK 293T cells. helicase ATP-binding domain, and, as such, is not known to possess Detergent extracts were prepared from the transfected cells, and another function. HLTF immune complexes were analyzed by immunoblotting We next mapped the residues in HIV-1 Vpr that are required for HLTF, Vpr, and the DCAF1 and DDB1 subunits of the for the effect on HLTF levels. Because Vpr and its paralogue Vpx CRL4DCAF1 E3. We found that HIV-1 Vpr directed the assembly of use their N-terminal regions to recruit novel protein substrates to a protein complex containing HLTF as well as DCAF1 and DDB1. CRL4DCAF1 E3 (15, 30, 44), we constructed a set of HIV-1 In contrast, neither the Vpr(H71R) variant, which retained a reduced Vpr point mutants in the N-proximal region and screened them by binding to HLTF, nor HLTF alone, nucleated such a DCAF1- using the previously described transient, dose-dependent HLTF containing complex. These observations indicate that Vpr mediates down-modulation assay. As shown in Fig. 5C, a double amino acid specific recruitment of HLTF to the CRL4 E3 complex that uses substitution E24R,R36P [Vpr(E24R,R36P)] diminished the ability of the DCAF1 substrate receptor, to which Vpr binds. Of note, HLTF Vpr to down-modulate HLTF, but not UNG2. These observations complexes formed in the absence of Vpr contained low levels of revealed that the E24R,R36P mutation selectively disrupts Vpr DDB1, which suggests that HLTF may physiologically participate in binding to HLTF, but does not grossly interfere with binding to DDB1 complexes that do not contain DCAF1. DCAF1 or recruitment of UNG2 to the CRL4DCAF1 E3 complex. Overall, these findings indicate that HIV-1 Vpr specifically To corroborate this possibility, we characterized the binding of binds to HLTF and recruits it to the DCAF1-DDB1 module of the Vpr(E24R,R36P) variant to HLTF and DCAF1. FLAG-HLTF was DCAF1 the CRL4 E3 complex, thereby directing HLTF for deg- coexpressed with HA-Vpr or HA-Vpr(E24R,R36P) in HEK 293T cells, radation by proteasome. and Vpr immune complexes were analyzed by immunoblotting. The

Fig. 4. HIV-1 Vpr recruits HLTF to the DDB1-DCAF1 module of the CRL4DCAF1 E3 complex for proteasome-dependent degradation. (A) Vpr targets HLTF for proteasome-dependent degradation. U2OS-iH1.Vpr cells (HIV-1 Vpr) and control U2OS cells (mock) were treated with doxycycline or not treated in the absence or presence of MG132 proteasome inhibitor (1 μg/mL) for 9 h. HLTF, MUS81, and Vpr levels in cell lysates were revealed by immunoblotting. TFIID provided a loading control. (B) Schematic representation of HIV-1–mediated recruitment of HLTF onto the CRL4DCAF1 E3 complex. The placement of HA and FLAG epitope tags on Vpr and HLTF, respectively, is indicated. (C) Vpr recruits HLTF to the DCAF1-DDB1 module of the CRL4DCAF1 E3 complex. FLAG-HLTF was transiently coexpressed with myc-tagged HIV-1 Vpr or Vpr(H71R) in HEK293T cells as indicated. Endogenous DCAF1, DDB1, and ectopic Vpr and HLTF were revealed in HLTF immune complexes and in detergent extracts by immunoblotting.

E3924 | www.pnas.org/cgi/doi/10.1073/pnas.1605023113 Hrecka et al. PNAS PLUS

Fig. 5. HIV-1 Vpr interaction with HLTF does not involve known functional elements in both proteins. (A) Summary of mutations in HLTF and their effects on Vpr-mediated down-modulation of HLTF levels. Schematic representation of the HLTF protein and HLTF deletion mutants shows the location of the HIRAN, RING, SNF2, and discontinuous DEXDc and HELICc helicase domains. The HLTF region mediating Vpr sensitivity is boxed. (B) HIV-1 Vpr does not act on known functional domains of HLTF. FLAG-HLTF and its variants, indicated in A, were transiently coexpressed with increasing doses of HA-tagged HIV-1 Vpr in HEK 293T cells, and HLTF and Vpr levels in cell extracts were revealed by immunoblotting. α-Tubulin provided a loading control. (C) The Vpr N terminus is required for depletion of HLTF levels. HIV-1 Vpr or Vpr(24R,36P) variant was coexpressed at increasing doses with HLTF (Upper) or UNG2 (Lower) in HEK293T cells, and cell extracts were analyzed as described earlier. (D) The E24R,R36P mutation selectively disrupts Vpr binding to HLTF. HA-Vpr and its variants were coexpressed with FLAG-HLTF in HEK 293T cells. The Vpr(H71R) variant that does not bind DCAF1 was used as a negative control. Endogenous DCAF1, DDB1, and ectopic Vpr and HLTF were revealed in Vpr immune complexes and detergent extracts by immunoblotting. MICROBIOLOGY Vpr and Vpr(E24R,R36P) variants efficiently coprecipitated the en- of U2OS.HLTF.KO cells in the absence of Vpr expression (Fig. 6C dogenously expressed DCAF1-DDB1 module of CRL4DCAF1 E3 and Fig. S5). Interestingly, MUS81 knockdown in U2OS.HLTF.KO complex (Fig. 5D). Vpr(H71R), which was analyzed in parallel as a cells was associated with an altered cell cycle profile with an in- negative control, did not associate with DDB1-DCAF1, and only crease in the G2-phase population compared with that for cells weakly associated with HLTF, as expected. Significantly, only WT subjected to nontargeting siRNA, even in the absence of Vpr (P < Vpr, but not the Vpr(E24R,R36P) variant, efficiently coprecipitated 0.01) (Fig. 6C). This observation indicated that a combined MUS81 HLTF. Notably, to our knowledge, the E24 and R36 residues have and HLTF deficiency alone could lead to the accumulation of cells not been reported to be important for any other known Vpr in- in G2 phase. teraction with cellular DNA repair machinery. These findings to- Next, we investigated whether MUS81 is required for the in- gether confirm that Vpr recruits HLTF to DCAF1 independent of duction of G2 arrest by HIV-1 Vpr in U2OS-iH1.Vpr cells. MUS81 its previously reported interactions with other DNA repair proteins. levels were depleted by RNAi 48 h before induction of Vpr ex- pression, and cell-cycle profiles were determined 2 d after induction. HLTF, MUS81, and the Induction of G2 Arrest by Vpr. We asked whether Strikingly, MUS81 depletion did not diminish the ability of Vpr to Vpr-mediated depletion of HLTF disrupts postreplication DNA arrest cells in G2. To the contrary, it slightly exacerbated the Vpr repair sufficiently to modulate the DNA damage checkpoint such phenotype in these cells, but the difference was not statistically that cells are arrested in the G2 phase of the cell cycle. To this end, significant. Notably, a similar effect was seen in HLTF-KO cells, in we tested the HIV-1 Vpr(E24R,R36P) variant for its ability to arrest which the difference conferred by the MUS81 knockdown was cells in G2 phase and determined whether HLTF and/or MUS81 statistically significant (P < 0.01). These observations reveal that the are required for the induction of G2 arrest by Vpr. U2OS-iH1. presence of MUS81 is probably not required for the induction of Vpr(E24R,R36P) cells were induced to express HLTF degradation- G2 arrest by Vpr. This evidence supports the model in which rep- defective Vpr(E24R,R36P) variant, and their cell cycle profile were lication stress resulting from combined depletion of MUS81 and A determined 2 d later. As shown in Fig. 6 ,HIV-1Vpr(E24R,R36P) HLTF contributes to the ability of Vpr to arrest cells at the DNA retained the ability to arrest U2OS cells in G2 phase. Similar ex- damage checkpoint in G2 phase. periments performed with U2OS HLTF-KO cells [U2OS.HLTF. KO-iH1.Vpr (45)] revealed that WT NL4-3 Vpr arrested these cells HLTF and UNG2 Are Common Targets of HIV-1 and SIVcpz Vpr Proteins. in G2 phase (Fig. 6B, panel 12). Thus, HLTF deficiency per se is not Experiments were performed to establish whether HLTF is a com- sufficient to trigger DNA-damage checkpoint, as reported in pre- mon target of primate lentiviral Vpr proteins. We focused on Vpr vious studies (45, 46), nor is HLTF required to mediate cell cycle proteins from HIV-1 and HIV-2, which represent two evolutionary arrest in G2 phase by Vpr. divergent branches of primate lentiviruses that adaptedtoreplicate As the data shown in Fig. 3C suggest a possible compensatory in human cells. Fig. 7A shows an amino acid sequence alignment for interaction between MUS81 and HLTF, we next tested the effect consensus Vpr proteins derived from the main groups of HIV-1 of RNAi-mediated MUS81 knockdown on cell cycle distribution (M, N, O) and SIVcpz viruses, isolated from two chimpanzee

Hrecka et al. PNAS | Published online June 22, 2016 | E3925 Fig. 6. The effects of HLTF and MUS81 depletion on HIV-1 Vpr-induced G2 arrest. (A) Vpr(E24R,R36P) arrests U2OS cells in G2 phase. Cell cycle profiles of U2OS cells induced with doxycycline [Dox (+)], or not [Dox (−)] to express HIV-1 NL4-3 Vpr, Vpr(E24R,R36P), or Vpr(H71R) for 48 h. The percentage fraction of cells in G1, S, and G2 phase is indicated, and panel numbers are shown in upper right corner. The abscissa is DNA content shown on a linear scale. The ordinate is DNA synthesis shown on a logarithmic scale (B) HIV-1 Vpr arrests U2OS.HLTF.KO cells in G2 phase. Cell-cycle profiles of U2OS.HLTF.KO cells induced with doxycycline [Dox (+)] or not [Dox (−)] to express HIV-1 NL4-3 Vpr for 48 h. (C) Vpr arrests MUS81-depleted U2OS and U2OS.HLTF.KO cells in G2 phase. Schematic rep- resentation of cell cycle profiles of parental (mock) U2OS (HLTF), U2OS.HLTF.KO cells (HLTF.KO), or their derivative cell lines carrying doxycycline-inducible HIV-1 Vpr transgenes (HIV-1 Vpr). The cells transfected with siRNA targeting MUS81 (MUS81) or nontargeting (scr) and induced (or not) with doxycycline 2 d later as indicated. Cell cycle profiles were determined 2 d after induction, and cells in G1, S, and G2/M phases were quantified as shown in A. Each bar represents averaged results from four replicates. The significance of the observed differences in G2-phase populations between the indicated conditions shown above the bars was calculated using an unpaired two-tailed Student’s t test with Welch’s correction (n = 4; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Representative results of three independent experiments are shown. subspecies (Ptt, Pts) from which HIV-1 originated after cross-species HIV-2/SIVsm lineage viruses encode a Vpr paralogue termed transmissions (47), as well as Vpr proteins from HIV-2 groups A and Vpx, which was lost during adaptation of ancient SIV to chim- B. Of note, to account for the considerable amino acid sequence panzees and is absent in the present HIV-1/SIVcpz branch of pri- divergence at the C terminus of HIV-2 group A Vpr proteins, two mate lentiviruses (48). Vpr and Vpx proteins usurp CRL4DCAF1 distinct consensus Vpr proteins, termed A1 and A2, were de- E3 and are highly adaptable, as revealed by their shared ability rived. Inspection of the alignment revealed that residues E24 and to reprogram this ubiquitin ligase toward a common SAMHD1 R36, which we found to be important for HLTF depletion by substrate in many SIV lineages (11). Therefore, we asked whether HIV-1 NL4-3 Vpr, are conserved in all consensus HIV-1 and HIV-2 Vpx has acquired the ability to program HLTF or UNG2 D SIVcpz Vpr sequences (Fig. 7A). Similarly, residue W54, which is degradation. As shown in Fig. 7 , neither the HIV-2 A nor B group required for UNG2 loading onto CRL4DCAF1 E3, was also pre- consensus Vpx protein decreased HLTF or UNG2 levels. Of note, sent in all HIV-1/SIVcpz consensus Vpr sequences. These ob- the Vpx proteins were functional as judged by their ability to deplete SAMHD1 (Fig. S6B). We conclude that HIV-2 lineage servations predicted that HLTF as well as UNG2 are common DCAF1 targets of HIV-1 and SIVcpz Vpr proteins. Vpr/Vpx proteins do not endow CRL4 E3 with specificity To test this prediction, we synthesized vpr for the con- toward HLTF or UNG2. A sensus Vpr proteins shown in Fig. 7 and characterized their ability – B HLTFandUNG2AreDepletedinHIV-1Infected Cells. To corroborate to deplete HLTF and UNG2. As shown in Fig. 7 ,alltestedHIV-1 the aforementioned findings, we characterized HLTF and UNG2 and SIVcpz Vpr proteins down-modulated HLTF and UNG2. levels in the context of HIV infection. As shown in Fig. 8 A and B, Their activities were comparable to those of the NL4-3 Vpr that we challenge of Jurkat T cells with VSV-G pseudotyped, Vpr-loaded used in the studies described earlier. Remarkably, even the HIV-1 virus-like particles (VLPs) or infection with a VSV-G ∼ SIVcpzPts Vpr, which is 30% divergent from HIV-1 M Vpr pseudotyped single-cycle HIV-1 NL4-3.GFP.R+ carrying an intact in the core region, was quite active against HLTF. vpr led to the depletion of HLTF and UNG2. Importantly, + + infection of primary CD4 T cells with NL4-3.GFP.R virus was HIV-2 Vpr and Vpx Do Not Modulate HLTF Nor UNG2 Levels. Sequence A also associated with a profound depletion of HLTF levels, indicat- alignment, shown in Fig. 7 , revealed that the key residues re- ing that endogenous Vpr expression is sufficient to remove HLTF quired for robust interaction with HLTF, as well as UNG2, are from natural HIV-1 target cells (Fig. 8C). In contrast, these effects not conserved in consensus HIV-2 Vpr proteins. Indeed, the were not seen in cells infected with control capsid-normalized HIV- consensus HIV-2 groups A and B Vpr proteins were inactive 1 VLP produced in the absence of Vpr, or with vpr-deficient NL4-3. – toward HLTF and UNG2 despite being expressed to levels much GFP.R HIV-1 (Fig. 8 A and B). higher than that of the NL4-3 Vpr (Fig. 7C). Importantly, the Next, we asked whether HIV-1 and HIV-2 indeed exert di- HIV-2 consensus Vpr proteins bound DCAF1 in coimmuno- vergent effects on HLTF and UNG2. Jurkat T cells were infected + precipitation assays, similar to HIV-1 NL4-3 Vpr, and therefore with two doses of normalized, single-cycle HIV-1 NL4-3.GFP.R , appeared to be functional (Fig. S6A). We conclude that only the and HIV-2 Rod having intact vpr and vpx genes (Fig. 8 D and E). HIV-1 and SIVcpz, but not HIV-2, Vpr proteins efficiently direct HIV-1 infection led to a dose-dependent depletion of HLTF and HLTF and UNG2 for proteasome-dependent degradation via UNG2 levels, as expected. In contrast, HIV-2 did not exert a CRL4DCAF1-H1.Vpr E3 ubiquitin ligase. comparable robust effect, even though the levels of both proteins

E3926 | www.pnas.org/cgi/doi/10.1073/pnas.1605023113 Hrecka et al. PNAS PLUS

Fig. 7. HLTF and UNG2 are depleted by HIV-1 and SIVcpz but not by HIV-2 Vpr proteins. (A) Alignment of consensus sequences of Vpr proteins from main HIV-1 (M, N, O), SIVcpz (Ptt, Pts), and HIV-2 (A1, A2, B) groups. Sequences are shown in one letter code. Residues required for binding to HLTF (E24, R36), UNG2 (W54), and DCAF1 (H71) are boxed. (B) HIV-1 and SIVcpz Vpr programs HLTF and UNG2 for degradation. FLAG-HLTF or FLAG-UNG2 were transiently coexpressed with increasing doses of the indicated consensus HA-epitope–tagged HIV-1 Vpr proteins. Levels of HLTF and Vpr in cell extracts were revealed by immunoblotting. α-Tubulin provided MICROBIOLOGY loading control. (C and D) Consensus HIV-2 Vpr and Vpx proteins do not modulate HLTF or UNG2 levels. FLAG-HLTF or FLAG-UNG2 were coexpressed with the in- dicated HA-epitope–tagged consensus HIV-2 Vpr or Vpx proteins. HLTF, UNG2, and Vpr/Vpx were analyzed as indicated earlier. appeared to be somewhat lower in cells infected with the highest more comprehensive description of the interactions between HIV-1 dose of HIV-2. In parallel, similar experiments were performed in Vpr and cellular DNA repair pathways. To this end, we performed THP-1 cells (Fig. 8F). Of note, these cells express SAMHD1 at high a proteomic screen, which led to the identification of HLTF DNA levels, but HLTF levels were not detectable by immunoblotting. We helicase as a specific target of the CRL4DCAF1-H1.Vpr E3 ubiquitin found that HIV-1 readily depleted UNG2 while having no effect on ligase. Of note, HLTF was also identified as HIV-1 Vpr target by SAMHD1 levels. In contrast, HIV-2 depleted SAMHD1 in a Vpx- Lahouassa et al. (49). dependent manner, as expected, whereas UNG2 levels were not Our evidence supports the possibility that HLTF is a direct affected. To corroborate these findings, we tested two ancestor target of HIV-1 Vpr for recruitment to the CRL4DCAF1 E3 and SIVmac251 and SIVmac239 viruses, each encoding an uninter- proteasome-dependent degradation. First, Vpr rapidly depletes rupted vpr gene. As shown in Fig. 8G, infection of THP-1 cells HLTF independently of cell cycle position, and this is not a resulted in almost complete depletion of SAMHD1 but had no downstream effect of DNA damage checkpoint activation. Second, detectable effect on UNG2 levels in these cells. We conclude that coimmunoprecipitation experiments show that Vpr bridges HLTF to HIV-1/SIVcpz lineage viruses counteract HLTF and UNG2 DNA the CRL4DCAF1 E3 complex. Third, Vpr-mediated depletion does repair proteins via the CRL4DCAF1 E3, whereas the HIV-2/SIVmac not require the HLTF RING domain, which was shown to mediate lineage viruses tested do not possess a comparable ability. proteasome-dependent HLTF degradation in response to DNA damage (41). These latter findings indicate that HLTF depletion is not Discussion a consequence of other Vpr interactions with DNA repair pathways. When replicating in primary, nondividing, target cells, primate Overall, our studies reveal a striking complexity of Vpr interac- lentiviruses are targeted by innate immune mechanisms that in- tions with cellular DNA repair machinery. This and previous reports troduce marks of damage into viral cDNA, thereby flagging it for together demonstrate that HIV-1/SIVcpz Vpr uses the CRL4DCAF1 processing by host-cell DNA repair enzymes. Hence, it is not E3 to remove, from infected cells, three key enzymes involved surprising that lentiviral accessory proteins intersect with cellular in distinct DNA repair pathways (15, 26, 32). Significantly, the mechanisms that modulate DNA repair, presumably to moder- recruitment of HLTF and UNG2, as well as Mus81 and UNG2, to ate the negative impact of DNA repair on virus genome stability CRL4DCAF1 E3 is mediated via different surfaces of the HIV-1 Vpr and/or priming an innate response to viral nucleic acids. In molecule. Although we have not yet separated HLTF and MUS81 HIV-1, the interactions with postreplication DNA repair processes binding surfaces on Vpr, RNAi studies revealed that expression of are coordinated via the CRL4DCAF1 E3 ubiquitin ligase, which is these two DNA repair proteins is not coordinated, thereby implying hijacked by Vpr (22, 26, 34). The present study aimed to gain a that Vpr depletes their levels independently of each other. Thus, the

Hrecka et al. PNAS | Published online June 22, 2016 | E3927 Fig. 8. Divergent effects of HIV-1 and HIV-2 on HLTF and UNG2 levels. (A and B) HIV-1 infection depletes HLTF and UNG2 levels. (A) Jurkat T cells were infected with + + − − HIV-1 VLP transcomplemented (Vpr) or not (−) with HIV-1 Vpr (HIV-1 VLP; Left) or a single-cycle HIV-1 NL4-3.GFP.R (vpr ), or R (vpr ) virus (HIV-1; Right). The levels of HLTF, UNG2, and α-tubulin loading control in extracts prepared from infected and control (marked “c”) cells 48 h post infection were revealed by immunoblotting. + – (B) HIV-1 VLP and HIV-1 NL4-3.GFP.R and R viruses used in A were normalized by immunoblotting for p24 capsid. Vpr was revealed with an α-HA antibody (HIV-1 + + VLP) or α-Vpr antibody (HIV-1). (C) HIV-1 depletes HLTF and MUS81 in primary CD4 T cells. CD4 T cells were activated with α-CD3/α-CD28 beads and transduced with HIV-1 NL4-3.GFP.R+ (or R–), and GFP-positive cells were isolated by cell sorting 2 d after infection and analyzed for expression of the indicated proteins by immu- noblotting. Twofold serial dilutions of control CD4+ T whole-cell lysates provided quantification standards. (D and E) Divergent effects of HIV-1 and HIV-2 on HLTF and + UNG2 levels. Jurkat T cells were infected with HIV-1 NL4-3.GFP.R (HIV-1) or HIV-2 ROD with intact vpr and vpx genes expressing an RFP marker. Two days after infection, reporter was revealed by FACS. The abscissa is GFP/RFP fluorescence shown on a logarithmic scale. The ordinate is forward scatter shown on a linear scale (D), and UNG2, SAMHD1, HLTF, and Lamin B loading control in cell extracts were visualized by immunoblotting (E). (F and G) THP-1 cells were infected + − with HIV-1 NL4-3.GFP.R [or R (−)] or HIV-2 ROD expressing Vpr and Vpx (vpr/vpx) or only Vpr [vpr/(−) or not infected (marked as “c”)] (F), or with SIVmac251 VLP or SIVmac239 (G) as indicated, and extracts were analyzed by immunoblotting. Asterisk indicates a nonspecific band revealed by the α-SAMHD1 antibody. effects on UNG2, HLTF, and MUS81 most likely reflect three in- also have different consequences for the fidelity of HIV-1 provirus dependently selected functions of HIV-1 Vpr. repair, which provides another tentative rationale for the removal of Viral accessory virulence factors usually redirect cellular E3 HLTF by HIV-1 Vpr. ubiquitin ligases to remove cellular proteins that exhibit direct or Notably, Vpr also disrupts one of the Holliday junctions indirect antiviral activity (50). Hence, the concerted removal of resolvases by depleting the MUS81 component of SLX1-SLX4/ several postreplication DNA repair enzymes by Vpr is consistent MUS81-EME1 (2) complex. This interaction was proposed to be with the notion that host cell DNA repair machinery restricts associated with untimely activation of MUS81-EME1 (2) nucle- steps in HIV-1 replication in host cells. Indeed, the key role for a ase activity to prevent an innate response to products of abortive dUTP-mediated antiretroviral host defense pathway is evident HIV-1 reverse transcription (26). Somewhat unexpectedly, our data from the fact that many retrovirus families captured a host show that depletion of MUS81 levels does not alleviate the ability of dUTPase gene during viral evolution (51, 52). Although HIV-1 Vpr to arrest cells in G2. Moreover, MUS81 and HLTF depletion, does not encode dUTPase, its Vpr protein effectively prevents although each insufficient on its own, together appear to facilitate UNG2-initiated uracil excision repair (31). This alternative mech- G2 arrest, consistent with the previously reported roles of these anism may allow HIV-1 to alleviative negative consequences of proteins in replication fork maintenance and restart, and with re- UNG2-mediated uracil excision and downstream pathways for the cent genetic studies of the Vpr–MUS81 interaction (45, 57, 58). integrity of HIV-1 cDNA (19). Thus, it appears that HIV-1 Vpr must perturb additional pathways The finding that HIV-1/SIVcpz Vpr specifically removes HLTF to trigger DNA damage checkpoint leading to G2 arrest. from infected cells suggests that it negatively impacts HIV-1 repli- The findings described here provide compelling evidence that cation. Possible scenarios are suggested by known HLTF functions. HIV-1 Vpr disrupts select DNA repair pathways via CRL4DCAF1 In particular, HLTF remodels replication forks that are stalled by E3 ubiquitin ligase. This in turn prompts speculation that Vpr DNA lesions into four-way branched DNA structures, thereby acts to delay the repair until after integration of the HIV-1 provirus providing an undamaged DNA template that allows for error-free into the host cell , an environment in which repair is bypass of the lesion and resumption of DNA replication. In- conventionally handled by alternative postreplication DNA repair terestingly, fork-like branched DNA structures resembling replica- pathways. Alternatively, Vpr may block the repair through these tion forks are transient intermediates in plus-strand displacement pathways altogether and thereby evade DNA repair-mediated re- synthesis that is carried out by retroviral reverse transcriptase (53, striction. Such scenarios, as well as the timing of the action of 54). Recognition and processing of such intermediates by HLTF HLTF, UNG2, and other aspects of DNA repair impacted by Vpr, could impede an ordered synthesis of the cDNA copy of the HIV-1 will be assessed in future studies. RNA genome. Although integration competent HIV-1 preintegra- Our comparative studies of the two main types of HIV un- tion complexes form in the cytoplasm, the completion of reverse expectedly revealed that HIV-1/SIVcpz Vpr proteins potently transcription does not appear to beaprerequisiteforHIV-1entry deplete HLTF and UNG2, whereas HIV-2 and their ancestral to the nucleus (55), where partially reverse-transcribed viral cDNA HIV-2sm lineage Vpr do not possess such robust abilities. Sig- can be accessed by DNA repair proteins. Alternatively, HLTF could nificantly, HIV-2/HIVsm viruses antagonize SAMHD1 dNTPase modulate processing of the integrated proviral DNA, as it is known via their Vpx proteins (6, 7) whereas HIV-1/SIVcpz do not to control the choice between two pathways of postreplication re- possess this ability. We propose that HIV-2 does not need to pair of DNA lesions at the replication fork: error-free, by template antagonize cellular DNA repair mechanisms in a manner com- switching, and error-prone, through recruitment of mutagenic parable to that seen with HIV-1, because it effectively counter- translesion DNA polymerases (45, 56). As these two distinct modes acts SAMHD1, which leads to an increase in cellular dNTP of DNA repair, error-free and error-prone, have different muta- concentrations. Through this mechanism, HIV-2 avoids mark- genic outcomes upon repair of chromosomal DNA, they would ing its cDNA for processing by DNA repair enzymes and the

E3928 | www.pnas.org/cgi/doi/10.1073/pnas.1605023113 Hrecka et al. resulting repair-mediated restriction and/or provides sufficient Transfections, Immunoprecipitations, and DDB1-DCAF1 Recruitment Assay. PNAS PLUS dNTP supply to support faithful repair in nondividing G1 cells, Transfections of HEK 293T cells and immunoprecipitations were performed unlike HIV-1. Regardless, the evidence presented here implies as described previously (34, 63). For HLTF recruitment assays, HEK 293T cells, × 7 that distinct lineages of primate lentiviruses use different strat- at 2 10 cells in four 10-cm plates per condition, were cotransfected with pCG plasmids expressing FLAG-tagged HLTF and appropriate HA-tagged egies to manage their genomes, protecting them from the ac- HIV-1 Vpr proteins in combinations. Whole-cell extracts were immunopre- quisition of DNA damage and/or from processing by DNA repair cipitated with FLAG-M2 beads (Sigma-Aldrich), and immune complexes were enzymes when replicating in dNTP-poor cellular environments. eluted by competition with FLAG-peptide under native conditions. HIV-1 Vpr The emergence of SIVcpz is thought to have involved re- immune complexes for MudPIT analyses were purified as we described combination between two SIV lineages from Old World Monkey previously (6, 63). (OWM) species (59), resulting in a deletion of the vpx gene and the ensuing loss of SAMHD1 antagonism by Vpx. SAMHD1 Cell Synchronization and Cell Cycle Analyses. U2OS-iH1.Vpr cells were syn- antagonism by Vpx is well-conserved in distinct SIV strains chronized in early S phase by double thymidine block. To reveal cell-cycle 5 isolated from OWM (11, 60), indicating the importance of profiles, aliquots of 1 × 10 cells were pulse-labeled with 5-ethynyl-2′- μ SAMHD1-exerted restriction on replication of primate lentivi- deoxyuridine (EdU; 10 M) for 60 min, and the incorporated EdU was de- ruses, and thereby raising the question of how the loss of tected by using a Click-iT Plus EdU Alexa Fluor 647 or Alexa Fluor 488 Flow Cytometry Assay Kit (Life Technologies). Cells were then stained with 2 SAMHD1 counteraction was compensated for in the HIV-1/ μg/mL 7AAD (Life Technologies) to reveal their DNA content, and analyzed SIVcpz lineage. One such mechanism was suggested by the with an LSRFortessa flow cytometer (BD Biosciences) and FlowJo software. A previous finding that cross-species transmission of SIV to total of 10,000 events were collected for each sample. Statistical analyses of chimpanzees was associated with an expansion of the range of cell cycle profiles based on quantification of cell populations in G1, S, ABOBEC3 proteins targeted by Vif (48). Our studies reveal that and G2/M phases in FlowJo were performed in GraphPad Prism software, the loss of Vpx in the HIV-1/SIVcpz lineage was also associated with P values calculated by unpaired two-tailed Student’s t test with with the acquisition/enhancement of an ability to disrupt DNA Welch’s correction. repair by HIV-1 Vpr via HLTF and UNG2, and suggest that + + the latter was to compensate for the inability to antagonize Isolation of Primary HIV-1–Infected CD4 T Cells by Cell Sorting. CD4 T cells obtained from human peripheral blood mononuclear cells by negative se- SAMHD1. This possibility makes sense mechanistically, as re- + lection using EasySep hCD4 T Cell Enrichment Kit (Stemcell Technologies) verse transcription, taking place in the dNTP-poor environment 6 were plated in 96-well plates at 1 × 10 cells per well and activated with of nondividing target cells, leads to incorporation of uracil and Dynabeads Human T-Activator CD3/CD28 (Invitrogen) in the presence of IL-2 possibly other marks of damage into viral cDNA, thereby flag- (30 U/mL) in full RPMI 1640 medium [supplemented with 10% (vol/vol) heat- ging it for processing and restriction by UNG2 and DNA inactivated FBS and antibiotics (63)]. Two days later, the cells were infected + − repair enzymes. with HIV-1 NL4-3.GFP.R or NL4-3.GFP.R virus and transferred into wells of a 24-well plate in a final volume of 500 μL per well of full RPMI 1640 medium Materials and Methods with IL-2 (30 U/mL). Two days post infection, cells were pooled and resus- 6 Cell Lines. HEK293T, U2OS, and U2OS.HLTF.KO cells (45) were maintained in pended in PBS + 1% BSA at 8 × 10 /mL. Dynabeads were removed and live DMEM (Life Technologies) supplemented with 10% (vol/vol) FBS, 2 mM GFP-positive cells were isolated by sorting on a FACSAria. Whole-cell lysates

L-glutamine, and penicillin/streptomycin in 5% (vol/vol) CO2 at 37 °C. CEM.SS were prepared from sorted and from uninfected control cells.

(61) (NIH AIDS Reagent Program), Jurkat, and THP-1 cells were maintained MICROBIOLOGY in RPMI 1640 medium supplemented as described earlier. CEM.SS-iH1.Vpr, Isolation of G1, S, and G2M Cells by Sorting. Cells (8 × 106) were stained with μ U2OS-iH1.Vpr, U2OS-iH1V.Vpr(E24R,R36P), and U2OS.HLTF.KO-iH1.Vpr cells 30 M Vybrant DyeCycle Green stain (Life Technologies), and G1, S, and G2/M harboring doxycycline-inducible HIV-1 NL4-3 vpr or other variant vpr populations (∼1 × 106 cell each) were sorted based on DNA content on a transgenes were engineered by using lentiviral Tet-On 3G Inducible Ex- FACSAria. Aliquots (105 cells) of the purified populations were reanalyzed by pression System (Clontech) and maintained in the aforementioned media FACS to assess their purity. The cells were lysed (62, 64), and cytoplasmic or supplemented with G418 (200 μg/mL) and puromycin (2 μg/mL). Ex- chromatin extracts were analyzed by immunoblotting. Detailed experi- pression was induced by addition of doxycycline (Sigma-Aldrich) to the mental procedures are described in SI Materials and Methods. culture medium. ACKNOWLEDGMENTS. We thank Jinwoo Ahn, Michael Emerman, Nicolas Immunoblotting and Antibodies. Typically, whole-cell (34), cytoplasmic, or chro- Manel, and Karlene Cimprich for sharing reagents; Chuanping Wang for matin extracts (62) were separated by SDS/PAGE and transferred to PVDF mem- technical assistance; Karlene Cimprich for stimulating discussions; Teresa Brose- branes for immunoblotting. Proteins were detected with appropriate primary nitsch for critical reading of the manuscript and editorial help; Angela Gronen- born for support; and Tomek Swigut for his help with statistical analyses. CEM.SS antibodies and immune complexes revealed with HRP-conjugated antibodies cells were obtained through the NIH AIDS Reagent Program, Division of AIDS, specific for the Fc fragment of mouse or rabbit IgG (Jackson ImmunoResearch National Institute of Allergy and Infectious Diseases, NIH, from Dr. Peter L. Nara. Laboratories) and enhanced chemiluminescence (GE Healthcare), or with fluo- This work was supported by NIH Grants AI077459 and AI100673 and a rescent antibodies to mouse or rabbit IgG (KPL) and Odyssey Infrared Imager P50GM082251 subcontract (to J.S.). The Flow Cytometry Facility at Case Western (Licor). SI Materials and Methods includes the list of antibodies used. Reserve University is supported by Center for AIDS Research Grant P30 AI036219.

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E3930 | www.pnas.org/cgi/doi/10.1073/pnas.1605023113 Hrecka et al. Supporting Information

Hrecka et al. 10.1073/pnas.1605023113 SI Materials and Methods sorted based on DNA content on a FACSAria. Aliquots (105 cells) Plasmid Expression Vectors and Synthetic Genes. pCG plasmids ex- of the purified populations were reanalyzed by FACS to assess their pressing epitope-tagged Vpr proteins UNG2, DDB1, and DCAF1 purity. The cells were lysed (62, 64), and cytoplasmic or chromatin were previously described (34). The human HLTF protein coding extracts were analyzed by immunoblotting. sequence was amplified by PCR from an EST clone (MHS6278- 202759242; Open Biosystems) and subclonedintopCGandretro- List of Antibodies Used for Immunoblotting. α-HLTF (A300-230A), viral MSCV(puro) vector encoding N-terminal myc-, HA-, or α-AKAP8 (A301-062A), α-ESCO2 (A301-689A), α-PWP1 (A303- FLAG-epitope tags. Vpr and HLTF deletion and point mutants 635A), α-RMBX2 (A302-222A), α-RuvBL2 (A302-536A-T), α-PRP19 were constructed by using QuikChange II mutagenesis kit, and (A300-101A-T), α-NONO (A300-587A-T), α-TRIP13 (A300-607A-T), mutations were confirmed by DNA sequencing. HIV-1 group M, N, and α-VCP (A300-588A-T) were from Bethyl Laboratories; α-TIF1β O, SIVcpzPtt, SIVcpzPts, and HIV-2 group A1, A2, and B consensus (4124), α-HP1α (2616), α-KU80 (2180), α-RAD18 (9040), α-MSH6 vpr genes were designed based on sequence information available at (5424), and PCNA (2586S) were from Cell Signaling; α-MUS81 (sc- Los Alamos National Laboratory databases (www..lanl.gov). 53382, sc-376661), α-TFIID (sc-204), α-DCAF1 (sc-376850), α-Cyclin Codon-optimized genes were synthesized by Genscript and Life A2 (sc-751), α-SHPRH (sc-69324), and α-tubulin (sc-5286) were from Sciences. Santa Cruz Biotechnology; α-tubulin (T5168) and α-Flag (M2) α + were from Sigma-Aldrich; -DDB1 (37-6200) was from Invitrogen; Lentiviral Expression Vectors and Viruses. HIV-1 NL4-3.GFP.R α α − -ZC3HAV1 (PA5-20986) was from Thermo Scientific; -Lamin and NL4-3.GFP.R proviral clones were constructed by re- + B1 (ab16048) was from Abcam; and α-UNG2 (TA503563) was placing the luciferase gene with GFP gene in pNL4-3.Luc.R − from OriGene. Concentrated α-HA [12CA5 (67)], α-Myc [9E10 and R constructs (65). HIV-1taqRFP (vif-, vpr-, nef-, env-) (68)], and α-HIV-1 CA [183-H12-5C (69)] were produced from proviral construct was provided by Nicolas Manel, Institut hybridomas by using CELLine Bioreactor flasks (Argos). The Curie, Paris. The psi packaging signal in HIV-1tagRFP was HIV-1 p24 Hybridoma 183-H12-5C was obtained through the inactivated by deleting the sequence “TACGCCAAAAATT- NIH AIDS Reagent Program, Division of AIDS, National Institute TTGACTAGCGGAGGCTAGAAGGAGAG” [HIV-1(psi−)]. of Allergy and Infectious Diseases, NIH, from Bruce Chesebro. HIV-1 VLP was produced from HEK293T cells transiently co- – transfected with HIV-1(psi ), VSV-G expression vector, and/or Immunoaffinity Purifications of Protein Complexes and MudPIT Analyses. pCG plasmid expressing HA-tagged NL4-3 Vpr. HIV-2.RFP (env-, CEM.SS-iH1.Vpr cells expressing a tandem HA-FLAG-epitope– Rod9) proviral clone with tagRFP reporter residing in nef was tagged HIV-1 NL4-3 Vpr (hfa-H.Vpr) under control of the Lenti-X provided by Michael Emerman, Fred Hutchinson Cancer Research Tet-On 3G Inducible Expression System (Clontech) and control Center, Seattle, and Nicolas Manel. HIV-2 vpr gene was inactivated CEM.SS cells (3 × 105 cells per milliliter; 4 L) were induced with by M1E,E3* mutation and vpx by M1T substitution, using standard μ molecular biology techniques. SIVmac251 VLP was produced from doxycycline (0.1 g/mL) for 9 h. Whole-cell extracts were prepared, + and tandem affinity purifications of Vpr and its associated proteins, pSIV3 construct possessing intact vpr and vpx genes (66). Virus ∼ particles were produced and concentrated as described previously from 5 g of wet cell mass, were performed as described previously (6, 63). Virus preparations were normalized by immunoblotting for (6). MudPIT analyses of purified protein complexes and calculation p24/p27 CA and/or Vpr, followed by quantification of fluorescent of distributed normalized spectral abundance factors were signals relative to a recombinant HIV-1 CA standard, using Odyssey previously described (70). imaging. Retroviral MSCV-based expression vectors were produced × 4 from HEK 293T cells (34, 64). For inducible expression, cDNAs RNAi. U2OS cells were plated in 12-well plates (2 10 cells per encoding various epitope-tagged Vpr proteins were subcloned into well; Becton Dickinson) in antibiotic-free DMEM supplemented pLVX-TRE3G vector (Lenti-X Tet-On 3G Inducible Expression with 10% (vol/vol) FBS. One day later, cells were transfected with – System; Clontech), and viruses were produced as described pre- siRNA to a 10 25 nM final concentration in 1 mL per well, using viously (34). Dharmafect (Invitrogen). siRNAs targeting MUS81 (AUGGU- CACCACUUCUUAAC, CAGCCCUGGUGGAUCGAUA) (26, Isolation of G1, S, and G2M Cells by Sorting. Cells (8 × 106) were 71), HLTF (L-006448-00-005), and control nontargeting siRNA stainedwith30μM Vybrant DyeCycle Green stain (Life Technol- pools “NT1” (D-001206-14-05) and “NT2” (D-001810-10), were ogies), and G1, S, and G2/M populations (∼1 × 106 cells each) were purchased from Dharmacon.

Hrecka et al. www.pnas.org/cgi/content/short/1605023113 1of5 Fig. S1. Search for proteins down-modulated by Vpr among Vpr-associated DNA repair proteins. (A) Cell-cycle profiles of CEM.SS-iH1.iVpr and U2OS-iH1.iVpr cells treated with doxycycline for 24 h or control cells cultured with etoposide (0.4 μM, 18 h) or nocodazole (100 ng/mL, 18 h). Percentage fractions of cells in G1, S, and G2/M phases are indicated in each panel. (B) Whole-cell extracts prepared from cells shown in A were immunoblotted with antibodies to the indicated proteins. Asynchronously dividing cells (indicated by “A”) were used as an additional control.

Hrecka et al. www.pnas.org/cgi/content/short/1605023113 2of5 Fig. S2. HLTF is expressed in HIV natural target cells. Whole-cell extracts prepared from the indicated human leukocyte populations were immunoblotted for HLTF, MUS81, and TFIID loading control.

Fig. S3. Vpr-mediated depletion of HLTF and MUS81 levels is independent of activation of DNA damage checkpoint responses. U2OS-iH1.Vpr and parental U2OS cells were cultured in the presence of doxycycline (100 ng/mL) and caffeine (0.75 mM, 1.5 mM) for 9 h or 24 h. Then, the cells were (A) stained with 7AAD and the cell cycle profiles were analyzed by FACS or (B) processed for analysis of HLTF, MUS81, Vpr, and TFIID loading control levels by immunoblotting.

Fig. S4. Transient dose-dependent assay for Vpr-induced DCAF1- and proteasome-dependent HLTF degradation. (A) FLAG-HLTF was transiently coexpressed with HIV-1 HA-Vpr in HEK 293T cells. Cells were cultured overnight in the presence or absence of MG132 (0.62 μM, 1.25 μM) or lactacystin (2.5 μM, 5 μM), proteasome inhibitors, and HLTF, Vpr, and α-tubulin loading control levels were revealed in whole-cell extracts by immunoblotting. (B) The ability of HIV-1 Vpr to deplete HLTF levels requires binding to DCAF1. The effects on HLTF levels of HIV-1 Vpr (NL4-3) and two variants, Vpr(H71R) or Vpr(W54R), that do not bind DCAF1 or do not degrade UNG2, respectively, were tested in transient coexpression assays.

Hrecka et al. www.pnas.org/cgi/content/short/1605023113 3of5 Fig. S5. RNAi-mediated depletion of MUS81 in U2OS and U2OS.HLTF.KO cells (relevant to Fig. 6). U2OS [HLTF(+/+)] and U2OS.HLTF.KO [HLTF(−/−)] cells were subjected to RNAi targeting MUS81, and whole-cell extracts analyzed by immunoblotting for the indicated proteins.

Fig. S6. Consensus HIV-2 Vpr and Vpx proteins are functional. (A) Consensus HIV-2 subtype A and B Vpr proteins bind the endogenous DDB1-DCAF1 module of CRL4DCAF1 E3 complex. Consensus HIV-2 HA-Vpr proteins were transiently expressed in HEK293T cells. HIV-1 NL4-3 Vpr and its H71R-substituted variant that does not bind DCAF1 were used as controls. Endogenous DCAF1, DDB1, and ectopic Vpr in α-HA-Vpr immune complexes and detergent extracts were revealed by immunoblotting. (B) Vpx proteins deplete SAMHD1 levels. HIV-2 HA-Vpr proteins were transiently coexpressed with SAMHD1 in HEK 293T cells. HIV-1 NL4-3 Vpr was expressed in parallel as a negative control. Cells were cultured overnight in the presence or absence of MG132 proteasome inhibitor (1 μg/mL), and SAMHD1, Vpr, Vpx and α-tubulin loading control levels were revealed in whole-cell extracts by immunoblotting.

Hrecka et al. www.pnas.org/cgi/content/short/1605023113 4of5 Table S1. Identification of HIV-1 Vpr-associated DNA repair proteins DNSAF

Gene MudPITα-Vpr 1 MudPITα-Vpr 2 Average MudPIT Mock Function/complex

HIV-1 Vpr 0.068256 0.132352 0.100304 ND DDB1 0.095255 0.057099 0.076177 ND CRL4.DCAF1 DDA1 0.083479 0.043864 0.063672 ND CRL4.DCAF1 DCAF1 0.060501 0.039376 0.049939 ND CRL4.DCAF1 CUL4A 0.001688 0.004629 0.003159 ND CRL4.DCAF1 CUL4B 0.002013 0.005168 0.00359 ND CRL4.DCAF1 ESCO2 0.001924 0.003542 0.002733 ND Cohesin acetylase TRIM28 0.00277 0.002081 0.002425 ND DNA damage, retrovirus silencing UNG 0.001567 0.001613 0.00159 ND BER MSH6 0.001520 0.001453 0.001487 ND MMR PCNA 0.000537 0.001831 0.001184 ND DNA replication and repair ANKRD17 0.001386 0.000902 0.001144 ND DNA replication, innate immunity VCP 0.000435 0.001401 0.000918 ND DSB repair RPS27L 0.001669 0.000517 0.001093 ND p53 inducible, checkpoint signaling NONO 0.000521 0.000415 0.000468 ND DSB repair, NHEJ RUVBL2 0.000076 0.000844 0.00046 ND DNA helicase, HR RFC3 0.000804 0.000071 0.000438 ND DNA replication, repair MMS19 0.000340 0.000527 0.000434 ND DNA replication, repair PRKDC 0.000314 0.000395 0.000354 ND NHEJ RFC4 0.000579 0.00006 0.00032 ND DNA replication, repair TRIP13 0.000162 0.000452 0.000307 ND NHEJ HLTF 0.000347 0.000237 0.000292 ND Error-free DNA repair KIF22 0.000474 0.000016 0.000245 ND Motor, spindle, cohesion RFC2 0.000198 0.000245 0.000222 ND DNA replication, repair RFC1 0.000336 0.000038 0.000187 ND DNA replication, repair RFC5 0.00022 0.000068 0.000144 ND DNA replication, repair PRPF19 0.000139 0.000086 0.000113 ND ATR, ATRIP recruitment

MudPIT identification of cellular proteins involved with DNA replication and/or repair that specifically copurify with HIV-1 Vpr from CEM.SS-iH1.Vpr cells. Protein complexes were purified by two sequential immunoprecipitations via HA-tag and then via FLAG-tag, each followed by competitive elution with peptide epitope and analyzed by MudPIT. Upper rows indicate dNSAF values for DDB1, DDA1, DCAF1, Cul4A, and Cul4B subunits of CRL4DCAF1 E3 complex. Lower rows (from ESCO2 down) indicate dNSAF values for DNA replica- tion/repair proteins that copurified with HIV-1 Vpr in two independent experiments (α-Vpr 1 and 2) and their means. None of these Vpr-associated proteins was detected in control immunopurifications from CEM.SS cells that did not express Vpr (Mock). ATR, ataxia telangiectasia related; ATRIP, ataxia telangiectasia-related interacting protein; BER, base excision repair; dNSAF, distributed normal- ized spectral abundance factor; DSB, double-stranded break; HR, homologous recombination; MMR, mismatch repair; ND, not de- tected; NHEJ, nonhomologous end-joining.

Hrecka et al. www.pnas.org/cgi/content/short/1605023113 5of5