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Home , APOBEC, APOBEC3A, Cytidine deaminase, HDAC1

Apobec3A maintains HIV-1 latency through recruitment of epigenetic silencing machinery to the long terminal repeat

Manabu Tauraa, Eric Songa, Ya-Chi Hob, and Akiko Iwasakia,c,1

aDepartment of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; bDepartment of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06520; and cHoward Hughes Medical Institute, Chevy Chase, MD 20815

Contributed by Akiko Iwasaki, December 13, 2018 (sent for review November 12, 2018; reviewed by Jeremy Luban and Daniel B. Stetson) HIV-1 integrates into the genome of target cells and establishes Results and Discussion latency indefinitely. Understanding the molecular mechanism of Apobec3A Suppresses HIV-1 Reactivation in Latently Infected Cell HIV-1 latency maintenance is needed for therapeutic strategies to Lines. To examine the effect of A3A on HIV-1 latency, we be- combat existing infection. In this study, we found an unexpected gan with siRNA knockdown and overexpression of A3A in HIV- role for Apobec3A ( MRNA editing catalytic 1 latently infected cell line J-Lat10.6. J-Lat10.6 cells are a Jurkat subunit 3A, abbreviated “A3A”) in maintaining the latency state cell line with a stably integrated HIV-1HXB2Δenv backbone that within HIV-1–infected cells. Overexpression of A3A in latently infected contains a GFP reporter in place of the nef gene. HIV- cell lines led to lower reactivation, while knockdown or knockout of 1 reactivation can be monitored through GFP expression and A3A led to increased spontaneous and inducible HIV-1 reactivation. HIV-1 mRNA and replication-defective viral particle release in A3A maintains HIV-1 latency by associating with proviral DNA at the the culture medium (28). We found that knockdown of A3A led ′ 5 long terminal repeat region, recruiting KAP1 and HP1, and impos- to spontaneous reactivation of HIV-1 in J-Lat10.6 latently infected ing repressive histone marks. We show that knockdown of A3A in cells (SI Appendix, Fig. S1 A and B). Overexpression of A3A latently infected human primary CD4 T cells enhanced HIV-1 suppressed phorbol myristate acetate (PMA)-induced HIV-1 reactivation. Collectively, we provide evidence and a mechanism SI Appendix D E

reactivation ( ,Fig.S1 and ). To confirm the role MICROBIOLOGY by which A3A reinforces HIV-1 latency in infected CD4 T cells. of A3A in the maintenance of HIV-1 latency, we generated A3A knockout J-Lat10.6 cells using CRISPR (Fig. 1A and SI Appendix, epigenetic regulation | TRIM28 | reactivation | viral latency | T cells Figs. S2 and S3 A–C). Genetic deletion of A3A resulted in in- creased spontaneous HIV-1 reactivation, measured at both the espite combination antiretroviral therapy (ART), HIV-1 mRNA and levels, compared with control guide RNA Dpersists in infected individuals as a latent and integrated (gRNA) CRISPR-transduced cells in both a knockout cell clone provirus (1–3). In resting CD4 T cells, the lack of active cellular and in three separate pools of A3A knockout cells (Fig. 1 B and C factors (4–9) and of HIV-1 Tat and its cellular and SI Appendix,Fig.S2A–D). PMA-induced HIV-1 reactivation cofactors (10–15) limits the initiation and elongation of viral was also enhanced in A3A knockout cells in a dose- and time- transcription, respectively (16, 17). Understanding the mecha- dependent manner (Fig. 1 D–F and SI Appendix,Fig.S3D–F). nism of latency maintenance is key to therapeutic approaches Likewise, pooled A3A knockdown in a separate J-Lat line, J-Lat6.3, aimed at a cure. Cellular restriction factors block various stages of the HIV-1 life cycle, including viral entry, reverse transcrip- Significance tion, nuclear transport, and virion release (18). However, host factors that maintain HIV-1 latency are less well understood. Human immunodeficiency virus 1 (HIV-1) infection causes a life- Apobec3A (apolipoprotein B MRNA editing enzyme catalytic long disease, due to the ability of the virus to integrate into the subunit 3A, abbreviated “A3A”) is one of the restriction factors host genome and establish latent infection. While research has that suppress HIV-1 primary infection in macrophages by its revealed a number of host restriction factors that block primary deaminase activity (19). A3A also suppresses other infection, much less is understood with regard to the host viruses, including hepatitis B virus, human papillomavirus, factors that promote or block reactivation of the integrated Epstein–Barr virus, cytomegalovirus, and herpes simplex virus proviral HIV-1. In this study, we show that a member of the type 1, by inducing the lethal mutation in viral genomic DNA by – Apobec3A (apolipoprotein B MRNA editing enzyme catalytic cytidine (20 23). Interestingly, although A3A has subunit 3A) family, A3A, suppresses HIV-1 reactivation by been reported to be a potent inhibitor of retroelements such as recruiting chromatin-modifying to impose repressive LINE-1, long terminal repeat (LTR), and Alu , marks around the long terminal repeat region. its inhibition mechanism is cytidine deamination-independent – Identification of host factors that control HIV-1 latency may and has not yet been elucidated (24 27). provide clues for therapeutic interventions needed to remove To understand the role of A3A in HIV-1 latency, we examined the viral reservoir from the infected individual. the effect of A3A overexpression or knockdown on HIV-1 reactivation. We found that A3A reinforced HIV-1 latency in Author contributions: M.T., E.S., Y.-C.H., and A.I. designed research; M.T. and E.S. per- HIV-1 latently infected cell lines. A3A suppressed HIV-1 formed research; M.T., E.S., and Y.-C.H. contributed new reagents/analytic tools; M.T., proviral transcription by binding to the LTR region and sub- E.S., and A.I. analyzed data; and M.T. and A.I. wrote the paper. sequently recruiting repressive complexes including KRAB- Reviewers: J.L., University of Massachusetts Medical School; and D.B.S., University associated protein 1 (KAP1) and heterochromatin protein 1 of Washington. (HP1), thereby inducing silencing through histone H3K9 meth- The authors declare no conflict of interest. ylation. We further demonstrate that A3A knockdown enhances Published under the PNAS license. HIV-1 reactivation in latently infected primary human CD4 1To whom correspondence should be addressed. Email: [email protected]. T cells. These results reveal an unexpected role of A3A in pre- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. venting reactivation of HIV-1 and promoting the maintenance of 1073/pnas.1819386116/-/DCSupplemental. the latency state.

www.pnas.org/cgi/doi/10.1073/pnas.1819386116 PNAS Latest Articles | 1of8 Downloaded by guest on September 30, 2021 A B C p24 ELISA Tat-Rev/18s A3A 23kDa 10 800 *** 700 8 *** p84 84kDa 600 Crispr-controlCrispr-A3 500 6 400 4 300 200 2 A 100 0 0 Crispr-controlWT Crispr-A3ACrispr-A3A Crispr-controlWT Crispr-A3 /ml)

A (pg HIV-1 p24 production HIV-1 Relative Tat-Rev mRNA Relative Tat-Rev

D p24 ELISA E Tat-Rev/18s 70,000 Crispr-control *** 2500 Crispr-control *** 60,000 Crispr-A3A Crispr-A3A *** 2000 50,000 *** 40,000 1500

production 30,000 *** 1000 24 20,000 p

1 500 10,000 n.s. (pg/ml) Relative Tat-Rev mRNA Relative Tat-Rev HIV- 0 0 0 1 10 100 (nM) 0 1 10 100 (nM) PMA PMA

PMA F control 1 10 100 (nM)

Crispr- 0.56 8.42 63.1 63.8 control

Crispr- 3.40 29.5 86.2 88.1 A3A SSC-A HIV-1-GFP

Fig. 1. Apobec3A regulates spontaneous HIV-1 reactivation. (A) A3A protein expression between CRISPR-control–transduced J-Lat10.6 (Crispr-control) and CRISPR-mediated A3A knockout J-Lat10.6 clones (Crispr-A3A). (B and C) HIV-1 p24 expression in culture medium and HIV-1 Tat-Rev mRNA expression between Crispr-control and Crispr-A3A J-Lat10.6 cell clones. Data are means ± SE (n = 3). ***P < 0.001 versus Crispr-control assessed by Student’s t test. (D–F) Crispr- control or Crispr-A3A J-Lat10.6 cell clones were treated with the indicated concentration of PMA for 6 or 18 h. HIV-1 reactivation was analyzed by p24 expression in culture medium at 18 h (D), HIV-1 Tat-Rev mRNA at 6 h (E), and flow cytometry at 18 h (F). Data are means ± SE (n = 3). ***P < 0.001 versus nontreated sample assessed by ANOVA with Tukey–Kramer’s test. n.s., not significant.

showed enhanced PMA-induced HIV-1 reactivation (SI Appendix, albeit slightly less effectively than wild-type (WT) A3A (Fig. Fig. S2 E and F). These results demonstrated that A3A suppresses 2A and SI Appendix,Fig.S4A and B). Further, PMA-induced HIV-1 in latently infected cell lines. HIV-1 reactivation was also suppressed by ectopic expression of A3A C106S, again slightly less effectively than WT A3A (SI Activity Is Not Required for HIV-1 Latency Enforcement Appendix,Fig.S4D). Thus, these data suggested that A3A sup- by A3A. Next, we examined the requirement of cytidine deaminase presses HIV-1 gene expression in largely a cytidine deaminase- activity of A3A in HIV-1 latency control. Some members of the independent manner. Apobec3 family of cytidine deaminases, such as A3G, exert their inhibitory effects on primary HIV-1 infection through incorporation A3A Binds to the NF-κB/Sp1–Binding Region of the HIV-1 LTR. To into virions, release into the cytosol, and deamination of newly syn- probe the mechanism by which A3A enforces latency, we ex- thesized viral cDNA in the target cells, thereby introducing lethal amined whether A3A binds the integrated HIV-1 genome. A3A mutations into the HIV-1 genome (27). In contrast, A3A is excluded was found in both the nucleus and cytosol (Fig. 3 C and D and SI from being incorporated into virions (26), and acts within the nucleus Appendix, Fig. S4C), consistent with previous reports (25, 29). To of infected cells (19). In addition, A3A inhibition of adeno-associated analyze the binding of A3A on the HIV-1 genome, we conducted virus and LTR and Alu retrotransposons is mediated in a ChIP-qPCR using an anti-A3A and primer set for the deamination-independent manner (24–26). Our analysis LTR region and HIV-1–coding gene segments within J- showed that the cytidine deaminase mutant A3A C106S (26) Lat10.6 cells (Fig. 2B). An anti-A3A antibody in ChIP-qPCR lacked deaminase activity but suppressed HIV-1 LTR activity, was validated by immunoprecipitation and Western blotting

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1819386116 Taura et al. Downloaded by guest on September 30, 2021 A B 1.2 WT gag vif tat vpu nef 0 GFP 9.7kb 1.0 ‡‡‡ C106S LTR pol1/2 vpr rev env LTR ††† 0.8 ‡‡‡ HIV-1 genome ††† 8 0.6 *** n.s. *** Isotype-control *** ††† 6 anti-A3A 0.4 *** 0.2 4 Activity Relative Luciferase 0 con 0.05 0.1 0.2 (μg) 2 * % of input DNA pK-A3A 0 LTR gag pol1 pol2 vif env tat nef

C HIV-1 LTR 0.7 DNA pull-down *** E 2% 0.6 Isotype-control Input anti-A3A 12345 0.5 ssDNA 0.4 (sense) 0.3 *** ssDNA (anti-sense) 0.2 *

% of input DNA 0.1 dsDNA 0 con 0.5 1 2 (μg) pK-A3A 1 2 3 4 5 Probe + + + + + + + + + + + + + + + D Un-transfected lysate - + - - + - - + - - + - - + - A3A-transfected lysate - - + - - + - - + - - + - - +

297 U3/R region of HXB2 (230bp) 526 MICROBIOLOGY 9382 9611 297 346 9382 1 9431 342 391 9427 2 9476 387 436 3 9472 432 9521 481 κB, Sp1 9517 4 9566 477 526 9562 5 9611

Fig. 2. Apobec3A binds to HIV-1 LTR and suppresses its activity. (A) 293T cells were transfected with 0.2 μg of pGL2-HIV-1 5′ LTR plasmid and cotransfected with the indicated dose of A3A WT or C106S plasmids. Forty-eight hours after transfection, relative luciferase activity was determined by comparison with relevant controls. Data are means ± SE (n = 3). ***P < 0.001, †††P < 0.001 versus control, ‡‡‡P < 0.001 versus WT assessed by ANOVA with Tukey–Kramer’s test. n.s., not significant. (B) Endogenous A3A binding to the HIV-1 genome was analyzed by performing ChIP-qPCR in J-Lat10.6 cells. A3A binding to separate HIV- 1 regions such as LTR, gag, pol1, pol2, vif, env, tat, and nef were examined using specific primers. Data are means ± SE (n = 3). ***P < 0.001, *P < 0.05 versus isotype control IgG assessed by ANOVA with Tukey–Kramer’s test. (C) Tzm-Bl cells were transfected with pK-A3A, and A3A binding to the HIV-1 LTR was analyzed by ChIP-qPCR. Data are means ± SE (n = 3). ***P < 0.001, *P < 0.05 versus isotype control IgG assessed by ANOVA with Tukey–Kramer’s test. (D) Nuclear extract from pcDNA3.1 or pK-A3A overexpressed 293T cells was incubated with five different biotinylated oligo-. Protein–DNA bindings were analyzed by EMSA. (E) Recombinant A3A was incubated with single-stranded (sense or antisense) or double-stranded five different biotinylated oligo-DNAs and streptavidin-coupled Dynabeads. A3A expression was examined by Western blotting.

(SI Appendix, Fig. S9A). Endogenous A3A specifically bound the (Fig. 2E). These results indicate that A3A directly binds the sense LTR of the integrated HIV-1 genome in J-Lat10.6 cells (Fig. strand of the NF-κB– and Sp1-binding region of the HIV-1 LTR. 2B). Similarly, ectopically expressed A3A detected by anti-A3A antibody, as well as V5-tagged A3A (A3A-V5) detected by anti- N-Terminal Region of A3A Is Required to Recruit KAP1 and Impose V5 antibody, also associated with the 5′ LTR in a 5′ LTR/Luc– Repressive Histone Marks on HIV-1 LTR. We next probed the integrated Tzm-Bl cell line (Fig. 2C and SI Appendix, Fig. S5A) mechanism by which A3A represses HIV-1 transcription. DNA and inhibited PMA-induced and Tat-induced LTR activity (SI methylation and repressive histone modifications are associated Appendix, Fig. S5 B–G). Together, these results showed that with silencing of proviruses (30–35). KAP1 is a well-known A3A binds to the LTR region of the integrated HIV-1 genome in regulator of endogenous retroviral transcription (36), and is re- latently infected cells. quired for HIV-1 LTR repression (37). KAP1 engages histone To narrow in on the specific region within the LTR bound by modifiers such as HP1 and histone methyltransferase SETDB1 A3A, we conducted an electrophoretic mobility-shift assay to induce di- and trimethylation of H3K9, silencing gene ex- (EMSA) to determine whether in either nontransfected pression by heterochromatin formation (37). However, a study or A3A-transfected cell lysates bind to five distinct dsDNA oli- has also demonstrated a possible role of KAP1 for gonucleotides spanning 230 bp within the U3 and R regions of HIV-1 transcription (38). To determine the role of KAP1 in the LTR. We observed a significant shift of the no. 2 oligonu- latency reactivation, we generated KAP1 knockout cells (SI cleotide when cell lysates were added. The no. 2 oligonucleotide Appendix, Fig. S6A). Genetic deletion of KAP1 resulted in both spans the NF-κB and Sp1 binding sites within U3 (Fig. 2D). enhanced spontaneous and PMA-induced HIV-1 reactivation in Moreover, a DNA pull-down assay indicated that recombinant J-Lat10.6 cells (SI Appendix, Fig. S6B). Therefore, in our hands, A3A specifically bound to the no. 2 sense single-stranded but not as well as reported by others (39–44), KAP1 has a repressive role antisense single-stranded or double-stranded oligonucleotide in HIV-1 transcription.

Taura et al. PNAS Latest Articles | 3of8 Downloaded by guest on September 30, 2021 A B C A3A 10% Isotype anti- J-Lat 10.6 control -HA input control A3A CE NE KAP1 KAP1 A3A IP: HA IgG A3A HDAC1 A3A A3A KAP1 β-actin A3A Input GAPDH p84

D E HIV-1 LTR 0.30 Isotype control *** 1 70 106 199 a.a. 0.25 A3A-HA ZDD anti-A3A 0.20 anti-HP1 anti-KAP1 * 0.15 25kDa 0.10 * * * % of input DNA 0.05 20kDa HA 0 IP: KAP1 control A3A # 15kDa * F HIV-1 LTR 10kDa 1.0 Isotype control *** KAP1 0.8 anti-A3A anti-H3K9-2me 0.6 * anti-H3K9-3me 25kDa 0.4 * 0.2 20kDa % of input DNA * * HA 0 control A3A * 15kDa 10% * input HIV-1 LTR 10kDa G 10 KAP1 Isotype control 8 anti-A3A GAPDH anti-KAP1 6 anti-H3K9-3me con WT ZDD 107-1991-106 4

2 ** *** * * :A3A % of input DNA # : expected size A3A 0 Crispr- Crispr- control A3A

Fig. 3. Apobec3A recruits KAP1 and HP1 to the HIV-1 LTR and induces heterochromatin formation. (A) 293T cells were transfected with A3A-HA. Forty-eight hours after transfection, total cell lysates were analyzed for protein interaction between A3A-HA and KAP1 by anti-HA immunoprecipitation and anti- KAP1 immunoblotting. (B) J-Lat10.6 cell lysate was analyzed for endogenous A3A and KAP1 interaction by anti-A3A immunoprecipitation and anti- KAP1 immunoblotting. (C) Nuclear (NE) and cytosol (CE) fractions from J-Lat10.6 were analyzed for A3A expression by Western blotting. (D) 293T cells were transfected with HA-tagged A3A-WT or A3A deletion mutants. Cell lysates were immunoprecipitated using an anti-KAP1 Ab and immunoblotted using an anti-HA antibody. (E and F) KAP1 and HP1 binding (E) and H3K9-2me and H3K9-3me histone modification (F) were examined by ChIP-qPCR targeting the LTR of HIV-1 in A3A-transfected Tzm-Bl cells. Data are means ± SE (n = 3). ***P < 0.001, *P < 0.05 versus control plasmid assessed by Student’s t test. (G) A3A, KAP1 binding, and H3K9-3me histone modifications were compared between Crispr-control and Crispr-A3A J-Lat10.6 cell clones. Data are means ± SE (n = 3). ***P < 0.001, **P < 0.01, *P < 0.05 versus WT assessed by Student’s t test.

Based on these data, we hypothesized that A3A binds to the 3D). Thus, these results indicated that the A3A N-terminal do- LTR and recruits KAP1 to suppress HIV-1 transcription through main, but not the ZDD or the C-terminal domains, is required imposing epigenetic silencing. To test this, we examined whether for KAP1 binding. A3A interacts with KAP1. Both immunofluorescence microscopy Based on the ability of A3A to recruit KAP1 to the LTR, we and immunoprecipitation analysis showed that A3A expressed next analyzed whether A3A promotes H3K9 methylation of the ectopically (in 293T cells; Fig. 3 A–C) and endogenously (in J- LTR. A3A overexpression in Tzm-Bl cells enhanced not only Lat10.6 cells; SI Appendix, Fig. S6 C and D) colocalized and A3A recruitment to the LTR but also increased KAP1 and interacted with KAP1. To identify the protein domains important HP1 recruitment (Fig. 3E). Consequently, A3A overexpression for this interaction, we constructed A3A mutants that lack the led to an increase in histone H3K9 di- and trimethylation on the -dependent catalytic domain (ZDD) or C- or N-terminal do- 5′ LTR (Fig. 3F). Conversely, KAP1 recruitment and H3K9 mains (Fig. 3D). All mutant A3A proteins were expressed in trimethylation of the LTR was reduced in the A3A knockout transfected cells, albeit at varying degrees (Fig. 3D). Immuno- J-Lat10.6 cell line (Fig. 3G). These results are consistent with precipitation of A3A mutants showed that the N-terminal deletion the idea that A3A binds to the LTR region and promotes of A3A rendered the molecule incapable of binding to KAP1 (Fig. KAP1 and HP1 recruitment, thereby imposing histone H3K9

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1819386116 Taura et al. Downloaded by guest on September 30, 2021 methylation and heterochromatin formation to repress HIV-1 man CD4 T cells (26). To examine A3A protein expression, we transcriptioninlatentlyinfectedcells. conducted intracellular flow cytometry analysis. A3A protein was expressed in CD4 T cells, albeit at a lower level than in mono- A3A Inhibits HIV-1 Reactivation in Latently Infected Human Primary cytes (SI Appendix, Fig. S7). Western blotting of lysates from CD4 T Cells. To probe the importance of these observations in monocytes, naïve CD4 T cells, and anti-CD3/anti-CD28–stimu- primary human CD4 T cells, we isolated and expanded periph- lated CD4 T cells revealed similar A3A protein expression in eral blood CD4 T cells from six healthy donors and transduced these cells (SI Appendix, Fig. S8). Additionally, fractionated ly- them with CRISPR-A3A lentivirus and pseudotyped single- sates from CD4 T cells showed nuclear A3A protein expres- round infectious NL4-3/Luc HIV-1 according to previous stud- sion, and microscopy analysis showed colocalization of A3A ies (45, 46). After culturing with IL-2 and puromycin for selec- and KAP1 in the nucleus of primary CD4 T cells (Fig. 4A tion of transduced cells, CD4 T cells were activated by treatment and SI Appendix,Fig.S9A). We also detected expression of with anti-CD3/anti-CD28 for 72 h. A previous report A3A at the protein and mRNA levels in latently infected pri- indicates that A3A mRNA is not expressed highly in naïve hu- mary CD4 T cells (Fig. 4 C and D and SI Appendix, Fig. S9B).

A B

Primary CD4 T cells n 2.0 * CE NE 1.5 Donor no.

1 2 3 4 5 6 1 2 3 4 5 6 protei A3A 1.0

HDAC1 0.5

β-actin Relative A3A 0 Crispr- Crispr- control A3A E C D Single-round Replication-competent MICROBIOLOGY * 2.5 2.0 n.s. 2.0 n.s. 2.0 1.5 1.5 1.5 1.0 1.0 1.0 0.5 0.5 0.5

Relative mRNA A3A 0 0 0 Relative integrated HIV-1 Crispr- Crispr- HIV-1 Relative integrated Crispr- Crispr- Crispr- Crispr- control A3A control A3A control A3A

F Single-round G Replication-competent *** *** n.s. n.s. ** *** 100000 * *** 4000 * ***

80000 3000 60000 2000 40000 1000 20000 Relative luciferase activity Relative luciferase activity 0 0 control CD3/28 control CD3/28 control CD3/28 control CD3/28 Crispr-control Crispr-A3A Crispr-control Crispr-A3A

Fig. 4. Apobec3A reinforces HIV-1 latency in primary human CD4 T cells. (A) CD4 T cells from six healthy donor PBMCs were expanded by incubation with CD3/28-Dynabeads antibodyand IL-2 with anti-human IL-12, anti-human IL-4, and TGF-β. Nuclear (NE) and cytosol (CE) lysates were fractionated and analyzed for A3A protein expression. Fractionation efficiency was confirmed by HDAC1 and β-actin expression. (B) Relative A3A protein expression was calculated vs. GAPDH protein expression in SI Appendix, Fig. S9A using ImageJ (NIH) and compared between CRISPR-control– and CRISPR-A3A–transduced CD4 T cells. *P < 0.05 versus control assessed by Student’s t test. (C) A3A mRNA expression was examined by RT-qPCR. Data are means ± SE (n = 6). *P < 0.05 versus control assessed by Student’s t test. (D and E) CD4 T cells from six healthy donor PBMCs were expanded by CD3/28-Dynabeads antibody and IL-2 with anti-human IL- 12, anti-human IL-4, and TGF-β. Expanded CD4 T cells were spinoculated with pseudotyped NL4-3/luciferase and CRISPR-control or CRISPR-A3A lentivirus and cultured in the presence of puromycin. Integrated HIV-1 gene copy was analyzed by Alu-HIV PCR assay before reactivation (D). Significance between Crispr- control and Crispr-A3A was analyzed by Student’s t test. HIV-1 reactivation was analyzed by fold change of luciferase activity between control and CD3/28- Dynabeads antibody stimulation (E). n.s., not significant. (F and G) CD4 T cells from six healthy donors were expanded by CD3/28-Dynabeads antibody and IL- 2 with anti-human IL-12, anti-human IL-4, and TGF-β, and spinoculated with replication-competent NL4-3/luciferase and Crispr-control or Crispr-A3A lentivirus. NL4-3/luciferase–infected cells were treated with raltegravir and nelfinavir to induced latency, and CRISPR-transduced cells were selected by puromycin treatment. Integrated HIV-1 gene copy was analyzed by Alu-HIV PCR assay before reactivation (F). ***P < 0.001, **P < 0.01, *P < 0.05 versus Crispr-control was assessed by ANOVA with Tukey–Kramer’s test. n.s., not significant between Crispr-control and Crispr-A3A was analyzed by Student’s t test. Replication- competent HIV-1 reactivation by CD3/28-Dynabeads antibody stimulation was examined by luciferase activity (G). Data are means ± SE (n = 6). ***P < 0.001, *P < 0.05 versus CRISPR-control assessed by ANOVA with Tukey–Kramer’s test. n.s., not significant.

Taura et al. PNAS Latest Articles | 5of8 Downloaded by guest on September 30, 2021 CRISPR-mediated A3A knockdown assessed by Western blot- repressive histone marks by recruiting KAP1 and HP1 in a cy- ting and by RT-qPCR of CD4 T-cell lysate and total mRNA tidine deaminase-independent manner. These data highlight the before reactivation showed ∼50% reduction in protein and relevance of A3A in HIV-1 latency maintenance and reac- mRNA expression (Fig. 4 B and C). An integration assay before tivation, and provide a pathway that can be targeted for future reactivation indicated that A3A knockdown did not affect HIV- therapeutic strategies. 1 integration efficiency (Fig. 4D). However, A3A knockdown enhanced NL4-3/Luc–derived luciferase activity upon engage- Materials and Methods ment of CD3 and CD28 in primary CD4 T cells (Fig. 4F and SI Reagents and Antibodies. PMA (phorbol 12-myristate 13-acetate) and LPS Appendix, Fig. S9C). Additionally, we confirmed these results in (Salmonella enterica serotype typhimurium) were purchased from Sigma- a widely used model of HIV-1 latency in primary human CD4 Aldrich. UltraPure salmon sperm DNA (15632011) was from Thermo Fisher T cells by using replication-competent NL4-3/Luc HIV-1 Scientific. Glycogen (AB00670-00020) was from AmericanBio. Lenti-X con- followed by ART combination treatment (47, 48). Similar to centrator was from Clontech. RediJect D-Luciferin Ultra Bioluminescent Substrate was purchased from PerkinElmer. Dynabeads Protein G, Dynabeads the replication-defective HIV-1 latency model (Fig. 4 D–F and SI Appendix C Human T-Activator CD3/CD28 (CD3/28-Dynabeads), and Dynabeads M-280 ,Fig.S9 ), the replication-competent model of HIV-1 Streptavidin were purchased from Thermo Fisher Scientific. Recombinant latency imposed by ART also demonstrated enhanced reac- A3A (TP320995) was purchased from OriGene. Anti-A3A (ab38641; for West- tivation when A3A was knocked down by CRISPR without ern blotting, immunofluorescence, and flow cytometry), anti-KAP1 (ab22553), changes in integration efficiency (Fig. 4 E and G and SI Ap- anti-H3K9 trimethylation (ab8898), and anti-V5 (ab9116) antibodies and nor- pendix,Fig.S9D). These experiments showed, through the use mal rabbit IgG (ab172730) were from Abcam. Anti-H3K9 dimethylation of two separate models, that A3A reinforces HIV-1 latency in (CS200587), anti-HP1gamma (CS203221), anti-Histone H3 (CS207299), anti– primary human CD4 T cells. NF-κB p65 (CS204359), and normal mouse IgG (12-371) antibodies were from –β Our study shows that A3A enforces latency by preventing Millipore. Anti-HA (H3663) and anti -actin (A1978) were from Sigma-Aldrich. HIV-1 reactivation through the recruitment of com- Anti-GAPDH (GTX627408-01) and anti-p84 (GTX70220-01) were from GeneTex. Anti-A3A (AP20219a, sc-130688; for IP and ChIP) and anti-HDAC1 (sc-7872) plex KAP1 and HP1 to the LTR. A3A binds to KAP1, and antibodies were from Abgent and Santa Cruz Biotechnology, respectively. promotes heterochromatin formation at the HIV-1 LTR. A3A HRP-conjugated anti-rabbit (7074) and anti-mouse (7076) antibodies were knockdown enhances the reactivation of HIV-1 in HIV-1 latently from Cell Signaling Technology. Brilliant Violet 605 anti-CD3 (300460), Alexa infected primary CD4 T cells. Thus, our study reveals a pre- 700 anti-CD4 (300526), and FITC anti-CD14 (367116) antibodies were from viously unknown role of A3A in suppressing HIV-1 reactivation BioLegend. Anti-rabbit Alexa Fluor 594 antibody was from Thermo Fisher in latently infected cells, and provides a potential target for in- Scientific. Anti-mouse Cy3 (715-165-151) and anti-rabbit Cy5 (111-176-144) tervention for reversing latency. We show direct binding of A3A antibodies were from Jackson ImmunoResearch. Anti–HIV-1 Tat rabbit anti- to a specific region within the HIV-1 LTR proviral DNA, namely serum (705) was provided by B. Cullen through the AIDS Research and Ref- the NF-κB and Sp1 binding sites. A3A bound to the sense strand erence Reagent Program, Division of AIDS, NIAID, NIH. DNA, and imposed blockade of HIV-1 reactivation independent of its cytidine deamination activity. A3A suppresses several ret- Plasmids, Cell Lines, Viruses, and siRNAs. pcDNA3.1-A3A(WT) was provided by – J. Ahn, University of Pittsburgh School of Medicine, Pittsburgh. pk-A3A WT roelements independent of its cytidine deamination activity (24 and C106S plasmids were provided by J. Moran, University of Michigan 26). Thus, A3A may function as an epigenetic modifier to reg- Medical School, Ann Arbor, MI. pGL2-LTR-Luc WT, ΔκB, ΔSp1, and ulate retroelement transcription in general. replication-competent NL4-3/Luc plasmids were provided by W. Greene, Our results suggest that A3A binding to the HIV-1 LTR in the Gladstone Institute of Virology and Immunology, San Francisco. pcDNA3.1- host genome occurs upon opening of the double-stranded DNA. A3A-V5/His plasmid was constructed by cutting the A3A-coding region from There are many ssDNA-binding proteins that perform essential pcDNA3.1-A3A using Kpn1 and Xho1 and inserting into a pcDNA3.1-V5/His functions during telomere synthesis, transcription, DNA repli- vector. ΔZDD, Δ107–199, and Δ1–106 A3A plasmids were generated by using cation, recombination, and repair (49). Other examples of a Q5 Site-Directed Mutagenesis Kit (New England Biolabs). The toxicity of transcriptional regulators that bind to single-stranded DNA in- the A3A constructs was analyzed by LDH assay (SI Appendix, Fig. S11 A and B). The oligonucleotide primers used for mutagenesis are shown in SI Ap- clude FUSE-binding protein 1 (FUBP1) (50), runt-related pendix, Table S1. CRISPR-A3A (K0105505), CRISPR-KAP1 (K2465405), and transcription factor (RUNX)-1 and RUNX-3 (51), purine-rich α β CRISPR-control (K010) lentiviral plasmids were from Applied Biological Ma- negative regulatory and (52), and myelin gene expression terials. pCMVR8.74 was provided by D. Torono through Addgene. pNL4-3. factor-2 (53). Since most transcriptional regulators and epige- Luc.R−E−, pHEF-VSVG, pCEP4-Tat, J-Lat10.6 cells, J-Lat6.3 cells, and Tzm-Bl netic modifiers bind to dsDNA, future studies are needed to cells were provided by N. Landau, L. J. Chang,E.Verdin,T.Folks,J.C.Kappes, determine the mechanism by which A3A manages ssDNA X. Wu, and Tranzyme, respectively, through the AIDS Research and Reference binding to genomic DNA at the steady state. The obligatory Reagent Program, Division of AIDS, NIAID, NIH. pRL-TK (E2241) plasmids were binding of A3A to ssDNA suggests a need for initial transcrip- purchased from Promega. 293T and 293FT cells were purchased from ATCC and tion of the LTR to recruit A3A, followed by KAP1 recruitment Invitrogen, respectively. ON-TARGETplus Human SMARTpool si-A3A (L-017432- and repression of proviral expression. 00-0005) and negative-control siRNA (con-si; D-001810-01-20) were purchased from GE Dharmacon. A crystal structure analysis of a complex of A3A with ssDNA bound in the revealed the residues within the ZDD ChIP-qPCR. The ChIP-qPCR procedure was described previously (56, 57). that confer specificity toward CC/TC motifs (54). A3A binds to Briefly, protein–DNA bound in cells was cross-linked by 1% formaldehyde target sequences with polythymidine oligomers containing cyti- treatment for 5 min. The reaction was stopped by adding 0.125 M glycine for dine bases, 5′-TTTTTTTCTTTTTTT-3′, with the highest affinity 5 min. Cells were washed with PBS and lysed using a cell lysis buffer (50 mM (55). Of note, our DNA pull-down analysis showed the strongest Hepes, pH 7.4, 1 mM EDTA, 85 mM KCl, 10% glycerol, 0.5% Nonidet P-40, binding of A3A to the oligonucleotide that contains the supplemented with protease inhibitor mixture). After centrifugation (1,200 × g, ACTTTC motif (SI Appendix, Table S4). We speculate that A3A 4 °C, 5 min), the nuclear fraction of cells was suspended in nucleus lysis may have affinity toward polythymidine containing cytidine that buffer (50 mM Tris·HCl, pH 8.0, 2 mM EDTA, 150 mM NaCl, 5% glycerol, 1% is found in the HIV-1 LTR and possibly other genomic DNA Triton-X 100, 0.1% SDS, supplemented with protease inhibitor cocktail). The sequences. Whether other nuclear Apobec3 protein families nuclear suspension was sonicated using a Bioruptor (30-s sonic and 30-s cooling cycle 35 times) to recover a fragmented nucleosome. For immuno- possess specific sequence affinity and regulate gene expression precipitation of the nucleosome, 2 to 5 μg of anti-A3A, anti-KAP1, anti-HP1γ, remains to be determined. anti-H3K9 trimethylation, anti-H3K9 dimethylation, and anti-Histone In conclusion, our study demonstrates that human A3A is H3 antibodies and isotype control IgG was added and rotated overnight at expressed in the nucleus by CD4 T cells and controls transcrip- 4 °C. After incubation with Dynabeads Protein G under rotation for 1 h at tion of latent HIV-1. A3A binds directly to the LTR and imposes room temperature (RT), beads were washed with ChIP wash buffer 1 (20 mM

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1819386116 Taura et al. Downloaded by guest on September 30, 2021 + Tris·HCl, pH 8.0, 150 mM NaCl, 1% Triton-X 100, 0.1% SDS, 2 mM EDTA) twice, naïve human CD4 T-cell enrichment kit (17555; Stemcell Technologies). CD4 ChIP wash buffer 2 (20 mM Tris·HCl, pH 8.0, 500 mM NaCl, 1% Triton-X 100, T cells were cultured with anti-human IL-12 (200 μg/mL), anti-human IL-4 0.1% SDS, 2 mM EDTA) twice, ChIP wash buffer 3 (20 mM Tris·HCl, pH 8.0, (200 μg/mL), TGF-β (50 μg/mL), and CD3/28-Dynabeads for 3 d; the Dynabeads 150 mM NaCl, 500 mM LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA) were then removed and cultured with IL-2 (30 IU/mL) for 4 d. CD4 T cells (5 × once, and then using TE buffer (10 mM Tris·HCl, pH 8.0, 1 mM EDTA) once. 105) were spinoculated with 50 ng of NL4-3/Luc pseudovirus and CRISPR-A3A After washing, the bead-bound nucleosome fraction was eluted using 150 μL lentivirus or control-CRISPR lentivirus by 800 × g for 2 h at RT. After spinocu- of elution buffer (1% SDS, 0.1 M NaHCO3) at 65 °C for 15 min twice. The eluted lation, cells were washed with prewarmed RPMI with IL-2 (30 IU/mL) and plated sample was de-cross-linked by adding 18 μLof5MNaClandincubatedat65°C on a 12-well plate. Two days after incubation, cells were treated with for 5 h. The DNA in the sample was precipitated by adding 2 μL 5 mg/mL 500 ng/mL puromycin for selection of CRISPR-transduced cells, and a glycogen and 2.5 times the volume of ethanol and incubated at −80 °C portion of the cells was used for Western blotting to check the knock- overnight. DNA was recovered by centrifugation and treated with Proteinase down efficiency of CRISPR-A3A. Five days after puromycin treatment, cells K for 1 to 2 h at 45 °C. Proteinase K-treated DNA was isolated by using a were reactivated using CD3/28-Dynabeads. Three days after reactivation, cells QIAquick PCR Purification Kit (Qiagen). For qPCR of immunoprecipitated DNA, were washed with PBS and lysed by passive lysis buffer for luciferase assay. targeting primers were used to analyze each protein-binding and histone HIV-1 reactivation efficiency was calculated by comparison with nonreactivated modification at HIV-1 . The oligonucleotide primers used for ChIP-qPCR samples. HIV-1 latent infection using NL4-3/Luc replication-competent virus was are listed in SI Appendix,TableS3. Specifically, the immunoprecipitation effi- carried out according to a previous report (47, 48). Briefly, CD4 T cells were ciency of the anti-A3A antibody was validated by immunoprecipitation and spinoculated with 50 ng of NL4-3/Luc pseudovirus and CRISPR-A3A lentivirus or Western blotting (SI Appendix,Fig.S12A). Histone H3 binding was analyzed by control-CRISPR lentivirus. Two days after incubation, CD4 T cells were treated ChIP-qPCR by using anti-Histone H3 antibody and primers for LTR (SI Appendix, with puromycin for selection of transduced cells. Three days later, CD4 T cells Fig. S10 B and C). were treated with 1 μM raltegravir and 0.5 μM nelfinavir for 4 d. Viability of HIV- 1–infected and ART-treated CD4 T cells was measured by auto cell counter (SI NL4-3/Luc Pseudovirus, NL4-3/Luc Virus, and CRISPR Lentivirus. NL4-3/Luc Appendix,Fig.S9E). CD4 T cells were treated with CD3/28-Dynabeads for 72 h, − −– pseudovirus was recovered from the culture medium of pNL4-3.Luc.R E , and HIV-1–driven luciferase activity was measured by luciferase assay compared – pCMVR8.74-, and pHEF-VSVG transfected 293T cells. NL4-3/Luc replication- with each nonstimulated control. competent virus was recovered from the culture medium of pNL4-3/Luc– transfected 293FT cells. Lentivirus encoding CRISPR-A3A, CRISPR-KAP1, Statistical Analysis. For statistical analysis, the data were analyzed by Stu- control-CRISPR gRNA, and CRISPR-Cas9 all in one was generated by follow- dent’s t test or ANOVA with either Tukey’s or Kramer’s multiple comparison ing the manufacturer’s recommended protocol (Applied Biological Mate- test (JMP software; SAS Institute), as indicated in each figure legend. A P rials). Virus in culture medium was concentrated by using a Lenti-X value of <0.05 is considered statistically significant. concentrator (Clontech), and viral titer was measured by using an HIV-

1 p24 ELISA. MICROBIOLOGY ACKNOWLEDGMENTS. We thank Alan N. Engelman (Harvard University) and Reuben Harris (University of Minnesota and HHMI) for critical reading of this HIV-1 Latently Infected Primary CD4 T-Cell Model. HIV-1 latent infection was manuscript. This work was supported by the Howard Hughes Medical done following a previous report (45). Briefly, peripheral blood mononuclear Institute (A.I.). M.T. was a Japan Society for the Promotion of Science Fellow cells (PBMCs) were isolated using Ficoll density gradient centrifugation and in part supported by the Kanzawa Medical Research Foundation and (Lymphoprep; Stemcell Technologies) from buffy coats of six healthy anon- Mochida Memorial Foundation for Medical and Pharmaceutical Research. ymous donors (New York Blood Center). Naïve CD4 T cells were isolated by a E.S. was supported in part by T32 MSTP Fellowship 2T32GM007205-41.

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