FEBS Letters 589 (2015) 2019–2025
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ZNF10 inhibits HIV-1 LTR activity through interaction with NF-jB and Sp1 binding motifs Hironori Nishitsuji a,c,⇑, Leila Sawada a, Ryuichi Sugiyama a,d, Hiroshi Takaku a,b,⇑ a
Department of Life and Environmental Sciences, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan Research Institute, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan c Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516, Japan d Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan b
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Article history: Received 2 March 2015 Revised 4 June 2015 Accepted 11 June 2015 Available online 19 June 2015 Edited by Ivan Sadowski Keywords: KRAB domain Zinc finger protein TRIM28 SETDB1 HP1-gamma HIV-1 LTR
a b s t r a c t Kruppel-associated box-containing zinc finger (KRAB-ZNF) genes constitute the single largest gene family of transcriptional repressors in the genomes of higher organisms. In this study, we isolated 52 cDNA clones of KRAB-ZFPs from U1 cell lines and screened them to identify which were capable of regulating HIV-1 gene expression. We identified 5 KRAB-ZFPs that suppressed P50% of HIV-1 LTR. Of the 5 identified KRAB-ZFPs, the expression of ZNF10 significantly enhanced the transcriptional repression activity of the LTR compared with other ZNFs. In addition, the depletion of endogenous ZNF10 led to the activation of HIV-1 LTR. The repressor activity of ZNF10 was required for TRIM28, SETDB1 and HP1-gamma binding. These results indicate that ZNF10 could be involved in a potent intrinsic antiretroviral defense. Ó 2015 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
1. Introduction The cellular tropism of HIV-1 replication has recently been found to be controlled by the 50 LTR of HIV-1 [1]. The HIV-1 LTR functions as a typical promoter, containing several different binding sites specific for cellular and viral regulatory proteins [2,3]. It is regulated not only by viral proteins (Tat, Nef, Vpu) but also by ubiquitously expressed host factors such as Sp1 and TFIID. Additionally, inducible transcription factors such as NF-jB, AP1, Sp1, NFAT, USF, and COUP play roles in LTR-driven gene expression. These inducible regulatory factors vary in response to stimuli such as hyperthermia, oxidative stress and infection [4–6]. Moreover, the Kruppel-associated box (KRAB) zinc finger proteins (ZNFs) have the potential to regulate HIV-1 gene expression.
Author contributions: H.N. and H.T.: conceived and supervised the study; H.N. and H.T.: designed experiments; H.N., L.S., and R.S.: performed experiments and analyzed data: H.T.: analyzed data. H.T. and H.N. wrote the manuscript. ⇑ Corresponding authors at: Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516, Japan (H. Nishitsuji). Research Institute, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan. Fax: +81 47 478 0407 (H. Takaku). E-mail addresses: [email protected] (H. Nishitsuji), [email protected], [email protected] (H. Takaku).
Several groups have reported that artificially engineered KRAB domain-containing zinc finger proteins that bind to HIV-1 sequences also induce proviral silencing [7–9]. Furthermore, host proteins such as OTK18 suppress HIV-1 Tat-induced LTR activation through the negative regulatory element (NRE) and ETS binding site (EBS). All of these LTR binding elements are important due to their therapeutic potential in the reactivation of HIV-1 in latently infected cells [10–12]. Reynolds et al. reported that a genetically engineered KRAB domain containing a C2H2-type zinc finger motif suppressed Tat-mediated HIV-1 LTR activity, thereby making it an attractive candidate for antiretroviral therapy [8]. In addition, endogenous OTK18, which contains 13 C2H2-type zinc finger motifs, inhibits HIV-1 replication. OTK18 was identified by differential display of mRNA from HIV-1-infected macrophages and was shown to interact with and suppress the NRE (255/238) and EBS (150/139) in the HIV-1 LTR [11,13]. More recently, we showed that ZNF350 (ZBRK1) is able to repress HIV-1 replication through binding the 145 to 126 region of the HIV-1 LTR and ZNF1 in conjunction with TRIM28 and HDAC2, thereby suppressing HIV-1 LTR-driven gene expression [14]. Interestingly, the methylation driven by SETDB1 did not influence this mechanism, which probably reflects the fact that each ZNF has a unique pattern of inhibition. TRIM28 (tripartite
http://dx.doi.org/10.1016/j.febslet.2015.06.013 0014-5793/Ó 2015 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
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motif-containing protein 28, also known as KRAB-associated protein 1, KAP1) is a well-characterized transcriptional repressor. The inhibition mediated by TRIM28 was proven to suppress endogenous retroviruses by recruiting the H3K9 methyltransferase ESET (also called SETDB1 or KMT1E) and heterochromatin protein 1 (HP1) in mouse ES cells [15,16]. TRIM28-mediated gene-specific transcriptional repression requires a ZNF protein such as ZFP809 to directly recognize integrated viral DNA [17]. Furthermore, TRIM28 inhibits HIV-1 integration through a cellular pathway targeting acetylated IN [18]. In this study, we investigated the role of KRAB-zinc finger proteins in the transcriptional repression of HIV-1. We found that ZNF10 highly represses HIV LTR-mediated transcription compared with other ZNFs. We also show that ZNF10 in conjunction with TRIM28, SETDB1 and HP1-gamma suppresses HIV-1 LTR-mediated transcription.
supernatant was incubated with 1 lg of anti-TRIM28 antibody and 40 ll of protein G magnetic beads for 2 h at 4 °C. The beads were then washed with PBS containing 0.02% TritonX-100, and the immunocomplexes were eluted by boiling in 20 ll of 5 sample buffer and analyzed by SDS–PAGE and Western blotting. 2.6. Luciferase assay The luciferase assay details are provided in the Supplementary Materials and Methods section. 2.7. Measurement of HIV-1 p24 antigen The p24 antigen measurement details are provided in the Supplementary Materials and Methods section. 2.8. HIV-1 challenge and culture assay
2. Materials and methods 2.1. Plasmids The details of the plasmid constructs used in this study are provided in the Supplementary Materials and Methods section.
The HIV-1 challenge and culture assay details are provided in the Supplementary Materials and Methods section. 3. Results and discussion 3.1. Screening results
2.2. Cells 293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 lg/ml streptomycin. MT4 and U1 cells were grown in RPMI 1640 supplemented with L-glutamine (Sigma Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (FBS) (Biosera, Nuaille, France) and 1% antibiotics (Sigma Aldrich). The cultures were maintained at 37 °C/5% CO2. On day 4, the U1 cells were collected and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and RNA was extracted from the pellets using RNeasy Plus Mini kits (Qiagen, Venlo, Netherlans). The RNA concentrations were measured with a spectrophotometer (Thermo Scientific, Wilmington, MA, USA), and RNA quality (absence of RNA degradation) was assessed by gel electrophoresis. The levels of HIV-1 p24 antigen were determined using chemiluminescence enzyme immunoassays (CLEIAs) (Lumipulsef, FUJIREBIO, Tokyo, Japan). 2.3. Western blotting The details of the western blotting analysis are provided in the Supplementary Materials and Methods section. 2.4. Small interfering RNA (siRNA) KRAB-ZNF (ZNF10, ZNF566, ZNF333, ZNF561, and ZNF324) mRNA was analyzed, and specific Stealth RNAi™ predesigned siRNAs were ordered (Life Technologies, Gland Island, NY, USA). siTRIM28 and siSETDB1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and siHP1-gamma was purchased from Sigma–Aldrich. 2.5. Immunoprecipitation 293T cells were transfected with 0.5 lg of pHA-ZNF10 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 48 h post-transfection, the transfected cells were harvested and suspended in 0.5 ml lysis buffer (20 mM Tris–HCl pH 7.5, 250 mM NaCl, 1 mM EDTA, 5% glycerol, 1% TritonX-100). The cell lysates were centrifuged at 15 000g for 20 min at 4 °C, after which the
We obtained the coding DNA sequences (CDSs) of 134 human ZNFs and queried the NCBI protein database. We then isolated 134 cDNAs encoding Kruppel-related zinc finger genes from a cell line latently infected with HIV-1 (U1). To evaluate the level of expression of the 134 ZNFs in U1 cells, we performed RT-PCR analysis. We observed no detectable mRNAs encoding 82 of the 134 ZNFs in U1 cells (data not shown). Therefore, we focused on the other 52 ZNFs as candidate transcriptional repressors of the HIV-1 LTR. Next, we determined whether these 52 ZNFs could inhibit HIV-1 LTR promoter activity. 293T cells were co-transfected with the KRAB-ZNF expression vectors and with the HIV-1 LTR-driven luciferase reporter plasmid. As shown in Fig. 1, ZNF10, ZNF566, ZNF333, ZNF561, and ZNF324 significantly suppressed P50% HIV-1 LTR activity compared with the empty vector control. By contrast, ZNF416, ZNF115, and ZNF41 potentially acted as positive regulators of HIV-1, promoting LTR-driven transcription (Fig. 1). Mysliwice et al. reported that ZFP496 functions as a transcriptional activator when it is tethered to DNA or when it is directly bound to the DNA-binding motif [19]. However, in this study we did not analyze the up-regulation of LTR-driven transcription. To further investigate the effect of KRAB-ZNF (ZNF10, ZNF566, ZNF333, ZNF561, and ZNF324)-mediated repression of the LTR, we used siRNAs specific for the five identified KRAB-ZNFs. The introduction of each specific siRNA induced efficient knockdown of expression of the corresponding protein (Fig. 2A). Knockdown of ZNF10 expression significantly enhanced the transcriptional activity of the LTR compared with the knockdown of other KRAB-ZNFs (Fig. 2B). The KRAB zinc finger protein 10 (ZNF10) contains two KRAB domains (A and B boxes) and nine zinc finger regions [20]. To evaluate the effect of ZNF10 in T cells, MT-4 cells were transduced with the control or ZNF10-specific short hairpin RNA (shRNA) vectors and then infected with HIV-1. Virus replication was then monitored by measuring the production of p24 in the supernatant every two days post-infection. The depletion of ZNF10 in MT-4 cells resulted in twofold more HIV-1 replication at four and six days post-infection (Fig. 2C). These results suggest that ZNF10 inhibits HIV-1 gene expression through transcriptional repression of the LTR.
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Fig. 1. The inhibitory effects of HIV-1 LTR-driven transcription by 52 ZNFs. 293T cells (4 105 cells) were transfected with 1 ng of pLTR-luc, 1 ng of pCMV-Renilla and 200 ng of pHA-ZNFs or p-HA (empty vector) using Lipofectamine 2000 (Invitrogen). At 48 h post-transfection, the levels of luciferase gene expression were determined by measuring the luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity. The results are representative of three independent experiments, and the error bars show the standard deviations from the mean values.
A
B
C
Fig. 2. (A) The knockdown of ZNF expression enhances the transcriptional activity of the HIV-1 LTR. 293T cells were treated with 70 nM of the indicated siRNA or control siRNA for 24 h prior to transfection with 5 ng of pLTR-Luc and 1 ng of pCMV-Renilla-Luc. At 48 h post-transfection, the protein lysates were subjected to SDS–PAGE and probed for the indicated protein. (B) The luciferase activity of the protein lysates in Fig. 2A was analyzed. Firefly luciferase activity was normalized to Renilla luciferase activity. (C) The effect of ZNF10 in T cells. MT-4 cells were infected with a lentiviral vector expressing shControl or shZNF10. At 5 days post-infection, the cells were infected with 2 ng of NL4–3 p24. Virus replication was monitored every 2 days after infection by measuring the amount of p24 viral antigen in the culture supernatant. The results are representative of three independent experiments, and the error bars show the standard deviations from the mean values.
3.2. Identification of potential ZNF10 response elements in the HIV-1 LTR To determine the LTR sequence responsible for the suppressive function of ZNF10, we generated deletion mutants of the LTR (Fig. 3A) [14]. The expression of ZNF10 repressed transcription from the 335 to +282 (Fig. 3C), 245 to +282 (Fig. 3D), and 106 to +282 LTR segments (Fig. 3E), as well as from the full-length LTR (Fig. 3B). These deletion mutants of the negative regulatory element (NRE) of HIV-1 LTR did not influence the suppressive effect of ZNF10. In addition, HIV-1 Tat was not required for the repressive activity of ZNF10 (Fig. 3H). A previous study has shown that transcriptional silencing of the HIV-1 LTR by HP1-gamma requires Sp1, P-TEFb (which leads to the
phosphorylation of the RNA polymerase IICTD by recruiting HIV-1 Tat to the TAR) and PCAF (which is known to possess histone acetyl transferase activity) [21]. To further determine the role of the cis-elements of the LTR in ZNF10 repression, we introduced mutations into the NF-jB or Sp1 binding sites in the LTR (Fig. 3A). There was no significant suppressive effect of ZNF10 upon mutations of the NF-jB- or Sp1-binding sites of the LTR (Fig. 3F and G). To determine the influence of ZNF10 and of the NF-jB- and Sp1-binding sites on HIV-1 LTR repression, 293T cells were transfected with pcDNA-HA or pcDNA-HA-ZNF10, pLTR-luc, pCMV-Renilla-luc, and pNF-jB-luc or pSp1-luc using Lipofectamine 2000 (Fig. 3I and J). Furthermore, we examined the over-expression of pNF-jB or pSp1 in HIV-1 LTR-Luc and ZNF10-expressing plasmid-transfected cells (Fig. 3K and L). The luciferase reporter assays showed that the
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Fig. 3. The region of ZNF10 that is responsible for the transcriptional repression of the HIV-1 LTR. (A) Schematic representation of the HIV-1 LTR-driven expression of firefly luciferase. The numbers shown are relative to the transcriptional start site nucleotide, +1. (B–G) 293T cells transfected with 1 ng of pCMV-Renilla-Luc and 200 ng of pHA or pHA-ZNF10, along with 5 ng of pLTR-Luc (B) or its corresponding mutants (C–G). The luciferase assay was performed as described in Fig. 1. (H) 293T cells were transfected with 1 ng of pCMV-Renilla-Luc, 5 ng of pLTR-Luc and 100 ng of pCMV-Tat, along with 200 ng of pHA or pHA-ZNF10. The luciferase assay was performed as described in Fig. 1. (I, J) 293T cells were transfected with 200 ng of pHA or pHA-ZNF10, 1 ng of pCMV-Renilla-luc and 10 ng of pNF-jB-luc (Agilent Technologies) or 10 ng of pSp1-luc (Qiagen) using Lipofectamine 2000. The luciferase assay was performed as described in Fig. 1. (K, L) 293T cells were transfected with 200 ng of pHA or pHA-ZNF10, 1 ng of pLTR-luc, 1 ng of pCMV-Renilla-luc and 200 ng of pcDNA-65(NF-jB) or 200 ng of pCIneo-Sp-1 using Lipofectamine 2000. The luciferase assay was performed as described in Fig. 1. The results are representative of three independent experiments, and the error bars show the standard deviations from the mean values.
NF-jB and Sp1 regions were required for the repression of the LTR by ZNF10 (Fig. 3I–L). These results suggest that the NF-jB and Sp1 sites within the LTR contain essential elements for the transcriptional repression of the LTR by ZNF10.
To address the binding of ZNF10 to the LTR, we performed an electrophoretic mobility shift assay (EMSA). Incubation of the LTR probe containing NF-jB and Sp1 binding sites with nuclear extract revealed a shifted band (Fig. S1). The LTR-ZNF10 complex
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Fig. 4. TRIM28, HP1-gamma and SETDB1 are required for the repressive activity of ZNF10. (A–D) 293T cells were treated with 70 nM of the indicated siRNA for 24 h prior to transfection with 5 ng of pLTR-Luc, 1 ng of pCMV-Renilla-Luc and 300 ng of pHA or pHA-ZNF10. At 48 h post-transfection, the protein lysates were subjected to SDS–PAGE and probed for the indicated protein. The luciferase activity of the protein lysates was also analyzed. Firefly luciferase activity was normalized to Renilla luciferase activity. (E) 293T cells were transfected with 500 ng of pHA-ZNF10. At 48 h post-transfection, the cell lysates were immunoprecipitated using an anti-HA antibody, followed by Western blotting with an anti-TRIM28 antibody. The results are representative of three independent experiments, and the error bars show the standard deviations from the means. (F) ZNF10 repression is reversed by HDAC inhibitors. 293T-LTR-Luc cells were transfected with 400 ng of pHA or pHA-ZNF10. At 24 h after transfection, the cells were treated with TSA for 24 h. The levels of luciferase gene expression were determined by measuring the luciferase activity. (G) U1 cells were treated with 5 ng/ml of PMA (Sigma Aldrich). After 48 h and 72 h, virus replication was monitored by measuring the amount of p24 viral antigen in the culture supernatant. The results are representative of three independent experiments, and the error bars show the standard deviation from the mean values. The protein lysates were subjected to SDS–PAGE and probed for the indicated protein.
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was observed to be supershifted by an anti-ZNF10 antibody. To further confirm this observation, a supershift was also performed using nuclear extracts from 293T cells expressing HA-ZNF10 and an anti-HA antibody. These results indicate that ZNF10 binds to the NF-jB and Sp1 binding sites of LTR. 3.3. Transcriptional repression of the HIV-1 LTR by ZNF10 requires TRIM28, HP1-gamma and SETDB1 Although the TRIM28/ZNF complex has been observed in the presence of different ZNFs [22–24], the role of other molecules in LTR-mediated transcriptional inhibition has been intensively studied. TRIM28 mediates gene silencing by recruiting the NuRD histone deacetylase (HDAC) complex, the SETDB1 histone methyltransferase and the heterochromatin-associated protein HP1-gamma to target promoters [25–29]. In a previous study, we demonstrated that ZBRK1 in conjunction with TRIM28 and HDAC2 suppresses HIV-1 LTR-driven gene expression [14]. To investigate the mechanism involved in ZNF10-mediated repression of the LTR, we transfected siRNAs specific for TRIM28, HP1-gamma or SETDB1 into empty plasmid-transfected or ZNF10-expressing plasmid-transfected cells. The introduction of each specific siRNA knocked down the expression of the corresponding protein (Fig. 4A–D). The expression of ZNF10 in siControl-transduced cells repressed LTR-driven transcription (Fig. 4A). By contrast, no repressive activity of ZNF10 was observed in the siTRIM28, siHP1-gamma, or siSETDB1-transduced cells (Fig. 4B–D). Furthermore, HA-ZNF10 was found to interact with endogenous TRIM28 (Fig. 4E). These results indicate that TRIM28, HP1-gamma and SETDB1 play essential roles in the repressive function of ZNF10. As previously described, TRIM28 interacts with Mi-2alpha and other components of the NuRD complex. Additionally, TRIM28-mediated silencing requires both its association with NuRD and HDAC activity. To test whether histone deacetylases play a role in the repression of LTR activity by ZNF10 in 293T-LTR-Luc cells, we used the pan-HDAC inhibitor trichostatin A (TSA). Treatment with 80 nM TSA significantly reversed the ZNF10-mediated repression of LTR-driven transcription in 293T-LTR-Luc cells (Fig. 4F). These results indicate that the ZNF10-mediated suppression of LTR activity requires HDAC activity. To investigate whether KFZ10 is influenced by HIV-1 latency, the effect of HIV-1 latency was examined in chronically infected promonocytic (U1) cells. After PMA treatment, the p24 antigen level was increased and the ZNF10 expression level was reduced in both cell lines (Fig. 4G). Protein degradation in cells is mediated by several protease systems. To investigate whether the ZNF10 reduction was due to proteasomal degradation, we treated PMA stimulated U1 cells with the proteasome inhibitor MG132 to prevent the virus-induced degradation of ZNF10. Our results indicated that ZNF10 expression was restored by proteasome inhibitors (Fig. S2). Proteasome inhibitors substantially prevented the degradation of ZNF10 in PMA stimulated U1 cells. Therefore, ZNF10 plays a prominent role in controlling the establishment and maintenance of latent HIV-1 provirus in unstimulated cells. In conclusion, we show that ZNF10 represses HIV LTR-mediated transcription to a greater extent than other ZNFs. We also show that ZNF10 in conjunction with TRIM28, SETDB1, and HP1-gamma suppresses HIV-1 LTR-mediated transcription. These results suggest that ZNF10 suppresses HIV-1 LTR-driven gene expression and may have potential as a novel antiviral therapeutic. Conflicts of interest The authors declare no conflicts of interest.
Acknowledgments We thank Dr. M. Abe, and Mr. H. Sato for their technical assistance. This work was supported in part by a Grant-in-Aid for Science Research (C) from the Japan Society for the Promotion of Science (JSPS), Japan; by a Grant-in-Aid for AIDS research from the Ministry of Health, Labor, and Welfare, Japan; and by a grant from the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2015.06. 013. References [1] Reed-Inderbitzin, E. and Maury, W. (2003) Cellular specificity of HIV-1 replication can be controlled by LTR sequences. Virology 314, 680–695. [2] Pereira, L.A., Bentley, K., Peeters, A., Churchill, M.J. and Deacon, N.J. (2000) A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter. Nucleic Acids Res. 28, 663–668. [3] Wu, Y. (2004) HIV-1 gene expression: lessons from provirus and nonintegrated DNA. Retrovirology 1, 13. [4] Ho, W.Z., Song, L. and Douglas, S.D. (1991) Human cytomegalovirus infection and trans-activation of HIV-1 LTR in human brain-derived cells. J. Acquir. Immun. Defic. Syndr. 4, 1098–1106. [5] Legrand-Poels, S., Hoebeke, M., Vaira, D., Rentier, B. and Piette, J. (1993) HIV-1 promoter activation following an oxidative stress mediated by singlet oxygen. J. Photochem. Photobiol. B 17, 229–237. [6] Roesch, F., Meziane, O., Kula, A., Nisole, S., Porrot, F., Anderson, I., Mammano, F., Fassati, A., Marcello, A., Benkirane, M. and Schwartz, O. (2012) Hyperthermia stimulates HIV-1 replication. PLoS Pathog. 8 (e100), 2792. [7] Herchenroder, O., Hahne, J.C., Meyer, W.K., Thiesen, H.J. and Schneider, J. (1999) Repression of the human immunodefi ciency virus type 1 promoter by the human KRAB domain results in inhibition of virus production. Biochim. Biophys. Acta 1445, 216–222. [8] Reynolds, L., Ullman, C., Moore, M., Isalan, M., West, M.J., Clapham, P., Klug, A. and Choo, Y. (2003) Repression of the HIV-1 5’ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. Proc. Natl. Acad. Sci. USA 100, 1615–1620. [9] Segal, D.J., Goncalves, J., Eberhardy, S., Swan, C.H., Torbett, B.E., Li, X. and Barbas 3rd., C.F. (2004) Attenuation of HIV-1 replication in primary human cells with a designed zinc finger transcription factor. J. Biol. Chem. 279, 14509–14519. [10] Horiba, M., Martinez, L.B., Buescher, J.L., Sato, S., Limoges, J., Jiang, Y., Jones, C. and Ikezu, T. (2007) OTK18, a zinc finger protein, regulates human immunodeficiency virus type I long terminal repeat through two distinct regulatory regions. J. Gen. Virol. 88, 236–241. [11] Margolis, D.M., Somasundaran, M. and Green, M.R. (1994) Human transcription factor YY1 represses human immunodeficiency virus type 1 transcription and virion production. J. Virol. 68, 905–910. [12] Buescher, J.L., Martinez, L.B., Sato, S., Okuyama, S. and Ikezu, T. (2009) YY1 and FoxD3 regulate antiretroviral zinc finger protein OTK18 promoter activation induced by HIV-1 infection. J. Neuroimmune Pharmacol. 4, 103–115. [13] Carlson, K.A., Leisman, G., Limoges, J., Pohlman, G.D., Horiba, M., Buescher, J., Gendelman, H.E. and Ikezu, T. (2004) Molecular characterization of a putative antiretroviral transcriptional factor, OTK18. J. Immunol. 172, 381–391. [14] Nishitsuji, H., Abe, M., Sawada, R. and Takaku, H. (2012) ZBRK1 represses HIV1 LTR-mediated transcription. FEBS Lett. 586, 3562–3568. [15] Rowe, H.M., Jakobsson, J., Mesnard, D., Rougemont, J., Reynard, S., Aktas, T., Maillard, P.V., Layard-Liesching, H., Verp, S., Marquis, J., Spitz, F., Constam, D.B. and Trono, D. (2010) KAP1 controls endogenous retroviruses in embryonic stem cells. Nature 463, 237–240. [16] Matsui, T., Leung, D., Miyashita, H., Maksakova, I.A., Miyachi, H., Kimura, H., Tachibana, M., Lorincz, M.C. and Shinkai, Y. (2010) Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature 464, 927–931. [17] Woled, D. and Goff, S.P. (2009) Embryonic stem cells use ZFP809 to silence retroviral DNAs. Nature 458, 1201–1204. [18] Allouch, A., Di Primio, C., Alpi, E., Lusic, M., Arosio, D., Giacca, M. and Cereseto, A. (2011) The TRIM Family Protein KAP1 Inhibits HIV-1 Integration. Cell Host Microbe 9, 484–495. [19] Mysliwiec, M.R., Kim, T.G. and Lee, Y. (2007) Characterization of zinc finger protein 496 that interacts with Jumonji/Jarid2. FEBS Lett. 581, 2633–2640. [20] Urrutia, R. (2003) KRAB-containing zinc-finger repressor proteins. Genome Biol. 4, 231.
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ZNF10 inhibits HIV-1 LTR activity through interaction with NF-jB and Sp1 binding motifs Hironori Nishitsuji a,c,⇑, Leila Sawada a, Ryuichi Sugiyama a,d, Hiroshi Takaku a,b,⇑ a
Department of Life and Environmental Sciences, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan Research Institute, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan c Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516, Japan d Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan b
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Article history: Received 2 March 2015 Revised 4 June 2015 Accepted 11 June 2015 Available online 19 June 2015 Edited by Ivan Sadowski Keywords: KRAB domain Zinc finger protein TRIM28 SETDB1 HP1-gamma HIV-1 LTR
a b s t r a c t Kruppel-associated box-containing zinc finger (KRAB-ZNF) genes constitute the single largest gene family of transcriptional repressors in the genomes of higher organisms. In this study, we isolated 52 cDNA clones of KRAB-ZFPs from U1 cell lines and screened them to identify which were capable of regulating HIV-1 gene expression. We identified 5 KRAB-ZFPs that suppressed P50% of HIV-1 LTR. Of the 5 identified KRAB-ZFPs, the expression of ZNF10 significantly enhanced the transcriptional repression activity of the LTR compared with other ZNFs. In addition, the depletion of endogenous ZNF10 led to the activation of HIV-1 LTR. The repressor activity of ZNF10 was required for TRIM28, SETDB1 and HP1-gamma binding. These results indicate that ZNF10 could be involved in a potent intrinsic antiretroviral defense. Ó 2015 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
1. Introduction The cellular tropism of HIV-1 replication has recently been found to be controlled by the 50 LTR of HIV-1 [1]. The HIV-1 LTR functions as a typical promoter, containing several different binding sites specific for cellular and viral regulatory proteins [2,3]. It is regulated not only by viral proteins (Tat, Nef, Vpu) but also by ubiquitously expressed host factors such as Sp1 and TFIID. Additionally, inducible transcription factors such as NF-jB, AP1, Sp1, NFAT, USF, and COUP play roles in LTR-driven gene expression. These inducible regulatory factors vary in response to stimuli such as hyperthermia, oxidative stress and infection [4–6]. Moreover, the Kruppel-associated box (KRAB) zinc finger proteins (ZNFs) have the potential to regulate HIV-1 gene expression.
Author contributions: H.N. and H.T.: conceived and supervised the study; H.N. and H.T.: designed experiments; H.N., L.S., and R.S.: performed experiments and analyzed data: H.T.: analyzed data. H.T. and H.N. wrote the manuscript. ⇑ Corresponding authors at: Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516, Japan (H. Nishitsuji). Research Institute, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan. Fax: +81 47 478 0407 (H. Takaku). E-mail addresses: [email protected] (H. Nishitsuji), [email protected], [email protected] (H. Takaku).
Several groups have reported that artificially engineered KRAB domain-containing zinc finger proteins that bind to HIV-1 sequences also induce proviral silencing [7–9]. Furthermore, host proteins such as OTK18 suppress HIV-1 Tat-induced LTR activation through the negative regulatory element (NRE) and ETS binding site (EBS). All of these LTR binding elements are important due to their therapeutic potential in the reactivation of HIV-1 in latently infected cells [10–12]. Reynolds et al. reported that a genetically engineered KRAB domain containing a C2H2-type zinc finger motif suppressed Tat-mediated HIV-1 LTR activity, thereby making it an attractive candidate for antiretroviral therapy [8]. In addition, endogenous OTK18, which contains 13 C2H2-type zinc finger motifs, inhibits HIV-1 replication. OTK18 was identified by differential display of mRNA from HIV-1-infected macrophages and was shown to interact with and suppress the NRE (255/238) and EBS (150/139) in the HIV-1 LTR [11,13]. More recently, we showed that ZNF350 (ZBRK1) is able to repress HIV-1 replication through binding the 145 to 126 region of the HIV-1 LTR and ZNF1 in conjunction with TRIM28 and HDAC2, thereby suppressing HIV-1 LTR-driven gene expression [14]. Interestingly, the methylation driven by SETDB1 did not influence this mechanism, which probably reflects the fact that each ZNF has a unique pattern of inhibition. TRIM28 (tripartite
http://dx.doi.org/10.1016/j.febslet.2015.06.013 0014-5793/Ó 2015 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
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motif-containing protein 28, also known as KRAB-associated protein 1, KAP1) is a well-characterized transcriptional repressor. The inhibition mediated by TRIM28 was proven to suppress endogenous retroviruses by recruiting the H3K9 methyltransferase ESET (also called SETDB1 or KMT1E) and heterochromatin protein 1 (HP1) in mouse ES cells [15,16]. TRIM28-mediated gene-specific transcriptional repression requires a ZNF protein such as ZFP809 to directly recognize integrated viral DNA [17]. Furthermore, TRIM28 inhibits HIV-1 integration through a cellular pathway targeting acetylated IN [18]. In this study, we investigated the role of KRAB-zinc finger proteins in the transcriptional repression of HIV-1. We found that ZNF10 highly represses HIV LTR-mediated transcription compared with other ZNFs. We also show that ZNF10 in conjunction with TRIM28, SETDB1 and HP1-gamma suppresses HIV-1 LTR-mediated transcription.
supernatant was incubated with 1 lg of anti-TRIM28 antibody and 40 ll of protein G magnetic beads for 2 h at 4 °C. The beads were then washed with PBS containing 0.02% TritonX-100, and the immunocomplexes were eluted by boiling in 20 ll of 5 sample buffer and analyzed by SDS–PAGE and Western blotting. 2.6. Luciferase assay The luciferase assay details are provided in the Supplementary Materials and Methods section. 2.7. Measurement of HIV-1 p24 antigen The p24 antigen measurement details are provided in the Supplementary Materials and Methods section. 2.8. HIV-1 challenge and culture assay
2. Materials and methods 2.1. Plasmids The details of the plasmid constructs used in this study are provided in the Supplementary Materials and Methods section.
The HIV-1 challenge and culture assay details are provided in the Supplementary Materials and Methods section. 3. Results and discussion 3.1. Screening results
2.2. Cells 293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 lg/ml streptomycin. MT4 and U1 cells were grown in RPMI 1640 supplemented with L-glutamine (Sigma Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (FBS) (Biosera, Nuaille, France) and 1% antibiotics (Sigma Aldrich). The cultures were maintained at 37 °C/5% CO2. On day 4, the U1 cells were collected and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and RNA was extracted from the pellets using RNeasy Plus Mini kits (Qiagen, Venlo, Netherlans). The RNA concentrations were measured with a spectrophotometer (Thermo Scientific, Wilmington, MA, USA), and RNA quality (absence of RNA degradation) was assessed by gel electrophoresis. The levels of HIV-1 p24 antigen were determined using chemiluminescence enzyme immunoassays (CLEIAs) (Lumipulsef, FUJIREBIO, Tokyo, Japan). 2.3. Western blotting The details of the western blotting analysis are provided in the Supplementary Materials and Methods section. 2.4. Small interfering RNA (siRNA) KRAB-ZNF (ZNF10, ZNF566, ZNF333, ZNF561, and ZNF324) mRNA was analyzed, and specific Stealth RNAi™ predesigned siRNAs were ordered (Life Technologies, Gland Island, NY, USA). siTRIM28 and siSETDB1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and siHP1-gamma was purchased from Sigma–Aldrich. 2.5. Immunoprecipitation 293T cells were transfected with 0.5 lg of pHA-ZNF10 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 48 h post-transfection, the transfected cells were harvested and suspended in 0.5 ml lysis buffer (20 mM Tris–HCl pH 7.5, 250 mM NaCl, 1 mM EDTA, 5% glycerol, 1% TritonX-100). The cell lysates were centrifuged at 15 000g for 20 min at 4 °C, after which the
We obtained the coding DNA sequences (CDSs) of 134 human ZNFs and queried the NCBI protein database. We then isolated 134 cDNAs encoding Kruppel-related zinc finger genes from a cell line latently infected with HIV-1 (U1). To evaluate the level of expression of the 134 ZNFs in U1 cells, we performed RT-PCR analysis. We observed no detectable mRNAs encoding 82 of the 134 ZNFs in U1 cells (data not shown). Therefore, we focused on the other 52 ZNFs as candidate transcriptional repressors of the HIV-1 LTR. Next, we determined whether these 52 ZNFs could inhibit HIV-1 LTR promoter activity. 293T cells were co-transfected with the KRAB-ZNF expression vectors and with the HIV-1 LTR-driven luciferase reporter plasmid. As shown in Fig. 1, ZNF10, ZNF566, ZNF333, ZNF561, and ZNF324 significantly suppressed P50% HIV-1 LTR activity compared with the empty vector control. By contrast, ZNF416, ZNF115, and ZNF41 potentially acted as positive regulators of HIV-1, promoting LTR-driven transcription (Fig. 1). Mysliwice et al. reported that ZFP496 functions as a transcriptional activator when it is tethered to DNA or when it is directly bound to the DNA-binding motif [19]. However, in this study we did not analyze the up-regulation of LTR-driven transcription. To further investigate the effect of KRAB-ZNF (ZNF10, ZNF566, ZNF333, ZNF561, and ZNF324)-mediated repression of the LTR, we used siRNAs specific for the five identified KRAB-ZNFs. The introduction of each specific siRNA induced efficient knockdown of expression of the corresponding protein (Fig. 2A). Knockdown of ZNF10 expression significantly enhanced the transcriptional activity of the LTR compared with the knockdown of other KRAB-ZNFs (Fig. 2B). The KRAB zinc finger protein 10 (ZNF10) contains two KRAB domains (A and B boxes) and nine zinc finger regions [20]. To evaluate the effect of ZNF10 in T cells, MT-4 cells were transduced with the control or ZNF10-specific short hairpin RNA (shRNA) vectors and then infected with HIV-1. Virus replication was then monitored by measuring the production of p24 in the supernatant every two days post-infection. The depletion of ZNF10 in MT-4 cells resulted in twofold more HIV-1 replication at four and six days post-infection (Fig. 2C). These results suggest that ZNF10 inhibits HIV-1 gene expression through transcriptional repression of the LTR.
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Fig. 1. The inhibitory effects of HIV-1 LTR-driven transcription by 52 ZNFs. 293T cells (4 105 cells) were transfected with 1 ng of pLTR-luc, 1 ng of pCMV-Renilla and 200 ng of pHA-ZNFs or p-HA (empty vector) using Lipofectamine 2000 (Invitrogen). At 48 h post-transfection, the levels of luciferase gene expression were determined by measuring the luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity. The results are representative of three independent experiments, and the error bars show the standard deviations from the mean values.
A
B
C
Fig. 2. (A) The knockdown of ZNF expression enhances the transcriptional activity of the HIV-1 LTR. 293T cells were treated with 70 nM of the indicated siRNA or control siRNA for 24 h prior to transfection with 5 ng of pLTR-Luc and 1 ng of pCMV-Renilla-Luc. At 48 h post-transfection, the protein lysates were subjected to SDS–PAGE and probed for the indicated protein. (B) The luciferase activity of the protein lysates in Fig. 2A was analyzed. Firefly luciferase activity was normalized to Renilla luciferase activity. (C) The effect of ZNF10 in T cells. MT-4 cells were infected with a lentiviral vector expressing shControl or shZNF10. At 5 days post-infection, the cells were infected with 2 ng of NL4–3 p24. Virus replication was monitored every 2 days after infection by measuring the amount of p24 viral antigen in the culture supernatant. The results are representative of three independent experiments, and the error bars show the standard deviations from the mean values.
3.2. Identification of potential ZNF10 response elements in the HIV-1 LTR To determine the LTR sequence responsible for the suppressive function of ZNF10, we generated deletion mutants of the LTR (Fig. 3A) [14]. The expression of ZNF10 repressed transcription from the 335 to +282 (Fig. 3C), 245 to +282 (Fig. 3D), and 106 to +282 LTR segments (Fig. 3E), as well as from the full-length LTR (Fig. 3B). These deletion mutants of the negative regulatory element (NRE) of HIV-1 LTR did not influence the suppressive effect of ZNF10. In addition, HIV-1 Tat was not required for the repressive activity of ZNF10 (Fig. 3H). A previous study has shown that transcriptional silencing of the HIV-1 LTR by HP1-gamma requires Sp1, P-TEFb (which leads to the
phosphorylation of the RNA polymerase IICTD by recruiting HIV-1 Tat to the TAR) and PCAF (which is known to possess histone acetyl transferase activity) [21]. To further determine the role of the cis-elements of the LTR in ZNF10 repression, we introduced mutations into the NF-jB or Sp1 binding sites in the LTR (Fig. 3A). There was no significant suppressive effect of ZNF10 upon mutations of the NF-jB- or Sp1-binding sites of the LTR (Fig. 3F and G). To determine the influence of ZNF10 and of the NF-jB- and Sp1-binding sites on HIV-1 LTR repression, 293T cells were transfected with pcDNA-HA or pcDNA-HA-ZNF10, pLTR-luc, pCMV-Renilla-luc, and pNF-jB-luc or pSp1-luc using Lipofectamine 2000 (Fig. 3I and J). Furthermore, we examined the over-expression of pNF-jB or pSp1 in HIV-1 LTR-Luc and ZNF10-expressing plasmid-transfected cells (Fig. 3K and L). The luciferase reporter assays showed that the
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Fig. 3. The region of ZNF10 that is responsible for the transcriptional repression of the HIV-1 LTR. (A) Schematic representation of the HIV-1 LTR-driven expression of firefly luciferase. The numbers shown are relative to the transcriptional start site nucleotide, +1. (B–G) 293T cells transfected with 1 ng of pCMV-Renilla-Luc and 200 ng of pHA or pHA-ZNF10, along with 5 ng of pLTR-Luc (B) or its corresponding mutants (C–G). The luciferase assay was performed as described in Fig. 1. (H) 293T cells were transfected with 1 ng of pCMV-Renilla-Luc, 5 ng of pLTR-Luc and 100 ng of pCMV-Tat, along with 200 ng of pHA or pHA-ZNF10. The luciferase assay was performed as described in Fig. 1. (I, J) 293T cells were transfected with 200 ng of pHA or pHA-ZNF10, 1 ng of pCMV-Renilla-luc and 10 ng of pNF-jB-luc (Agilent Technologies) or 10 ng of pSp1-luc (Qiagen) using Lipofectamine 2000. The luciferase assay was performed as described in Fig. 1. (K, L) 293T cells were transfected with 200 ng of pHA or pHA-ZNF10, 1 ng of pLTR-luc, 1 ng of pCMV-Renilla-luc and 200 ng of pcDNA-65(NF-jB) or 200 ng of pCIneo-Sp-1 using Lipofectamine 2000. The luciferase assay was performed as described in Fig. 1. The results are representative of three independent experiments, and the error bars show the standard deviations from the mean values.
NF-jB and Sp1 regions were required for the repression of the LTR by ZNF10 (Fig. 3I–L). These results suggest that the NF-jB and Sp1 sites within the LTR contain essential elements for the transcriptional repression of the LTR by ZNF10.
To address the binding of ZNF10 to the LTR, we performed an electrophoretic mobility shift assay (EMSA). Incubation of the LTR probe containing NF-jB and Sp1 binding sites with nuclear extract revealed a shifted band (Fig. S1). The LTR-ZNF10 complex
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Fig. 4. TRIM28, HP1-gamma and SETDB1 are required for the repressive activity of ZNF10. (A–D) 293T cells were treated with 70 nM of the indicated siRNA for 24 h prior to transfection with 5 ng of pLTR-Luc, 1 ng of pCMV-Renilla-Luc and 300 ng of pHA or pHA-ZNF10. At 48 h post-transfection, the protein lysates were subjected to SDS–PAGE and probed for the indicated protein. The luciferase activity of the protein lysates was also analyzed. Firefly luciferase activity was normalized to Renilla luciferase activity. (E) 293T cells were transfected with 500 ng of pHA-ZNF10. At 48 h post-transfection, the cell lysates were immunoprecipitated using an anti-HA antibody, followed by Western blotting with an anti-TRIM28 antibody. The results are representative of three independent experiments, and the error bars show the standard deviations from the means. (F) ZNF10 repression is reversed by HDAC inhibitors. 293T-LTR-Luc cells were transfected with 400 ng of pHA or pHA-ZNF10. At 24 h after transfection, the cells were treated with TSA for 24 h. The levels of luciferase gene expression were determined by measuring the luciferase activity. (G) U1 cells were treated with 5 ng/ml of PMA (Sigma Aldrich). After 48 h and 72 h, virus replication was monitored by measuring the amount of p24 viral antigen in the culture supernatant. The results are representative of three independent experiments, and the error bars show the standard deviation from the mean values. The protein lysates were subjected to SDS–PAGE and probed for the indicated protein.
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was observed to be supershifted by an anti-ZNF10 antibody. To further confirm this observation, a supershift was also performed using nuclear extracts from 293T cells expressing HA-ZNF10 and an anti-HA antibody. These results indicate that ZNF10 binds to the NF-jB and Sp1 binding sites of LTR. 3.3. Transcriptional repression of the HIV-1 LTR by ZNF10 requires TRIM28, HP1-gamma and SETDB1 Although the TRIM28/ZNF complex has been observed in the presence of different ZNFs [22–24], the role of other molecules in LTR-mediated transcriptional inhibition has been intensively studied. TRIM28 mediates gene silencing by recruiting the NuRD histone deacetylase (HDAC) complex, the SETDB1 histone methyltransferase and the heterochromatin-associated protein HP1-gamma to target promoters [25–29]. In a previous study, we demonstrated that ZBRK1 in conjunction with TRIM28 and HDAC2 suppresses HIV-1 LTR-driven gene expression [14]. To investigate the mechanism involved in ZNF10-mediated repression of the LTR, we transfected siRNAs specific for TRIM28, HP1-gamma or SETDB1 into empty plasmid-transfected or ZNF10-expressing plasmid-transfected cells. The introduction of each specific siRNA knocked down the expression of the corresponding protein (Fig. 4A–D). The expression of ZNF10 in siControl-transduced cells repressed LTR-driven transcription (Fig. 4A). By contrast, no repressive activity of ZNF10 was observed in the siTRIM28, siHP1-gamma, or siSETDB1-transduced cells (Fig. 4B–D). Furthermore, HA-ZNF10 was found to interact with endogenous TRIM28 (Fig. 4E). These results indicate that TRIM28, HP1-gamma and SETDB1 play essential roles in the repressive function of ZNF10. As previously described, TRIM28 interacts with Mi-2alpha and other components of the NuRD complex. Additionally, TRIM28-mediated silencing requires both its association with NuRD and HDAC activity. To test whether histone deacetylases play a role in the repression of LTR activity by ZNF10 in 293T-LTR-Luc cells, we used the pan-HDAC inhibitor trichostatin A (TSA). Treatment with 80 nM TSA significantly reversed the ZNF10-mediated repression of LTR-driven transcription in 293T-LTR-Luc cells (Fig. 4F). These results indicate that the ZNF10-mediated suppression of LTR activity requires HDAC activity. To investigate whether KFZ10 is influenced by HIV-1 latency, the effect of HIV-1 latency was examined in chronically infected promonocytic (U1) cells. After PMA treatment, the p24 antigen level was increased and the ZNF10 expression level was reduced in both cell lines (Fig. 4G). Protein degradation in cells is mediated by several protease systems. To investigate whether the ZNF10 reduction was due to proteasomal degradation, we treated PMA stimulated U1 cells with the proteasome inhibitor MG132 to prevent the virus-induced degradation of ZNF10. Our results indicated that ZNF10 expression was restored by proteasome inhibitors (Fig. S2). Proteasome inhibitors substantially prevented the degradation of ZNF10 in PMA stimulated U1 cells. Therefore, ZNF10 plays a prominent role in controlling the establishment and maintenance of latent HIV-1 provirus in unstimulated cells. In conclusion, we show that ZNF10 represses HIV LTR-mediated transcription to a greater extent than other ZNFs. We also show that ZNF10 in conjunction with TRIM28, SETDB1, and HP1-gamma suppresses HIV-1 LTR-mediated transcription. These results suggest that ZNF10 suppresses HIV-1 LTR-driven gene expression and may have potential as a novel antiviral therapeutic. Conflicts of interest The authors declare no conflicts of interest.
Acknowledgments We thank Dr. M. Abe, and Mr. H. Sato for their technical assistance. This work was supported in part by a Grant-in-Aid for Science Research (C) from the Japan Society for the Promotion of Science (JSPS), Japan; by a Grant-in-Aid for AIDS research from the Ministry of Health, Labor, and Welfare, Japan; and by a grant from the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2015.06. 013. References [1] Reed-Inderbitzin, E. and Maury, W. (2003) Cellular specificity of HIV-1 replication can be controlled by LTR sequences. Virology 314, 680–695. [2] Pereira, L.A., Bentley, K., Peeters, A., Churchill, M.J. and Deacon, N.J. (2000) A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter. Nucleic Acids Res. 28, 663–668. [3] Wu, Y. (2004) HIV-1 gene expression: lessons from provirus and nonintegrated DNA. Retrovirology 1, 13. [4] Ho, W.Z., Song, L. and Douglas, S.D. (1991) Human cytomegalovirus infection and trans-activation of HIV-1 LTR in human brain-derived cells. J. Acquir. Immun. Defic. Syndr. 4, 1098–1106. [5] Legrand-Poels, S., Hoebeke, M., Vaira, D., Rentier, B. and Piette, J. (1993) HIV-1 promoter activation following an oxidative stress mediated by singlet oxygen. J. Photochem. Photobiol. B 17, 229–237. [6] Roesch, F., Meziane, O., Kula, A., Nisole, S., Porrot, F., Anderson, I., Mammano, F., Fassati, A., Marcello, A., Benkirane, M. and Schwartz, O. (2012) Hyperthermia stimulates HIV-1 replication. PLoS Pathog. 8 (e100), 2792. [7] Herchenroder, O., Hahne, J.C., Meyer, W.K., Thiesen, H.J. and Schneider, J. (1999) Repression of the human immunodefi ciency virus type 1 promoter by the human KRAB domain results in inhibition of virus production. Biochim. Biophys. Acta 1445, 216–222. [8] Reynolds, L., Ullman, C., Moore, M., Isalan, M., West, M.J., Clapham, P., Klug, A. and Choo, Y. (2003) Repression of the HIV-1 5’ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. Proc. Natl. Acad. Sci. USA 100, 1615–1620. [9] Segal, D.J., Goncalves, J., Eberhardy, S., Swan, C.H., Torbett, B.E., Li, X. and Barbas 3rd., C.F. (2004) Attenuation of HIV-1 replication in primary human cells with a designed zinc finger transcription factor. J. Biol. Chem. 279, 14509–14519. [10] Horiba, M., Martinez, L.B., Buescher, J.L., Sato, S., Limoges, J., Jiang, Y., Jones, C. and Ikezu, T. (2007) OTK18, a zinc finger protein, regulates human immunodeficiency virus type I long terminal repeat through two distinct regulatory regions. J. Gen. Virol. 88, 236–241. [11] Margolis, D.M., Somasundaran, M. and Green, M.R. (1994) Human transcription factor YY1 represses human immunodeficiency virus type 1 transcription and virion production. J. Virol. 68, 905–910. [12] Buescher, J.L., Martinez, L.B., Sato, S., Okuyama, S. and Ikezu, T. (2009) YY1 and FoxD3 regulate antiretroviral zinc finger protein OTK18 promoter activation induced by HIV-1 infection. J. Neuroimmune Pharmacol. 4, 103–115. [13] Carlson, K.A., Leisman, G., Limoges, J., Pohlman, G.D., Horiba, M., Buescher, J., Gendelman, H.E. and Ikezu, T. (2004) Molecular characterization of a putative antiretroviral transcriptional factor, OTK18. J. Immunol. 172, 381–391. [14] Nishitsuji, H., Abe, M., Sawada, R. and Takaku, H. (2012) ZBRK1 represses HIV1 LTR-mediated transcription. FEBS Lett. 586, 3562–3568. [15] Rowe, H.M., Jakobsson, J., Mesnard, D., Rougemont, J., Reynard, S., Aktas, T., Maillard, P.V., Layard-Liesching, H., Verp, S., Marquis, J., Spitz, F., Constam, D.B. and Trono, D. (2010) KAP1 controls endogenous retroviruses in embryonic stem cells. Nature 463, 237–240. [16] Matsui, T., Leung, D., Miyashita, H., Maksakova, I.A., Miyachi, H., Kimura, H., Tachibana, M., Lorincz, M.C. and Shinkai, Y. (2010) Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature 464, 927–931. [17] Woled, D. and Goff, S.P. (2009) Embryonic stem cells use ZFP809 to silence retroviral DNAs. Nature 458, 1201–1204. [18] Allouch, A., Di Primio, C., Alpi, E., Lusic, M., Arosio, D., Giacca, M. and Cereseto, A. (2011) The TRIM Family Protein KAP1 Inhibits HIV-1 Integration. Cell Host Microbe 9, 484–495. [19] Mysliwiec, M.R., Kim, T.G. and Lee, Y. (2007) Characterization of zinc finger protein 496 that interacts with Jumonji/Jarid2. FEBS Lett. 581, 2633–2640. [20] Urrutia, R. (2003) KRAB-containing zinc-finger repressor proteins. Genome Biol. 4, 231.
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