Human T lymphotropic virus type 1 (HTLV-1) belongs to the deltaretrovirus family and has been linked to multiple diseases. However, the innate host defense against HTLV-1 is unclear. In this study, we report that the expression of Ku70, a known DNA sensor against DNA viruses, could be induced by HTLV-1 infection in HeLa, PMA-differentiated THP1 cells, primary human monocytes, and human monocyte-derived macrophages. In these cells, the overexpression of Ku70 inhibited the HTLV-1 protein expression, whereas the knockdown of Ku70 promoted the HTLV-1 protein expression. Furthermore, the overexpression of Ku70 enhanced the cellular response to HTLV-1 infection, whereas Ku70 knockdown yielded the opposite effect. Additionally, Ku70 was found to interact with HTLV-1 reverse transcription intermediate ssDNA90. ssDNA90 stimulation induced Ku70 expression and Ku70 promoted ssDNA90-triggered innate immune responses. Finally, HTLV-1 infection enhanced the association between Ku70 and stimulator of IFN genes, suggesting that stimulator of IFN genes was involved in Ku70-mediated host defenses against HTLV-1 infection. Taken together, our findings suggest a new sensor that detects HTLV-1 reverse transcription intermediate and controls HTLV-1 replication. These findings may provide new angles to understand host defenses against HTLV-1 infection and HTLV-1–associated diseases.

The innate immune responses triggered by viral components are important for the host defense against viral infection, including type I IFN secretion and other proinflammatory cytokine production (1). The recognition of viral components is essential to the initiation of innate immune responses, which is completed by a series of pattern recognition receptors (2). The nucleic acids are relatively conserved in viruses and suitable for innate immune recognition (3). Whereas RIG-I–like receptors, including retinoic acid–inducible gene I, melanoma differentiation-associated gene 5, and laboratory of genetics and physiology 2, are responsible for cytosolic viral RNA recognition (4), the cytosolic DNA forms of viruses are detected by an array of molecules identified as cytosolic DNA sensors, such as DNA-dependent activator of IFN regulatory factors, RNA polymerase III, IFN-γ–inducible factor 16, DExD/H-box helicase 41, Ku70, stimulator of IFN genes (STING), cyclic GMP–AMP (cGAMP) synthase (cGAS), and so on (5, 6).

During the life cycle of retroviruses, several types of nucleic acid structures were produced by reverse transcription, including the RNA/DNA intermediates, the ssDNA intermediates, and the final dsDNA (7). The accumulation of these nucleic acid structures provides the opportunity for the host cells to sense viral invasion and initiate innate immune responses through DNA sensors. Indeed, it has been reported that cGAS and TLR9 are responsible for the recognition of RNA/DNA intermediates (8, 9). cGAS is also characterized as a sensor of reverse-transcribed DNA from HIV, murine leukemia virus, and SIV (10). Another DNA sensor, IFN-γ–inducible factor 16, was proved to detect the DNA forms from the lentiviral replication (11). These studies indicated that DNA sensors might play a role in other retrovirus infections.

Human T lymphotropic virus type 1 (HTLV-1) belongs to the deltaretrovirus family and infects ∼10–20 million people worldwide (12), leading to multiple diseases, including aggressive blood cancer, adult T cell leukemia/lymphoma (13), and the chronic, progressive neurologic and inflammatory disease termed HTLV-1–associated myelopathy/tropical spastic paraparesis (14). HTLV-1 can establish persistent infection in humans for many years and cause diverse clinical diseases after long periods of virus latency (15). It has been reported that free HTLV-1 induces a TLR7-dependent innate immune response in killer plasmacytoid dendritic cells (16) and that transfected HTLV-1 reverse transcription intermediate (RTI) ssDNA90 triggers STING–IFN regulatory factor (IRF)3-dependent antiviral responses in monocytes (17). Considering that HTLV-1 infects many types of cells (18), other sensors may be involved in host defenses against HTLV-1 infection.

Ku70 is well studied for its role in dsDNA break repair pathway in the complex with Ku80 and catalytic subunit DNA-PKcs known as DNA-dependent protein kinase (DNA-PK) (19, 20). Interestingly, Ku70 appears relatively more abundant than DNA-PKcs (21), suggesting that Ku70 may be involved in other processes other than DNA repair. Given the fact that Ku70 binds dsDNA, which is also an important pathogen-associated molecular pattern from viruses, Ku70 has the potential to be a DNA sensor. Several groups have reported that Ku70 detects dsDNA and induces innate host defenses against DNA viruses (22). Zhang et al. (23) reported that Ku70 was a DNA sensor and induced IFN-λ1 activation other than IFN-β in an IRF1/IRF7-dependent manner. Ferguson et al. (24) revealed another mechanism by which Ku70 regulated the host defenses against DNA viruses. They identified the DNA-PK complex as a DNA sensor, which triggered IRF3-dependent innate immune responses, including the production of IFN-β (24). Li et al. reported that Ku sensed hepatitis B virus DNA and induced chemokine secretion in an IRF1-dependent pathway (25).

In this study, we found that Ku70 could be induced by HTLV-1 infection and inhibited HTLV-1 replication. Ku70 associated with HTLV-1 RTI ssDNA90 and enhanced ssDNA90 or HTLV-1 induced type I IFN responses through STING-dependent signaling pathway. Taken together, our findings suggest Ku70 is a DNA sensor for HTLV-1 RT products and may contribute to our understanding of the host defenses against HTLV-1 infection.

Human Ku70 was amplified by PCR using cDNA from MT2 cocultured HeLa cells and was subsequently cloned into a Flag-pcDNA3 vector (Invitrogen). Hemagglutinin (HA)-STING was obtained as described previously (26). Human Ku80 was amplified by PCR using cDNA from MT2 cocultured HeLa cells and was subsequently cloned into a Flag-pcDNA3 vector (Invitrogen). Flag-MyD88 was amplified by PCR using cDNA from LPS-treated bone marrow–derived dendritic cells and was subsequently cloned into a Flag-pcDNA3 vector (Invitrogen). The 90-base-long HTLV-1 ssDNA90 is the reverse complement of the 5′ untranslated region (315–404) of complete HTLV-1 genome (National Center for Biotechnology Information) and was synthesized by Sangon Biotech. The sequence was as follows: 5′-CTGTGTACTAAATTTCTCTCCTGGAGAGTGCTATAGAATGGGCTGTCGCTGGCTCCGAGCCAGCAGAGTTGCCGGTACTTGGCCGTGGGC-3′. The scrambled ssDNA90 as a control was also synthesized by Sangon Biotech. The sequence was as follows: 5′-ATTCAGCTCACGGCGTCGAGTGCTGCTCGATGGCTCCTTAGTCCTGCTAAGTCGAGGTGGCTAATCCGGTAGTCGGTCGGATGGAATTCG-3′. HSV60 (tlrl-hsv60n) and 2′3′-cGAMP (tlrl-nacga23) were obtained from InvivoGen. The following Abs were used for immunoblot analysis or immunoprecipitation: anti-Flag (F3165; Sigma-Aldrich), anti-Ku70 (ab83501; Abcam), anti–HTLV-1 p19 (ab9080; Abcam), anti-Tax (sc-57872; Santa Cruz Biotechnology), anti–p-IRF3 (4947; Cell Signaling Technology), anti-IRF3 (sc-9082; Santa Cruz Biotechnology), anti–p-p65 (3033; Cell Signaling Technology), anti-p65 (10745-1-AP; Proteintech), anti-STING (19851-1-AP; Proteintech), anti-gm130 (610822; BD Biosciences), anti-calnexin (C4731; Sigma-Aldrich), anti-cGAS (26416-1-AP; Proteintech), anti–β-catenin (51067-2-AP; Proteintech), anti–p-STING (85735, Ser366; Cell Signaling Technology), anti–p-β-catenin (5651, Ser552; Cell Signaling Technology), and anti–β-actin (60008-1; Proteintech). The PMA (S1819) was purchased from Beyotime Biotechnology.

HeLa cells were cultured in DMEM. PBMCs were enriched from donor blood using Ficoll density gradient separation, and human monocytes were separated by their adherence to the culture plate. MT2, human monocytes, and THP1 cells were grown in RPMI 1640. PMA-differentiated THP1 (PMA-THP1) cells referred to THP1 cells that were pretreated with 100 ng/ml PMA for 24 h. Differentiation of monocytes to macrophages was achieved by culturing in DMEM in the presence of 10 ng/ml M-CSF (PeproTech) for 6 d. All cells were supplemented with 10% FBS (Life Technologies), 4 mM l-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin under humidified conditions with 5% CO2 at 37°C. Transfection of HeLa and human monocytes was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Transfection of ssDNA90 and small interfering RNA (siRNA) into THP1 cells was performed with Lipofectamine 2000 (Invitrogen). Transfection of plasmids into THP1 cells was performed using electroporator (CUY21EX; BEX) according to the manufacturer’s instructions.

PMA-THP1 cells were infected with HSV-1 (KOS strain, multiplicity of infection of 10) for 1.5 h and subsequently washed with PBS and cultured in fresh media. An HSV-1 viral titer was determined by a plaque-forming assay on Vero cells.

The supernatants of overnight seeded MT2 cells were collected and ultracentrifuged for 2 h at 30,000 × g at 4°C. The viral pellet was resuspended in RPMI 1640, and HTLV-1 particles were quantified by gag p19 ELISA assays (ZeptoMetrix). A total of 250,000 purified monocytes were incubated with 2 mg of HTLV-1 for the indicated periods of time in RPMI 1640.

Immunoprecipitation and immunoblot analyses were performed as described previously (26). In short, cells were transfected with various combinations of plasmids or siRNA. At 24 h after the transfection, the cell lysates were prepared in lysis buffer containing 1% (v/v) Nonidet P-40, 20 mM Tris-HCl (pH 8), 10% (v/v) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate, and a protease inhibitor mixture (Roche). After centrifugation for 20 min at 14,000 × g, supernatants were collected and incubated with the indicated Ab together with protein A/G Plus–agarose immunoprecipitation reagent (Santa Cruz Biotechnology) at 4°C for 3 h or overnight. After three washes, the immunoprecipitates were boiled in SDS sample buffer for 10 min and analyzed by immunoblot. For endogenous coimmunoprecipitation experiments, the cell lysates of HeLa cells were incubated with indicated Abs and analyzed by immunoblot.

Total RNA was extracted from the cultured cells with TRIzol reagent (Invitrogen) as described by the manufacturer. All gene transcripts were quantified by real-time PCR with SYBR Green quantitative PCR master mix using an ABI 7500 Fast real-time PCR system (Applied Biosystems). The relative fold induction was calculated using the 2−△△Ct method. The primers used for real-time PCR were as follows: Tax, forward, 5′-ATACCCAGTCTACGTGTTTGGAG-3′, reverse, 5′-CCGATAACGCGTCCATCGATG-3′; p19, forward, 5′-CACCCCTTTCCCTTTCATTCACGA-3′, reverse, 5′-CCGGCCGGGGTATCCTTTT-3′; px, forward, 5′-CAAAGTTAACCATGCTTATTATCAGC-3′, reverse, 5′-ACACGTAGACTGGGTATCCGAA-3′; Env, forward, 5′-CCATCGTTAGCGCTTCCAGCCCC-3′, reverse, 5′-CGGGATCCTAGCGTGGGAACAGGT-3′; IL-6, forward, 5′-GAGGATACCACTCCCCAACAGACC-3′, reverse, 5′-AAGTGCATCATCGTTGTTCATACA-3′; IFN-β, forward, 5′-CACGACAGCTCTTTCCATGA-3′, reverse, 5′-AGCCAGTGCTCGATGAATCT-3′; TNF-α, forward, 5′-GGCGTGGAGCTGAGAGATAAC-3′, reverse, 5′-GGTGTGGGTGAGGAGCACAT-3′; RANTES, forward, 5′-TACACCAGTGGCAAGTGCTC-3′, reverse, 5′-ACACACTTGGCGGTTCTTTC-3′; ISG56, forward, 5′-GCCATTTTCTTTGCTTCCCCTA-3′, reverse, 5′-TGCCCTTTTGTAGCCTCCTTG-3′; IFN-λ, forward, 5′-TTTTCTAGACGGCAGGAAGGCCATGGC-3′, reverse, 5′-TCTAGACCTGGCCATGTAATGCCCCAAT-3′; GAPDH, forward, 5′-TCAACGACCACTTTGTCAAGCTCA-3′, reverse, 5′-GCTGGTGGTCCAGGTCTTACT-3′.

Ku70-Silencer select predesigned siRNA was obtained from Invitrogen. The siRNA sequences used were as follows: K1, forward, 5′-GAGUGAAGAUGAGUUGACATT-3′, reverse, 5′-UGUCAACUCAUCUUCACUCTG-3′; K2, forward, 5′-GACAUAUCCUUGUUCUACATT-3′, reverse, 5′-UGUAGAACAAGGAUAUGUCAA-3′.

The Silencer select negative control siRNA was purchased from Invitrogen (catalog no. 4390843). STING–Stealth–RNA interference was designed by the Invitrogen BLOCKiT RNAi Designer. The siRNA sequences used were as follows: forward, 5′-GGCCCGGAUUCGAACUUACAAUCAG-3′, reverse, 5′-CUGAUUGUAAGUUCGAAUCCGGGCC-3′.

The cGAS–Stealth–RNA interference was designed by the Invitrogen BLOCKiT RNAi Designer. The siRNA sequences used were as follows: forward, 5′-GCACGUGAAGAUUUCUGCACCUAAU-3′, reverse, 5′-AUUAGGUGCAGAAAUCUUCACGUGC-3′.

The negative control siRNA was purchased from Invitrogen (catalog no. 12935300).

THP1, human monocytes, human monocyte-derived macrophages (hMDMs), or HeLa cells were transfected with siRNA using Lipofectamine 2000 according to the manufacturer’s instructions. At 24 h after transfection, the cells were used for further experiments.

After indicated transfection or stimulation, HeLa cells were fixed with 4% paraformaldehyde in PBS and permeabilized with Triton X-100 and then blocked with 1% BSA in PBS. Nuclei were stained with DAPI.

The data are presented as the mean ± SD from at least three independent experiments. The statistical comparisons between the different treatments were performed using the unpaired Student t test, and p < 0.05 was considered statistically significant.

To investigate whether Ku70 plays a role during HTLV-1 infection, we first examined the expression pattern of Ku70 in HTLV-1–infected HeLa or PMA-THP1 cells (a human macrophage-like cell line). HeLa and PMA-THP1 cells were cocultured with MT2 (HTLV-1–transformed T cell line) cells. Immunoblot assays demonstrated that endogenous Ku70 protein was markedly upregulated in both the HeLa and PMA-THP1 cells after coculture with MT2 cells (Fig. 1A). Furthermore, the amount of Ku70 increased in a dose-dependent fashion with the amount of added MT2 cells (Fig. 1B). Then, we transfected HeLa cells with HTLV-1 RTI ssDNA90, and immunoblot assays demonstrated that the Ku70 expression was upregulated with the increasing amounts of ssDNA90 (Fig. 1C). Finally, we found that Ku70 expression was induced in both HeLa and PMA-THP1 cells by ssDNA90 stimulation, although the expression patterns were somehow different in time course assays (Fig. 1D). Taken together, these data suggested that Ku70 was induced by HTLV-1 infection and HTLV-1 RTI ssDNA90 stimulation.

FIGURE 1.

Ku70 expression was induced by HTLV-1 infection. (A) HeLa (top) or PMA-THP1 cells (bottom) were cocultured with MT2 cells for the indicated periods of time. Afterwards, the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays. (B) HeLa cells were cocultured with MT2 cells at a ratio of 1:0, 1:0.01, 1:0.1, and 1:1 for 24 h. Afterwards, the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays. (C) HeLa cells were transfected with indicated doses of ssDNA90 for 24 h. Afterwards, the cells were lysed for immunoblot assays. (D) HeLa (top) or PMA-THP1 (bottom) cells were transfected with 0.5 μg/ml ssDNA90 for the indicated periods of time. Afterwards, the cells were lysed for immunoblot assays. β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments.

FIGURE 1.

Ku70 expression was induced by HTLV-1 infection. (A) HeLa (top) or PMA-THP1 cells (bottom) were cocultured with MT2 cells for the indicated periods of time. Afterwards, the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays. (B) HeLa cells were cocultured with MT2 cells at a ratio of 1:0, 1:0.01, 1:0.1, and 1:1 for 24 h. Afterwards, the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays. (C) HeLa cells were transfected with indicated doses of ssDNA90 for 24 h. Afterwards, the cells were lysed for immunoblot assays. (D) HeLa (top) or PMA-THP1 (bottom) cells were transfected with 0.5 μg/ml ssDNA90 for the indicated periods of time. Afterwards, the cells were lysed for immunoblot assays. β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments.

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To determine the functions of Ku70, HeLa cells overexpressing Ku70 were cocultured with MT2 cells, and the expression levels of HTLV-1 proteins were investigated. Real-time PCR results indicated that the expression levels of HTLV-1 proviral transcripts for Tax, p19, Env, and px were impaired, and immunoblot assays demonstrated that Tax and p19 levels were decreased at protein levels in the presence of Ku70 (Fig. 2A, 2B). We confirmed these results in PMA-THP1 cells, and the real-time PCR results indicated that Ku70 overexpression led to decreased expression levels of HTLV-1 proviral transcripts for Tax and p19 in MT2-cocultured PMA-THP1 cells (Fig. 2C). Taken together, these data suggested that Ku70 expression decreased HTLV-1 protein expression.

FIGURE 2.

Effects of Ku70 overexpression on HTLV-1 protein expression. (A) HeLa cells were transfected with empty vector (Vec) or Flag tagged-Ku70. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for real-time PCR analyses. (B) HeLa cells were transfected with empty vector (−) or Flag–tagged Ku70 (+). At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays. (C) PMA-THP1 cells were transfected with empty vector (Vec) or Flag-tagged Ku70. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for real-time PCR analyses. β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

FIGURE 2.

Effects of Ku70 overexpression on HTLV-1 protein expression. (A) HeLa cells were transfected with empty vector (Vec) or Flag tagged-Ku70. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for real-time PCR analyses. (B) HeLa cells were transfected with empty vector (−) or Flag–tagged Ku70 (+). At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays. (C) PMA-THP1 cells were transfected with empty vector (Vec) or Flag-tagged Ku70. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for real-time PCR analyses. β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

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To investigate the role of endogenous Ku70 in HTLV-1 infection, we examined the effects of knockdown of Ku70 on HTLV-1 protein expression. We designed two pairs of siRNA oligonucleotides specific for Ku70 RNA (K1 and K2) and a control siRNA (KC). The results showed that K2 efficiently silenced endogenous Ku70 expression in HeLa cells (Fig. 3A). K2 was then used for subsequent experiments. As shown in Fig. 3B, in immunoblot assays, the knockdown of endogenous Ku70 increased the expression levels of Tax and p19 in HeLa cells after cocultured with MT2 cells (Fig. 3B). Consistently, real-time PCR assays indicated that the expression of HTLV-1 proviral transcripts for Tax, p19, EnV, and px was enhanced after the knockdown of Ku70 in MT2-cocultured HeLa cells (Fig. 3C). Similar results were observed in MT2-cocultured PMA-THP1 cells (Fig. 3D, 3E). Taken together, these data suggested that endogenous Ku70 limited HTLV-1 protein expression.

FIGURE 3.

Effects of Ku70 knockdown on HTLV-1 protein expression. (A) HeLa cells were transfected with control siRNA (KC) or Ku70-specific siRNA (K1 and K2). At 24 h after transfection, the cells were lysed for immunoblot assays. (B and C) HeLa cells were transfected with KC or K2. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays (B) or real-time PCR analyses (C). (D and E) PMA-THP1 cells were transfected with KC or K2. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays (D) or real-time PCR analyses (E). β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SD (n = 3). *p < 0.05.

FIGURE 3.

Effects of Ku70 knockdown on HTLV-1 protein expression. (A) HeLa cells were transfected with control siRNA (KC) or Ku70-specific siRNA (K1 and K2). At 24 h after transfection, the cells were lysed for immunoblot assays. (B and C) HeLa cells were transfected with KC or K2. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays (B) or real-time PCR analyses (C). (D and E) PMA-THP1 cells were transfected with KC or K2. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays (D) or real-time PCR analyses (E). β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SD (n = 3). *p < 0.05.

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Given that Ku70 is involved in DNA and DNA viruses triggered innate immune responses, including IFN-β, IL-6, and some chemokines, we examined whether Ku70 affected HTLV-1 infection–triggered antiviral responses. HeLa cells were transfected with Ku70 and then cocultured with MT2 cells. Real-time PCR assays indicated that the expression levels of IFN-β and TNF-α were enhanced in the presence of Ku70 (Fig. 4A). Western blot assays suggested that the phosphorylation of IRF3 and p65 was increased in presence of Ku70 (Fig. 4B). However, Ku80, which usually forms a heterodimer with Ku70, did not affect the expression levels of Tax, p19, and IFN-β in MT2-cocultured HeLa cells (Supplemental Fig. 1). We next determined whether endogenous Ku70 regulated HTLV-1 infection–induced IFN-β production. We found that knockdown of Ku70 caused decreased IFN-β expression in MT2-cocultured HeLa cells (Fig. 4C). Consistently, the phosphorylation of IRF3 and p65 was also impaired in Ku70-silenced HeLa cells after coculture with MT2 (Fig. 4D). Similar results were observed in MT2-cocultured PMA-THP1 cells. After coculture with MT2 cells, PMA-THP1 cells transfected with Ku70 siRNA K2 showed impaired expression levels of IFN-β, TNF-α, and an IFN-stimulated gene (ISG56), with decreased phosphorylation levels of IRF3 and p65 (Fig. 4E, 4F). Taken together, these data suggested that Ku70-enhanced HTLV-1 infection triggered antiviral responses.

FIGURE 4.

Effects of Ku70 on antiviral responses against HTLV-1 infection. (A and B) HeLa cells were transfected with empty vector (Vec) or Flag-tagged Ku70. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for real-time PCR analyses (A) or immunoblot assay (B). (C and D) HeLa cells were transfected with KC or K2. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for real-time PCR analyses (C) or immunoblot assay (D). (E and F) PMA-THP1 cells were transfected with KC or K2. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for real-time PCR analyses (E) or immunoblot assay (F). β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SD (n = 3). *p < 0.05.

FIGURE 4.

Effects of Ku70 on antiviral responses against HTLV-1 infection. (A and B) HeLa cells were transfected with empty vector (Vec) or Flag-tagged Ku70. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for real-time PCR analyses (A) or immunoblot assay (B). (C and D) HeLa cells were transfected with KC or K2. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for real-time PCR analyses (C) or immunoblot assay (D). (E and F) PMA-THP1 cells were transfected with KC or K2. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for real-time PCR analyses (E) or immunoblot assay (F). β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SD (n = 3). *p < 0.05.

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Next, we tried to determine the role of Ku70 in HTLV-1 RTI ssDNA90-induced antiviral responses and found that the knockdown of Ku70 inhibited ssDNA90-induced expression of IFN-β in HeLa cells, with decreased phosphorylation levels of IRF3 and p65 (Fig. 5A, 5B). Consistently, the knockdown of Ku70 also inhibited ssDNA90-induced expression of IFN-β in PMA-THP1 cells, with decreased phosphorylation levels of IRF3 and p65 (Fig. 5C, 5D). Then we tried to determine the roles of Ku70 during some other DNA or virus stimulation. Interestingly, Ku70 expression could be induced by ssDNA90 transfection or HTLV-1 infection but not by an irrelevant ssDNA (scrambled ssDNA90), HSV60 (the double-stranded 60-bp oligonucleotide derived from the HSV-1 genome), or HSV-1 stimulation (Fig. 5E). Furthermore, Ku70 knockdown only inhibited ssDNA90-induced IFN-β production and had little effect on HSV60- or cGAMP-induced IFN-β production (Fig. 5F). Taken together, these data suggested that Ku70-enhanced HTLV-1 RTI ssDNA90 induced antiviral responses.

FIGURE 5.

Effects of Ku70 knockdown on ssDNA90-triggered innate immune responses. (A and B) HeLa cells were transfected with KC or K2. At 24 h after transfection, the cells were transfected with 0.5 μg/ml ssDNA90 for another 8 h. Then the cells were lysed for real-time PCR analyses (A) or immunoblot assay (B). (C and D) PMA-THP1 cells were transfected with KC or K2. At 24 h after transfection, the cells were transfected with 0.5 μg/ml ssDNA90 for another 8 h. Then the cells were lysed for real-time PCR analyses (C) or immunoblot assay (D). (E) PMA-THP1 cells were stimulated with 0.5 μg/ml scrambled ssDNA90, 0.5 μg/ml HSV60, 0.5 μg/ml ssDNA90, HTLV-1 (by coculture with MT2 cells), or HSV-1 for 24 h. Then the cells were lysed for immunoblot assays. (F) PMA-THP1 cells were transfected with KC or K2. At 24 h after transfection, the cells were stimulated with 0.5 μg/ml scrambled ssDNA90, 0.5 μg/ml ssDNA90, 0.5 μg/ml HSV60, or 1 μg/ml cGAMP for 8 h. Then the cells were lysed for real-time PCR analyses. β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01. Scram, scrambled ssDNA90.

FIGURE 5.

Effects of Ku70 knockdown on ssDNA90-triggered innate immune responses. (A and B) HeLa cells were transfected with KC or K2. At 24 h after transfection, the cells were transfected with 0.5 μg/ml ssDNA90 for another 8 h. Then the cells were lysed for real-time PCR analyses (A) or immunoblot assay (B). (C and D) PMA-THP1 cells were transfected with KC or K2. At 24 h after transfection, the cells were transfected with 0.5 μg/ml ssDNA90 for another 8 h. Then the cells were lysed for real-time PCR analyses (C) or immunoblot assay (D). (E) PMA-THP1 cells were stimulated with 0.5 μg/ml scrambled ssDNA90, 0.5 μg/ml HSV60, 0.5 μg/ml ssDNA90, HTLV-1 (by coculture with MT2 cells), or HSV-1 for 24 h. Then the cells were lysed for immunoblot assays. (F) PMA-THP1 cells were transfected with KC or K2. At 24 h after transfection, the cells were stimulated with 0.5 μg/ml scrambled ssDNA90, 0.5 μg/ml ssDNA90, 0.5 μg/ml HSV60, or 1 μg/ml cGAMP for 8 h. Then the cells were lysed for real-time PCR analyses. β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01. Scram, scrambled ssDNA90.

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We confirmed the role of Ku70 in HTLV-1–infected primary human monocytes and primary hMDMs. We examined the expression of Ku70 in primary human monocytes after coculture with MT2 cells. As shown in Fig. 6A, Ku70 had a strong expression in MT2-cocultured human monocytes whereas Ku70 could not be detected in untreated human monocytes. Then we silenced Ku70 in human monocytes by K2 and investigated its effects on host defenses against HTLV-1 infection. Western blot assays showed that Ku70-silenced human monocytes had a higher expression level of p19 and a decreased IRF3 phosphorylation level after coculture with MT2 cells (Fig. 6B). Consistently, real-time PCR assays indicated that the expression of HTLV-1 proviral transcripts for Tax, p19, and px was enhanced after the knockdown of Ku70 in MT2-cocultured human monocytes (Fig. 6C). Furthermore, the expression levels of IFN-β, RANTES, TNF-α, IL-6, and IFN-λ were decreased in Ku70-silenced human monocytes after coculture with MT2 cells (Fig. 6C). Then we investigated the role of Ku70 in primary hMDMs infected by cell-free HTLV-1. Western blot assays showed that the expression level of endogenous Ku70 protein was markedly upregulated in hMDMs after cell-free HTLV-1 infection (Fig. 6D). Additionally, the knockdown of endogenous Ku70 increased the expression level of p19 and decreased the IRF3 phosphorylation level in hMDMs after cell-free HTLV-1 infection (Fig. 6E). Taken together, these data suggested that Ku70 affected HTLV-1 replication and innate host defenses against HTLV-1 infection in primary human monocytes and hMDMs.

FIGURE 6.

Effects of Ku70 knockdown on antiviral responses in primary human monocytes and hMDMs. (A) Human monocytes were cocultured with MT2 cells for 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays. (B and C) Human monocytes were transfected with KC or K2. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot analyses (B) or real-time PCR assay (C). (D) hMDMs were infected with HTLV-1 for 24 h. Then the cells were lysed for immunoblot assays. (E) hMDMs were transfected with KC or K2. At 24 h after transfection, the cells were infected with HTLV-1 for 24 h. Then the cells were lysed for immunoblot assays. β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001.

FIGURE 6.

Effects of Ku70 knockdown on antiviral responses in primary human monocytes and hMDMs. (A) Human monocytes were cocultured with MT2 cells for 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assays. (B and C) Human monocytes were transfected with KC or K2. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot analyses (B) or real-time PCR assay (C). (D) hMDMs were infected with HTLV-1 for 24 h. Then the cells were lysed for immunoblot assays. (E) hMDMs were transfected with KC or K2. At 24 h after transfection, the cells were infected with HTLV-1 for 24 h. Then the cells were lysed for immunoblot assays. β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments and are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001.

Close modal

When considering that Ku70 can bind dsDNA and has been reported as a DNA sensor for DNA viruses, we wondered whether Ku70 could bind ssDNA and act as a DNA sensor for the retrovirus HTLV-1. ssDNA90 was marked by biotin and transfected into HeLa cells with Flag-Ku70. Coimmunoprecipitation assays revealed that Ku70 interacted with ssDNA90 (Fig. 7A). We confirmed this result by a competition experiment and found that the association of biotin-ssDNA90 with Ku70 could be competed by ssDNA90 in a dose-dependent manner (Fig. 7B). Furthermore, endogenous Ku70 association with ssDNA90 was detected in HeLa cells (Fig. 7C). Taken together, these data suggested that Ku70 interacted with ssDNA90 and could be a sensor for HTLV-1 RTI.

FIGURE 7.

Ku70 sensed ssDNA90 and induced IFN-β production through STING. (A) HeLa cells were transfected with 2 μg of Flag-Ku70. At 24 h after transfection, the cells were transfected with 1 μg of ssDNA90 or 1 μg of biotinylated ssDNA90 for 8 h. The cell lysates were immunoprecipitated (IP) with streptavidin beads and immunoblotted (IB) with anti-Flag. (B) HeLa cells were transfected with 1 μg of Flag-Ku70. At 24 h after transfection, the cells were transfected with 0.5 μg of biotinylated ssDNA90 and increasing concentrations of ssDΝΑ90 (0, 0.25, 0.5, and 1 μg) for 8 h. The cell lysates were immunoprecipitated with streptavidin beads and immunoblotted with anti-Flag. (C) HeLa cells were transfected with 1 μg of ssDNA90 or 1 μg of biotinylated ssDNA90 for 8 h. The cell lysates were immunoprecipitated with streptavidin beads and immunoblotted with anti-Ku70. (D) HeLa cells were transfected with Flag-Ku70. At 24 h after transfection, the cells were cocultured with MT2 cells or left untreated as indicated for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed. The cell lysates were immunoprecipitated with anti-STING and immunoblotted with anti-Flag or anti-STING as indicated. (E) HeLa cells were were cocultured with MT2 cells or left untreated as indicated for 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed. The cell lysates were immunoprecipitated with anti-STING and immunoblotted with anti-Ku70 or anti-STING as indicated. (F) HeLa cells were transfected with expressing plasmids for HA-STING and Flag-Ku70. At 24 h after transfection, HeLa cells were transfected with 1 μg of ssDNA90 or left untreated for another 8 h. Then the cells were prepared for confocal microscopy. Nuclei were stained with DAPI. Original magnification ×400. (G) HeLa cells were transfected with control siRNA (SC) or STING-specific siRNA (ST). Twenty-four hours later, the cells were transfected with Flag-Ku70. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assay. β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments.

FIGURE 7.

Ku70 sensed ssDNA90 and induced IFN-β production through STING. (A) HeLa cells were transfected with 2 μg of Flag-Ku70. At 24 h after transfection, the cells were transfected with 1 μg of ssDNA90 or 1 μg of biotinylated ssDNA90 for 8 h. The cell lysates were immunoprecipitated (IP) with streptavidin beads and immunoblotted (IB) with anti-Flag. (B) HeLa cells were transfected with 1 μg of Flag-Ku70. At 24 h after transfection, the cells were transfected with 0.5 μg of biotinylated ssDNA90 and increasing concentrations of ssDΝΑ90 (0, 0.25, 0.5, and 1 μg) for 8 h. The cell lysates were immunoprecipitated with streptavidin beads and immunoblotted with anti-Flag. (C) HeLa cells were transfected with 1 μg of ssDNA90 or 1 μg of biotinylated ssDNA90 for 8 h. The cell lysates were immunoprecipitated with streptavidin beads and immunoblotted with anti-Ku70. (D) HeLa cells were transfected with Flag-Ku70. At 24 h after transfection, the cells were cocultured with MT2 cells or left untreated as indicated for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed. The cell lysates were immunoprecipitated with anti-STING and immunoblotted with anti-Flag or anti-STING as indicated. (E) HeLa cells were were cocultured with MT2 cells or left untreated as indicated for 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed. The cell lysates were immunoprecipitated with anti-STING and immunoblotted with anti-Ku70 or anti-STING as indicated. (F) HeLa cells were transfected with expressing plasmids for HA-STING and Flag-Ku70. At 24 h after transfection, HeLa cells were transfected with 1 μg of ssDNA90 or left untreated for another 8 h. Then the cells were prepared for confocal microscopy. Nuclei were stained with DAPI. Original magnification ×400. (G) HeLa cells were transfected with control siRNA (SC) or STING-specific siRNA (ST). Twenty-four hours later, the cells were transfected with Flag-Ku70. At 24 h after transfection, the cells were cocultured with MT2 cells for another 24 h. Then the cells were washed with PBS three times to remove MT2 cells and lysed for immunoblot assay. β-Actin was used as a loading control in the immunoblot assays. The data are representative of three independent experiments.

Close modal

It has been reported that DNA-PK interacts with the STING-dependent signaling pathway, leading to IFN-β production (24). Therefore we investigated the role of STING in Ku70-induced IFN-β production. Flag-Ku70 was transfected in HeLa cells, and coimmunoprecipitation experiments showed that Ku70 associated with STING in the same complex and this association was enhanced after the cells were cocultured with MT2 cells (Fig. 7D). We confirmed this result by detecting endogenous association between Ku70 and STING. As shown in Fig. 7E, the Ku70 expression level was higher after HTLV-1 infection and the amount of Ku70 associated with STING was increased. Additionally, confocal microscopy indicated that Flag-Ku70 translocated from the nucleus to the cytoplasm where it colocalized with HA-STING in HeLa cells after ssDNA90 stimulation (Fig. 7F). It has been reported that activated STING translocated from endoplasmic reticulum, through Golgi apparatus, and to the perinuclear microsomes (27). Therefore, we investigated the effects of Ku70 knockdown on STING translocation. As shown in Supplemental Fig. 2, ssDNA90 transfection could induce STING translocation to Golgi, and Ku70 knockdown decreased ssDNA90-induced STING translocation to some extent. Next, we examined the effects of STING knockdown on the function of Ku70 during HTLV-1 infection. We found that in STING-silenced HeLa cells, Ku70 overexpression had a weaker effect on IRF3 phosphorylation and p19 expression than in the cells transfected with control STING siRNA (Fig. 7G). Additionally, we detected whether the roles of Ku70 in HTLV-1 infection were dependent on cGAS. We examined the effects of Ku70 overexpression on HTLV-1 protein expression in cGAS-silenced HeLa cells. As shown in Supplemental Fig. 3, cGAS knockdown did not affect the effects of Ku70 on HTLV-1 protein expression. It is known that not only STING but also MyD88 and β-catenin serve as signal mediators to induce IFN-β in DNA-sensing pathways (28, 29). We investigated the roles of MyD88 and β-catenin during HTLV-1 infection. As shown in Supplemental Fig. 4A, although the phosphorylation of β-catenin was increased after HTLV-1 infection, the knockdown of Ku70 had no significant effect on the phosphorylation of β-catenin. Then we investigated the role of MyD88 during HTLV-1 infection. HeLa cells overexpressing MyD88 were cocultured with MT2 cells, and the expression levels of IFN-β and Tax were investigated. Real-time PCR results indicated that MyD88 had no significant effect on the expression levels of IFN-β and Tax (Supplemental Fig. 4B). Furthermore, no significant interaction was detected between Ku70 and MyD88 (Supplemental Fig. 4C). Taken together, these data suggested that Ku70 induced IFN-β production in a STING-dependent pathway during HTLV-1 infection.

DNA-PK components are expressed in most cancerous and normal tissues, whereas they are absent in macrophages and lymphoid cells (30). Our data showed that HeLa cells expressed Ku70 at a low level and no Ku70 expression was detected in the macrophage-like cell lines PMA-THP1 or in the primary human monocytes. However, upon coculture with HTLV-1–transformed MT2 cells or stimulation with HTLV-1 RTI ssDNA90, all of these cells had a relatively much stronger expression of Ku70. Additionally, cell-free HTLV-1 infection could induce the expression of Ku70 in the primary hMDMs. Interestingly, Ku70 expression could not be induced by scrambled ssDNA90, HSV60, or HSV-1 stimulation, suggesting that some specificity might exist in the roles of Ku70 during viral infection. Therefore it was meaningful to explore the function of Ku70 in regulating HTLV-1 infection. Interestingly, the expression patterns of Ku70 in HeLa and PMA-THP1 cells were somehow different in time course assays. In HeLa cells, the high expression of Ku70 continued 24 h after stimulation. In PMA-THP1 cells, the Ku70 expression could not be detected in the cells without stimulation and peaked at 8 h after HTLV-1 infection or ssDNA90 transfection, following a very low level at 24 h after stimulation. These data suggested that the expression of Ku70 might be tightly regulated in innate immune cells.

Several groups reported that Ku70 triggers innate host defenses against DNA viruses upon sensing viral dsDNA in the cytoplasm (2325). However, in the DNA virus adenovirus, Ku70 was found to associate with the viral genome and to be important for viral growth (31). In the present study, we reported that, apart from dsDNA, Ku70 could detect the HTLV-1 RTI ssDNA90 and inhibited HTLV-1 replication, suggesting a role of Ku70 in antiretroviral innate immunity. Coimmunoprecipitation assays revealed that both exogenous and endogenous Ku70 interacted with ssDNA90. Ku70 overexpression inhibited HTLV-1 protein expression whereas Ku70 knockdown enhanced HTLV-1 protein expression in HeLa and PMA-THP1 cells. We confirmed these data in human primary cells, and similar results were observed in primary human monocytes and hMDMs.

Type I IFNs, including IFN-α and IFN-β, have been used for the treatment of HTLV-1–associated diseases (32, 33), suggesting the important role of type I IFNs in HTLV-1 infection. Our findings demonstrated that Ku70 positively regulated HTLV-1–induced IFN-β production. IFN-β production and IRF3 phosphorylation were increased in the presence of Ku70 and decreased in Ku70-silenced HeLa or PMA-THP1 cells after HTLV-1 infection or HTLV-1 RTI ssDNA90 stimulation. We also observed that Ku70 overexpression enhanced TNF-α production and the p65 phosphorylation whereas Ku70 knockdown impaired these processes. The effects of Ku70 on the antiviral responses were more significantly in primary human monocytes, with the impaired production of a serious of cytokines, such as IFN-β, TNF-α, IL-6, and RANTES, following the knockdown of Ku70. Interestingly, our data showed that HTLV-1 infection induced the expression of IFN-λ and Ku70 knockdown resulted in decreased expression of IFN-λ, which has been demonstrated to be important in restricting viral infections (34). It is suggested that IFN-λ3 expression was significantly higher in HTLV-1–associated myelopathy/tropical spastic paraparesis patients than in HTLV-1 asymptomatic carriers (35). However, the role of IFN-λ in HTLV-1 infections is not well understood and needs further examinations.

STING has been demonstrated to be critical in DNA-PK–induced IRF3-dependent innate responses triggered by dsDNA and DNA viruses (24). Because Ku70 affected ssDNA90 or HTLV-1 stimulated IFN-β production and the IRF3 phosphorylation, we examined whether STING was involved in Ku70-mediated antiviral responses. Our findings indicated that STING was associated Ku70 during HTLV-1 infection. It has been reported that STING translocated from endoplasmic reticulum, through Golgi apparatus, and to the perinuclear microsomes upon activation (27). Our data showed that Ku70 translocated from the nucleus to the cytoplasm where it colocalized with STING in HeLa cells after ssDNA90 stimulation. Furthermore, STING knockdown could attenuate the effects of Ku70 on IRF3 phosphorylation and HTLV-1 infection, suggesting that STING was involved in Ku70-mediated antiviral defenses.

In the life cycle of retroviruses, several DNA-containing nucleic acid structures are produced. Our results suggested that the ssDNA could be detected by Ku70 and triggered the innate antiviral responses. However, the linear retroviral dsDNA migrates into the nucleus where only a fraction of it integrates into the host genome (7). Considering that Ku70 could bind dsDNA from DNA viruses, Ku70 has the potential to recognize the dsDNA from HTLV-1. Further studies may need to clarify this item.

Taken together, our research demonstrated a new DNA sensor against retrovirus, which detected HTLV-1 RTI ssDNA and induced antiviral responses, including the production of IFN-β, IFN-λ, TNF-α, and other cytokines. These findings may expand our knowledge on host antiviral innate immunity and may provide clues for designing novel therapeutic interventions to treat HTLV-1–associated diseases.

This work was supported by National Natural Science Foundation of China Grants 31400776, U1504811, and 31600697, Universities of Henan Province Key Scientific Research Project Grants 15A310023 and 16A31003, and by Xinxiang Medical University Scientific Research Grants 2013QN116 and 2014QN156.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • cGAMP

    cyclic GMP–AMP

  •  
  • cGAS

    cyclic GMP–AMP synthase

  •  
  • DNA-PK

    DNA-dependent protein kinase

  •  
  • HA

    hemagglutinin

  •  
  • hMDM

    human monocyte-derived macrophage

  •  
  • HTLV-1

    human T lymphotropic virus type 1

  •  
  • IRF

    IFN regulatory factor

  •  
  • PMA-THP1

    PMA-differentiated THP1

  •  
  • RTI

    reverse transcription intermediate

  •  
  • siRNA

    small interfering RNA

  •  
  • STING

    stimulator of IFN genes.

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:
1102
1107
.

The authors have no financial conflicts of interest.

Supplementary data