Targeting the gene encoding human T-cell leukemia virus type 1 basic zip factor via CRISPR/Cas9 potentially mitigates viral infection

Targeting the gene encoding human T-cell leukemia virus type 1 basic zip factor via CRISPR/Cas9 potentially mitigates viral infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting the gene encoding human T-cell leukemia virus type 1 basic zip factor via CRISPR/Cas9 potentially mitigates viral infection Yuki Hashikura, Misaki Izaki, Kazumi Umeki, Mami Azeta, Katsumi Kawano, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6562052/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Herein, we investigated the role of an essential transcription factor in the human T-cell leukemia virus type 1 (HTLV-1) provirus, the HTLV-1 basic zip factor (HBZ), in HTLV-1 infections and adult T-cell leukemia/lymphoma (ATL). We designed five synthetic guide RNAs (sgRNAs) targeting HBZ and introduced them into ATL and HTLV-1 infected cell lines using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Of all sgRNAs, sgRNA 171 was the most efficient in introducing mutations at the target site as 70–80% of Cas9/sgRNA 171-transfected host cells contained mutations. Various types of mutations, including deletions, substitutions, insertion, and combinations, were detected in the Cas9/sgRNA 171-treated cells. Based on the predicted peptide sequence, most mutant clones were assumed to inactivate the HBZ mRNA. The mRNA levels of the transactivator from the X-gene region ( tax ) increased after HBZ editing by Cas9/sgRNA 171. No off-target effects were observed in the four human genome regions partially homologous to the sgRNA 171 target sequence. Furthermore, ST-1 cells transfected with Cas9/sgRNA 171 displayed significantly reduced proliferation. These findings suggest that the HBZ mRNA might be crucial for the survival of HTLV-1-infected cells, including ATL, providing insights into the molecular pathogenesis of the HTLV-1 provirus. Health sciences/Diseases/Haematological diseases/Haematological cancer/Lymphoma/Non hodgkin lymphoma/T cell lymphoma Health sciences/Diseases/Infectious diseases/Viral infection human T-cell leukemia virus type-1 HTLV-1 basic zip factor CRISPR/Cas9 antivirus targeted therapies synthetic guide RNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis/uveitis [1–4]. HTLV-1 mainly targets the CD4 + T-lymphocytes. Around 3–5% of HTLV-1 carriers may develop ATL after a long latency [5]. The viral proteins, transactivator from the X-gene region (Tax) and HTLV-1 basic zip factor (HBZ), are significantly involved in the proliferation and tumorigenesis of HTLV-1 infected cells [6, 7]. Tax induces cell proliferation and resistance to apoptosis in HTLV-1 infected cells [8–12]. Conversely, Tax acts as an antigen recognized by cytotoxic T-lymphocytes (CTLs). Therefore, host CTLs can eliminate HTLV-1 infected cells [13], resulting in a decrease in these cells after long-term latent infection [13]. Tax expression can also be attenuated by partial loss of provirus, nonsense mutations, and 5′ long terminal repeat (LTR) promoter region methylation [14–17]. Furthermore, HBZ downregulates Tax, which activates the transcription of the viral genes via the 5′LTR [18]. The decrease in Tax expression might be a potential mechanism via which HTLV-1 infected cells escape the host immune system, allowing the infection to persist throughout life [19]. HBZ, expressed constitutively in all ATL cells, promotes the proliferation of the infected cells [20]. Single-molecule RNA fluorescence in situ hybridization (FISH) studies showed that 30–80% of HTLV-1-infected peripheral blood mononuclear cells are HBZ -positive [21]. HBZ mRNA is a bifunctional RNA that encodes the protein and acts as an RNA [22]. Several studies have suggested that the HBZ mRNA might be essential for the proliferation, suppression of apoptosis, and production of cytokines/chemokines in HTLV-1 infected cells [23–24]. Furthermore, HBZ mRNA can exist in spliced or unspliced forms. The spliced mRNA suppresses the functions of Tax more effectively than the unspliced form [25]. Transgenic mice expressing HBZ have been shown to develop T-cell lymphoma. Based on these observations, the HBZ mRNA has been implicated in the carcinogenesis of HTLV-1-infected cells [26]. Thus, although the roles of HBZ in the pathogenesis of HTLV-1 infection and its associated diseases have been investigated, much remains unclear. In this study, we aimed to investigate the roles and functions of HBZ in ATL cell lines using synthetic guide RNAs (sgRNAs). These sgRNAs were designed to introduce mutations in the sequence adjacent to the translation initiation site of the spliced HBZ mRNA and into HBZ using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated HBZ gene knockout resulted in an increase in Tax expression and a decrease in cell proliferation. Therefore, the functions of HBZ in HTLV-1 infected cells and its potential as a therapeutic target can be studied using the CRISPR/Cas9 system with an efficient sgRNA. Methods Cell lines For this study, we used three ATL cell lines: MT-1, TL-Om1, and ST-1. MT-1 was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB 1209 Lot No. 12172007, Osaka, Japan) [27]. TL-Om1 and ST-1 were kindly gifted by Kazuo Sugamura [28] and Hiroo Hasegawa (Department of Laboratory Medicine, Nagasaki University School of Medicine), respectively [29]. MT-1 was grown at 37°C in a humidified atmosphere with 5% CO 2 in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum with 5 μg/mL penicillin, 5 μg/mL streptomycin, and 10 μg/mL neomycin. The ST-1 and TL-Om1 cell lines were grown at 37°C in a humidified atmosphere with 5% CO 2 in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum and 10 μg/mL recombinant human IL2 (PeproTech, Cranbury, USA) with 5 μg/mL penicillin, 5 μg/mL streptomycin, and 10 μg/mL neomycin. Preparation and transfection of sgRNA The sgRNAs were designed using the CRISPR/Cas9 guide RNA design software CRISPRdirect (https://crispr.dbcls.jp/) to search for candidate sgRNA sequences within the HBZ sequence (Accession no. AB219938). We designed five different sgRNAs by selecting a region with minimal homology to the human genomic DNA based on the idea that a frameshift mutation can efficiently inactivate HBZ if a deletion can be introduced in a region close to the N-terminus of the HBZ . One of these sgRNAs, sgRNA 171, was predicted to cleave the translation start site of HBZ . The remaining sgRNAs (sgRNA 255, sgRNA 258, sgRNA 395, and sgRNA 405) were presumed to cleave downstream of the translation start site (Table 1). A protospacer adjacent motif was present in these sgRNA sequences. TrueGuide™ sgRNA N.C. (Thermo Fisher Scientific, Tokyo, Japan) was used as the negative control (NC). Cas9 and sgRNA (Cas9/sgRNA) were transfected into the cell lines using the Invitrogen™ Neon™ Transfection System kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions. Electroporation was performed using a Neon Transfection System with a single pulse at 1,650 V for 20 ms. The cell solution was added to a 24-well plate and incubated at 37°C for 6 days under 5% CO 2 . Confirmation of the mutations The mutations created by CRISPR/Cas9 were screened using the Genomix Cleavage Detection Kit (Thermo Fisher Scientific). After culturing Cas9/sgRNA-transfected cells for 48 h, the cell pellets were collected by centrifugation at 1,500 rpm for 5 min and lysed using Cell Lysis Buffer (50 µL) containing 4% protein degrader by heated initially at 68°C for 15 min, followed by 95°C for 10 min. Then, 2 μL of this solution was used as a template for PCR. The primers used for sgRNA 171 target region amplification were forward primer—HTLV-8396F: 5′-CAGACTAAGGCTCTGACGTC-3′ and reverse primer—HTLV-8886R: 5′-AGACGTAGAGTTGAGCAAGC-3′. The primers for sgRNA 255 and sgRNA 258 target region amplification were forward primer—HTLV-7171F: 5′-CAAGCACAGCTTCCTCCTCC-3′ and reverse primer—HTLV-7518R: 5′-TGAGCCGATAACGCGTCCATC-3′. The primers for sgRNA 395 and sgRNA 405 target region amplification were forward primer—HTLV-6737F: 5′-CAAATCCTCCTTCTCCTGCA-3′, reverse primer—HTLV-7181R: 5′-AGCTGTGCTTGACGGTTTGC-3′. A suitable NC was selected for each of the three sgRNA target regions. The PCR was performed under the following conditions: one cycle of 95°C for 10 min, 40 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 20 s, and extension at 72°C for 30 s. Subsequently, 6 µL of the detection buffer was added to 3 µL of the PCR product and heated at 95°C. The mixture was cooled at 6°C per minute from 85°C to 25°C. After adding 1 µL of the detection enzyme, the mixture was heated at 37°C for 60 min. PCR products, including the mismatch base pair, were recognized by this enzyme, followed by cleavage to form two short bands. The PCR products digested with the detection enzyme were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining. Concurrently, non-digested PCR products were cloned to identify the mutations in the HTLV-1-infected cells. Then, they were subjected to a sequence assay using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Massachusetts, USA) and ABI Prism 3500 Genetic Analyzer (Applied Biosystems ThermoFisher, California, United States), according to the manufacturer’s instructions. Cell proliferation assay after CRISPR/Cas9 transfection Cells treated with CRISPR/Cas9 were collected and counted every 2 days after CRISPR/Cas9 transfection to assess cell proliferation. After staining the cells using trypan blue (Nacalai Tesque, Kyoto, Japan), viable cells were counted using a hemocytometer (NanoEntek, Seoul, Korea). The accuracy of the cell count of 5 × 10 5 /mL cells were 1.4 × 10 4 /mL standard deviation and 2.78% coefficient of variation. Quantification of HBZ and tax/rex mRNA These cells were collected every 2 days and transfected with Cas9/sgRNA 171. Total RNA was extracted from these cells using the TRIzol® Reagent (Life Technology Japan, Tokyo, Japan), according to the manufacturer’s instructions. The extracted RNA was reverse transcribed into complementary DNA using an M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, USA) with random primers (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. Reverse transcription real-time PCR (RT-qPCR) was performed to detect HTLV-1 tax /rex and HBZ mRNA levels. The PCR data was processed and analyzed based on the method described by Hashikura et al. [30]. RT-qPCR was performed in a duplicate manner using Light Cycler 2.0 (Roche Diagnostics, Mannheim, Germany). Relative quantification of HTLV-1 tax/rex and HBZ mRNA was performed using GAPDH mRNA as an internal control. HBZ and tax/rex mRNA were tested for significant differences from Cas9/sgRNA N.C. transfection. Western blot analysis of HBZ and Tax The cells transfected with Cas9/sgRNA 171 were collected every 2 days and then lysed using an sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.01% bromophenol blue, 10 mM β-β-mercaptoethanol) containing a protease inhibitor mixture (Thermo Fisher Scientific, Waltham, USA). The resultant cell lysates were heated to 98°C for 5 min in the sample buffer and separated on 8% SDS-polyacrylamide gels. Then, they were electroblotted onto polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Little Chalfont, UK). The primary antibodies used in this study were the mouse anti-Tax antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the rat anti-HBZ monoclonal antibody, mAb 91-1 [31]. As a loading control, mouse anti-α-tubulin antibody (Abcam, Cambridge, MA, USA) was used. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (horse antimouse IgG and goat antirat IgG; Cell Signaling Technology, Inc. Danvers, MA, USA). An enhanced chemiluminescence detection system was used to detect the protein signals (Cytiva, Tokyo, Japan), and they were measured by a luminescent image analyzer FUSION SOLO.7S. EDGE (VILBER, Marne-la-Vallée, France). The expression level of HBZ was measured as a value corrected using the optical density (O.D.) ratio of HBZ and Tubulin. Statistical analysis Parametric tests (Student t-test) were used to compare the differences in cell proliferation and RNA expression between the Cas9/sgRNA 171-treated cells and the NCs. P-values <0.05 were considered statistically significant. Statistical analyses were performed using SPSS Statistics 20 (IBM, Chicago, IL, USA). Results Evaluation of the mutation efficiency of the sgRNAs using the cleavage assay Among the five sgRNAs tested, Cas9/sgRNA 171 was the most effective at introducing mutations in HBZ , as shown by the cleavage assay results (Fig. 1; Supplementary Figure S1). Furthermore, Cas9/sgRNA 171 induced mutations at the methionine-encoding sequence of the HBZ translation start site. Consequently, sgRNA 171 was selected for inactivating HBZ in the following studies. Analysis of CRISPR/Cas9-induced mutations in LTR containing HBZ After transfecting cells with Cas9/sgRNA 171 and culturing them for 2 days, the mutations introduced in these cells were detected using the Genomix Cleavage Detection Kit. Mutations were observed in the translation initiation site of HBZ in the Cas9/sgRNA171-transfected cells (Table 2; Supplementary Figure S2). Furthermore, the genomic DNA extracted from the cells were subcloning and sequenced 2 days after the transfection. The results revealed various mutations, such as deletions and insertions in 24/30 clones (80%) in MT-1, 22/30 clones (73%) in TL-Om1, and 21/30 clones (70%) in ST-1 cell lines (Table 2). However, the mutation rate 6 days after transfection was reduced to 5/30 clones (17%) and 11/30 clones (37%) in the MT-1 and TM-Om1 cells, respectively. More than half of the sequences had no mutations (Table 2). Conversely, sequencing results revealed mutations in 19/30 clones (63%) in the ST-1 cells 6 days after transfection, which is comparable to that observed 2 days after transfection (Table 2). To confirm that Cas9/sgRNA 171 does not cleave human genomic DNA, we examined its off-target effects in the four human genome regions with partial homology to the sgRNA 171 sequences (12 of the 20 bases of sgRNA 171 matched). The results showed no insertions and deletions in any of the four regions. As far as we investigated, no recombination with the off-target regions in human genome DNA was observed (Supplementary Figure S3) Cell proliferation after CRISPR/Cas9 transfection The proliferation of CRISPR/Cas9-transfected cells was analyzed by counting cells at 2, 4, and 6 days after the transfection. MT-1 and TL-Om1 transfected with Cas9/sgRNA 171 exhibited suppressed proliferation compared with those transfected with Cas9/sgRNA NC, although the difference was not significant (Fig. 2a, b). The cell proliferation of Cas9/sgRNA 171-transfected ST-1 cells was markedly suppressed compared to those transfected with the NC (Fig. 2c). Expression of HBZ mRNA and tax/rex mRNA after transfection of Cas9/sgRNA 171 The mRNA levels of HBZ and tax/rex were investigated using RT-qPCR. The HBZ mRNA levels in the MT-1 and TL-Om1 cells decreased significantly until 4 days after transfection compared to those transfected with Cas9/sgRNA NC (p < 0.05) (Fig. 3a, b). However, this difference was non-significant 6 days after transfection (Fig. 3a, b). The HBZ mRNA levels in the Cas9/sgRNA 171-transfected ST-1 cells were significantly decreased compared with those transfected with Cas9/sgRNA NC until 6 days after transfection (Fig. 3c). In contrast, tax/rex mRNA levels in Cas9/sgRNA 171-treated MT-1 cells increased significantly till 6 days after transfection compared with the Cas9/sgRNA NC-transfected cells (Fig. 3d). While the tax/rex mRNA levels in TL-Om1 cells were below the detection limit until 4 days after CRISPR/Cas9 transfection. However, these levels increased significantly 6 days after transfection compared with those transfected with Cas9/sgRNA NC (Fig. 3e). No significant difference was seen in the tax/rex mRNA levels in ST-1 cells transfected with Cas9/sgRNA 171 and Cas9/sgRNA NC (Fig. 3f). Expression of HBZ and Tax protein after transfection of Cas9/sgRNA 171 The protein expression levels of HBZ and Tax in all three cell lines transfected with Cas9/sgRNA 171 were detected using Western blotting (Fig. 4a; Supplementary Figure S4–6). HBZ expression decreased in all cell lines after CRISPR/Cas9 transfection (Fig. 4b–d). While the tax mRNA levels showed an upward trend, its protein levels could not be detected using Western blotting (Fig. 4a). Discussion In this study, CRISPR/Cas9 was used to knock out the HBZ gene and suppress the expression of the corresponding mRNA and protein. The newly designed sgRNA 171 induced a mutation in the codon at the translation initiation site of spliced-type HBZ mRNA (Supplementary Figure S2), suppressing the HBZ mRNA and protein expression in all three cell lines. In ST-1 cells, which contain one copy of the HTLV-1 provirus per cell, cell proliferation was suppressed by HBZ gene knockout, whereas this inhibition was not observed in the MT-1 and TL-Om1 cell lines, in which multiple HTLV-1 proviruses were integrated per cell. This might be because the suppression of HBZ expression by CRISPR/Cas9 might upregulate tax expression in the MT-1 and TL-Om1 cell lines, suggesting that HBZ expression might affect cell proliferation and tax expression in the HTLV-1-infected cell lines. Two days after transfection with Cas9/sgRNA 171, the mutation efficiency observed in MT-1, TL-Om1, and ST-1 cells was approximately 70%. Deletions, substitutions, and insertions were observed in 80%, 73%, and 70% of the cells, respectively. The mutation introduction efficiency was comparable to that reported in a previous study in which CRISPR/Cas9 was transfected into activated human T cells. The results of this study were also consistent with ours [32]. Using DNA sequencing, we examined the off-target effects by analyzing the cleavage of similar DNA sequences. However, no off-target effects were observed with Cas9/sgRNA 171, indicating that this system can efficiently and specifically induce mutations in HBZ and control its expression. Other studies have shown that CRISPR/Cas9-based gene editing of the LTR region in the provirus of the human immunodeficiency virus (HIV), a retrovirus similar to HTLV-1, has low off-target effects [33]. These results suggest that provirus-specific DNA sequences nonhomologous to the human genome are ideal targets for CRISPR/Cas9 to achieve virus-specific genome editing in cells infected with retroviruses, including HTLV-1 [34]. The proliferation of ST-1 cells transfected with Cas9/sgRNA 171 was suppressed. However, this suppressive effect was lower in the MT-1 and TL-Om1 cells than the ST-1 cells. Spliced HBZ mRNA has been reported to suppress cell death and enhance cell proliferation [23, 25]. The proliferation ability of the MT-1 and TL-Om1 cell lines, which do not have genetic mutations in HBZ , can be attributed to the expression of the spliced HBZ mRNA in these cells. The three cell lines used in this study have been reported to have different numbers of proviruses integrated into the genome of each cell [35–37]. MT-1 and TL-Om1 have approximately 3 and 1.78 copies of provirus per cell, respectively, whereas ST-1 has one copy per cell. Therefore, the efficiency of Cas9/sgRNA 171 in introducing HBZ mutations per cell in MT-1 and TL-Om1 cells, which contain multiple proviruses per cell, may be relatively lower than in ST-1. Hence, HBZ mRNA expression was maintained in MT-1 and TL-Om1 cells, leading to cell proliferation. In this study, a significant decrease in HBZ mRNA was observed in the three cell lines 2 days after the transfection with Cas9/sgRNA 171. This might be due to the suppression of HBZ transcription. Furthermore, the deletion or insertion of the translation initiation codon sequence of HBZ by Cas9/sgRNA 171 might result in the generation of premature translation-termination codons due to a frameshift in HBZ , leading to the expression of mutant HBZ mRNA. The mRNA quality control mechanism of the host also might eliminate this mutant HBZ mRNA [38–40]. Reports have shown that the proproliferative effect of HBZ on HTLV-1-infected cells depends on the HBZ mRNA structure [20]. The Cas9/sgRNA 171-induced mutations in HBZ might also affect its RNA structure and suppress cell proliferation. Therefore, we speculated that HTLV-1-infected cells in which HBZ was completely knocked out were unable to proliferate and underwent cell death, whereas those in which HBZ was partially knocked out maintained HBZ expression and could survive and proliferate. As no mutations were introduced into the HBZ region of the proviruses in cells containing multiple proviruses, the HBZ mRNA levels were maintained, allowing the cells to proliferate. Furthermore, in HTLV-1-infected cells in which the HBZ gene was knocked out using Cas9/sgRNA, the expression of tax mRNA increased slightly, consistent with previous reports showing that HBZ regulates Tax expression [41]. This study has several limitations. First, the efficiency of inducing mutations in HBZ within the HTLV-1-infected cells was low. Although electroporation was selected as the transfection technique in this study, other transfection methods that are more efficient in inducing gene mutations in HTLV-1-infected cell lines should be considered. In the future, the transfection of CRISPR/Cas9 using a viral vector should be investigated to verify whether HBZ gene mutations can be introduced into various HTLV-1-infected cell lines containing multiple proviruses in the host genome. Second, as the initiation codon for HBZ , the target of sgRNA 171, is located in the 3′LTR, it is not easy to completely distinguish the 5′LTR from the 3′LTR for analysis and evaluation. Therefore, if mutations are introduced not only in the HBZ gene but also in the 5′LTR, they might be included in this analysis. Third, we have not yet verified whether HBZ can be edited by Cas9/sgRNA 171 in HTLV-1-infected cells from patients. HBZ mRNA and protein have been shown to be involved in the onset of ATL [20,24,42]. Therefore, future studies are required to determine whether Cas9/sgRNA 171 can also control the proliferation of HTLV-1-infected and ATL cells derived from patients. In conclusion, we used gene editing technology using CRISPR/Cas9 and a newly designed sgRNA 171 to introduce mutations into HBZ and suppress the expression of HBZ mRNA and protein. Our results demonstrated that knocking out HBZ resulted in the suppression of cell proliferation in HTLV-1-infected cell lines. Furthermore, the target retroviral and proviral gene sequences were specific to the retrovirus-infected cells and did not overlap with any region within the human genome, reducing the off-target effects and achieving successful gene modification using CRISPR/Cas9. Therefore, HBZ gene editing technology using CRISPR/Cas9 might be a potential therapeutic approach for HTLV-1 infections and ATL. Declarations Acknowledgments The authors would like to thank Ms. Y. Kaseda (Miyazaki University) for technical support and assistance. Author contributions Y.H. and M.I. contributed equally to this work. Conceptualization, Y.H., M.I. and K.U.; methodology, Y.H., M.I. and K.U.; software, Y.H., M.I. and K.U.; validation, Y.H., M.I. and K.U.; formal analysis, Y.H., M.I. and K.U.; investigation, Y.H., M.I., K.U., M.A. and K.K.; resources, Y.H., M.I., H.H. and M.S.; data curation, Y.H. and M.I.; writing—original draft preparation, Y.H., M.I. and K.U.; writing—review and editing, Y.H., M.I. and K.U.; visualization, Y.H. and M.I.; supervision, K.U.; project administration, K.U.; funding acquisition, Y.H., M.I. and K.U. All authors have read and agreed to the published version of the manuscript. Competing interests We have no disclosure Data availability Experimental data are provided within the manuscript or supplementary information files. The sequence datasets analyzed during the current study are available in the DDBJ repository, LC874449, LC874450, LC874451, LC874452, LC874453, LC874454, LC874455, LC874456, LC874457, LC874458, LC874459, LC874460, LC874461, LC874462, LC874463, LC874464, LC874465, LC874466, LC874467, LC874468, LC874469, LC874470, LC874471, LC874472, LC874473, LC874474, LC874475, LC874476, LC874477, LC874478, LC874479, LC874480, LC874481, LC874482, LC874483, LC874484, LC874485, LC874486, LC874487, LC874488, LC874489, LC874490, LC874491, LC874492, LC874493, LC874494, LC874495, LC874496, LC874497, LC874498, LC874499, LC874500, LC874501, LC874502, LC874503, LC874504, LC874505, LC874506, LC874507, LC874508, LC874509, LC874510, LC874511, LC874512, LC874513, LC874514, LC874515, LC874516, LC874517, LC874518, LC874519, LC874520, LC874521, LC874522, LC874523, LC874524, LC874525, LC874526, LC874527, LC874528, LC874529, LC874530, LC874625, LC874626, LC874627, LC874628, LC874629, LC874630, LC874631, LC874632, LC874633, LC874634, LC874635 and LC874636. Ethics declarations Informed consent Not applicable Ethics approval Not applicable Funding This study was supported by JSPS KAKENHI Grant Number 23K15371 and a Grant for Clinical Research from Miyazaki University Hospital. References Poiesz, B. J. et al. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. U. S. A. 77 , 7415-7419 (1980). Hinuma, Y. et al. Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc. Natl. Acad. Sci. U. S. A. 78 , 6476-6480 (1981). Osame, M. et al. HTLV-I associated myelopathy, a new clinical entity. Lancet . 1 , 1031-1032 (1986). Gessain, A et al. Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis. Lancet. 2 , 407-410 (1985). Tajima, K. et al. Epidemiological features of HTLV-I carriers and incidence of ATL in an ATL-endemic island: a report ofae community-based cooperative study in Tsushima, Japan. Int. J. Cancer 40 , 741-746 (1987). Giam, C. Z. & Semmes, O. J. HTLV-1 infection and adult T-cell leukemia/lymphoma-A tale of two proteins: tax and HBZ. Viruses 8 , 161 (2016). Matsuoka, M. & Jeang, K. T. Human T-cell leukemia virus type 1 (HTLV-1) and leukemic transformation: viral infectivity, Tax, HBZ and therapy. Oncogene 30 , 1379-1389 (2011). Takahashi, M. et al. HTLV-1 Tax oncoprotein stimulates ROS production and apoptosis in T cells by interacting with USP10. Blood 122 , 715-725 (2013). Tattermusch, S. & Bangham, C. R. HTLV-1 infection: what determines the risk of inflammatory disease? Trends Microbiol. 20 , 494-500 (2012). Kawata, S., Ariumi, Y. & Shimotohno, K. p21(Waf1/Cip1/Sdi1) prevents apoptosis as well as stimulates growth in cells transformed or immortalized by human T-cell leukemia virus type 1-encoded tax. J. Virol. 77 , 7291-7299 (2003). Chlichlia, K. et al. Immediate effects of reversible HTLV-1 tax function: T-cell activation and apoptosis. Oncogene 10 , 269-277 (1995). Kitajima, I. et al. Induction of apoptosis in murine clonal osteoblasts expressed by human T-cell leukemia virus type I tax by NF-kappa B and TNF-alpha. J. Bone Miner. Res. 11 , 200-210 (1996). Kannagi, M., Ohashi, T., Harashima, N., Hanabuchi, S. & Hasegawa, A. Immunological risks of adult T-cell leukemia at primary HTLV-I infection. Trends Microbiol. 12 , 346-352 (2004). Tamiya, S. et al. Two types of defective human T-lymphotropic virus type I provirus in adult T-cell leukemia. Blood 88 , 3065-3073 (1996). Furukawa, Y., Kubota, R., Tara, M., Izumo, S. & Osame, M. Existence of an escape mutant in the HTLV-I tax during the development of adult T-cell leukemia. Blood 97 , 987-993 (2001). Fan, J. et al. APOBEC3G generates nonsense mutations in human T-cell leukemia virus type 1 proviral genomes in vivo. J. Virol. 84 , 7278-7287 (2010). Taniguchi, Y. et al. Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms. Retrovirology 2 , 64 (2005). Gaudray, G. et al. The complementary strand of the human T-cell leukemia virus type 1 RNA genome encodes a bZIP transcription factor that downregulates viral transcription. J. Virol. 76 , 12813-12822 (2002). Yasunaga. J. Strategies of human T-cell leukemia virus type 1 for persistent infection: implications for the leukemogenesis of adult T-cell leukemia-lymphoma. Front. Microbiol. 11, 979 (2020). Satou, Y., Yasunaga, J., Yoshida, M. & Matsuoka, M. HTLV-I basic leucine zipper factor gene mRNA supports proliferation of adult T cell leukemia cells. Proc. Natl. Acad. Sci. U. S. A. 103 , 720-725 (2006). Miura, M. et al. Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modeling. PLoS Pathog. 15 , e1008164 (2019). Toyoda, K. & Matsuoka, M. Functional and pathogenic roles of retroviral antisense transcripts. Front. Immunol. 13 , 875211 (2022). Mitobe, Y., Yasunaga, J., Furuta, R. & Matsuoka, M. HTLV-1 bZIP factor RNA and protein impart distinct functions on T-cell proliferation and survival. Cancer Res. 75 , 4143-4152 (2015). Ma, G. et al. Human retroviral antisense mRNAs are retained in the nuclei of infected cells for viral persistence. Proc. Natl. Acad. Sci. U. S. A. 118 , e2014783118 (2021). Yoshida, M., Satou, Y., Yasunaga, J., Fujisawa, J. & Matsuoka M. Transcriptional control of the spliced and unspliced human T-cell leukemia virus type 1 bZIP factor (HBZ) gene. J. Virol. 82 , 9359-9368 (2008). Satou, Y. et al. HTLV-1 bZIP factor induces T-cell lymphoma and systemic inflammation in vivo. PLoS Pathog. 7 , e1001274 (2011). Miyoshi, I. et al. A novel T-cell line derived from adult T-cell leukemia. Gan 71 , 155-156 (1980). Sugamura, K. et al. Cell surface phenotypes and expression of the viral antigens of various human cell lines carrying the human T-cell leukemia virus. Int. J. Cancer 34 , 221-228 (1984). Yamada, Y. et al. Interleukin-15 (IL-15) can replace the IL-2 signal in IL-2-dependent adult T-cell leukemia (ATL) cell lines: expression of IL-15 receptor alpha on ATL cells. Blood 91 , 4265-4272(1998). Hashikura, Y. et al. The diversity of the structure and genomic integration sites of HTLV-1 provirus in MT-2 cell lines. Hum. Cell 29 , 122-129 (2016). Shiohama, Y. et al. Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status. Retrovirology 13 , 29 (2016). Seki, A. & Rutz, S. Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. J. Exp. Med. 215 , 985-997 (2018). Hu, W. et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc. Natl. Acad. Sci. U. S. A. 111 , 11461-11466 (2014). Panfil, A. R., Green, P. L. & Yoder, K. E. CRISPR genome editing applied to the pathogenic retrovirus HTLV-1. Front. Cell Infect. Microbiol. 10 , 580371 (2020). Levin, M. C. et al. PCR-in situ hybridization detection of human T-cell lymphotropic virus type 1 (HTLV-1) tax proviral DNA in peripheral blood lymphocytes of patients with HTLV-1-associated neurological disease. J. Virol. 70 , 924-933 (1996). Kuramitsu, M. et al. Identification of TL-Om1, an adult T-cell leukemia (ATL) cell line, as a reference material for quantitative PCR for human T-lymphotropic virus 1. J. Clin. Microbiol. 53 , 587-596 (2015). Hata, T. et al. Multi-clonal expansion of unique human T-lymphotropic virus type-I-infected T cells with high growth potential in response to interleukin-2 in prodromal phase of adult T cell leukemia. Leukemia 13 , 215-221 (1999). Isken, O. & Maquat, L. E. Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. Genes Dev. 21 , 1833-1856 (2007). Baker, K. E. & Parker, R. Nonsense-mediated mRNA decay: terminating erroneous gene expression. Curr. Opin. Cell. Biol. 16 , 293-299 (2004). Chang, Y. F., Imam, J. S. & Wilkinson, M. F. The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 76 , 51-74 (2007). Matsuoka, M. & Mesnard, J. M. HTLV-1 bZIP factor: the key viral gene for pathogenesis. Retrovirology. 17 , 2 (2020). Miyazaki, M. et al. Preferential selection of human T-cell leukemia virus type 1 provirus lacking the 5' long terminal repeat during oncogenesis. J. Virol. 81 , 5714-5723 (2007). Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files 4SupplementaryInformationver.2.pdf Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Nov, 2025 Reviews received at journal 19 Sep, 2025 Reviews received at journal 13 Sep, 2025 Reviewers agreed at journal 29 Aug, 2025 Reviewers agreed at journal 28 Aug, 2025 Reviewers invited by journal 19 Aug, 2025 Editor assigned by journal 12 Aug, 2025 Editor invited by journal 25 Jun, 2025 Submission checks completed at journal 25 Jun, 2025 First submitted to journal 30 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6562052","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":505654353,"identity":"a3bf5d47-0da9-45a6-8f21-cc2119ff8c8f","order_by":0,"name":"Yuki Hashikura","email":"","orcid":"","institution":"University of Miyazaki of Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Hashikura","suffix":""},{"id":505654354,"identity":"df54235e-9802-4adb-99a9-bd0069da3126","order_by":1,"name":"Misaki Izaki","email":"","orcid":"","institution":"University of Miyazaki of Hospital","correspondingAuthor":false,"prefix":"","firstName":"Misaki","middleName":"","lastName":"Izaki","suffix":""},{"id":505654355,"identity":"2458c70d-4afd-45ef-b999-ac655e51508b","order_by":2,"name":"Kazumi Umeki","email":"","orcid":"","institution":"University of Miyazaki","correspondingAuthor":false,"prefix":"","firstName":"Kazumi","middleName":"","lastName":"Umeki","suffix":""},{"id":505654356,"identity":"d90a6b9a-da37-4bcf-bfa8-b3654c773439","order_by":3,"name":"Mami Azeta","email":"","orcid":"","institution":"Kyushu University of Health and Welfare","correspondingAuthor":false,"prefix":"","firstName":"Mami","middleName":"","lastName":"Azeta","suffix":""},{"id":505654357,"identity":"07ecdc90-dbaa-4757-8280-9babf804ae50","order_by":4,"name":"Katsumi Kawano","email":"","orcid":"","institution":"University of Miyazaki of Hospital","correspondingAuthor":false,"prefix":"","firstName":"Katsumi","middleName":"","lastName":"Kawano","suffix":""},{"id":505654358,"identity":"65b02754-02bd-4040-8b9b-f4a0f9352156","order_by":5,"name":"Hiroo Hasegawa","email":"","orcid":"","institution":"Nagasaki University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hiroo","middleName":"","lastName":"Hasegawa","suffix":""},{"id":505654359,"identity":"7fa95e1c-8589-45cb-8cf3-4df733f4c44a","order_by":6,"name":"Mineki Saito","email":"","orcid":"","institution":"Kawasaki Medical School","correspondingAuthor":false,"prefix":"","firstName":"Mineki","middleName":"","lastName":"Saito","suffix":""},{"id":505654360,"identity":"f75f058c-f88b-41bd-9578-1b8cf68d6fda","order_by":7,"name":"Kunihiko Umekita","email":"data:image/png;base64,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","orcid":"","institution":"University of Miyazaki of Hospital","correspondingAuthor":true,"prefix":"","firstName":"Kunihiko","middleName":"","lastName":"Umekita","suffix":""}],"badges":[],"createdAt":"2025-04-30 07:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6562052/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6562052/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89985612,"identity":"04ee99bc-7268-4031-a473-b59ebccfef21","added_by":"auto","created_at":"2025-08-27 06:49:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1052874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCleavage detection assay results indicating the mutations introduced by different Cas9/sgRNA pairs\u003c/strong\u003e. Electrophoresis results of the genomic sequences obtained from \u003cstrong\u003ea\u003c/strong\u003e) MT-1, \u003cstrong\u003eb)\u003c/strong\u003e TL-Om1, and \u003cstrong\u003ec)\u003c/strong\u003eST-1 cells after transfection with different Cas9/sgRNAs. These results reveal that sgRNA 171 was most efficient at inducing mutations. The presence of two smaller bands shows the successful cleavage of the PCR products, including the mismatch base pair. The original photographs in Figure 1 are provided in Figures S1(a), (b), and (c).\u003c/p\u003e","description":"","filename":"3aFig.1CleavagedetectionassayresultsindicatingthemutationsintroducedbydifferentCas9sgRNApairs..tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562052/v1/24343e60fb3ee0c3ccd924c2.jpg"},{"id":89985614,"identity":"cdec9287-bff6-4a91-9615-7cee32380ca2","added_by":"auto","created_at":"2025-08-27 06:49:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":904377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Cas9/sgRNA 171 transfection on cell proliferation.\u003c/strong\u003e \u003cstrong\u003ea-c)\u003c/strong\u003e The numbers of live MT-1, TL-Om1, and ST-1 cells after transfection with Cas9/sgRNA 171 and Cas9/sgRNA NC were assessed using trypan blue staining. Means ± standard error of mean from three independent experiments are shown. N.S. not significant; *p \u0026lt; 0.05; **p \u0026lt; 0.01. The proliferation of MT-1 and TL-Om1 cells transfected with Cas9/sgRNA 171 was suppressed compared with those transfected with Cas9/sgRNA NC, but no significant difference was observed. Cas9/sgRNA 171-transfected ST-1 cells exhibited markedly inhibited cell proliferation compared with those transfected with Cas9/sgRNA NC.\u003c/p\u003e","description":"","filename":"3bFig.2EffectofCas9sgRNA171transfectiononcellproliferation..tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562052/v1/41232c137d3fe6f1d5e4165d.jpg"},{"id":89985615,"identity":"a85c0e25-1d65-488d-bcb3-37c23f33d8b2","added_by":"auto","created_at":"2025-08-27 06:49:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1222952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReal-time reverse transcriptase PCR results showing the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHBZ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etax/rex\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mRNA levels in cells transfected with Cas9/sgRNA\u003c/strong\u003e. Means ± SE obtained from three independent experiments are shown. Significant differences were evaluated by comparing the results with those from cells transfected with Cas9/sgRNA NC. N.S.: not significant; *p \u0026lt; 0.05; **p \u0026lt; 0.01. †indicates below the detection limit for quantification. \u003cstrong\u003ea–c)\u003c/strong\u003e The \u003cem\u003eHBZ\u003c/em\u003e mRNA levels in MT-1, TL-Om1, and ST-1 cells transfected with Cas9/sgRNA 171 and Cas9/sgRNA NC were reduced significantly until 4 days after transfection. Conversely, the \u003cem\u003eHBZ\u003c/em\u003e mRNA levels in ST-1 cells were reduced significantly until 6 days after transfection. \u003cstrong\u003ed–f)\u003c/strong\u003eThe \u003cem\u003etax\u003c/em\u003e/\u003cem\u003erex\u003c/em\u003e mRNA levels in MT-1, ST-1, and TL-Om1 cells transfected with Cas9/sgRNA 171 and Cas9/sgRNA NC. The \u003cem\u003etax/rex \u003c/em\u003emRNA levels were increased significantly in the Cas9/sgRNA-transfected MT-1 cells, while those in the TL-Om1 cells were below the detection limit for quantification until 4 days after transfection. However, these levels increased significantly 6 days after Cas9/sgRNA transfection. No significant differences were seen in the \u003cem\u003etax/rex\u003c/em\u003e mRNA levels in the ST-1 cells transfected with Cas9/sgRNA 171 and Cas9/sgRNA NC.\u003c/p\u003e","description":"","filename":"3cFig.3RealtimereversetranscriptasePCRresultsshowingtheHBZandtaxmRNAlevelsincellstransfectedwithCas9sgRNA..tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562052/v1/a28259f7570d88dc457c7881.jpg"},{"id":89985618,"identity":"37138a4b-b6cb-40af-999f-5dec8e3da8b8","added_by":"auto","created_at":"2025-08-27 06:49:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1243527,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Cas9/sgRNA 171 transfection on the protein expression of HBZ and Tax\u003c/strong\u003e. \u003cstrong\u003ea) \u003c/strong\u003eWestern blotting results showing the expression of HBZ and Tax after Cas9/sgRNA 171 transfection in MT-1, TL-Om1, and ST-1 cell lines. The molecular weights of the bands were consistent with those reported previously. HBZ was reduced in all cells after transfection with Cas9/sgRNA 171, while Tax was undetectable in all cell lines. Tubulin was used as the loading control.\u003cstrong\u003e b-d) \u003c/strong\u003eThe optical density (O.D.) ratios of tubulin to HBZ in MT-1, TL-Om1, and ST-1 cell lines are shown. HBZ expression levels were measured as values corrected using the tubulin signal intensity. Original Western blot images of the three cell lines are provided in Figure S4, Figure S5, and Figure S6.\u003c/p\u003e","description":"","filename":"3dFig.4EffectofCas9sgRNA171transfectionontheproteinexpressionofHBZandTax..tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562052/v1/87ebaf3589dcc1e697b08160.jpg"},{"id":89988738,"identity":"d439f277-202b-45ca-a2cc-0999d032565a","added_by":"auto","created_at":"2025-08-27 07:05:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5128442,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6562052/v1/dd5a9d72-2028-4a27-b054-54d8079b37cb.pdf"},{"id":89985619,"identity":"80689785-a90a-4204-991f-0e3a5b9f5043","added_by":"auto","created_at":"2025-08-27 06:49:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":572397,"visible":true,"origin":"","legend":"","description":"","filename":"4SupplementaryInformationver.2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6562052/v1/fac6665024abceb4d90eb1ff.pdf"},{"id":89987209,"identity":"d227f7d7-3246-4b34-ad28-9ebbe4f03f8a","added_by":"auto","created_at":"2025-08-27 06:57:44","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28191,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6562052/v1/195155a142d92a7fb8c2850d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting the gene encoding human T-cell leukemia virus type 1 basic zip factor via CRISPR/Cas9 potentially mitigates viral infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis/uveitis [1\u0026ndash;4]. HTLV-1 mainly targets the CD4\u0026thinsp;+\u0026thinsp;T-lymphocytes. Around 3\u0026ndash;5% of HTLV-1 carriers may develop ATL after a long latency [5]. The viral proteins, transactivator from the X-gene region (Tax) and HTLV-1 basic zip factor (HBZ), are significantly involved in the proliferation and tumorigenesis of HTLV-1 infected cells [6, 7]. Tax induces cell proliferation and resistance to apoptosis in HTLV-1 infected cells [8\u0026ndash;12]. Conversely, Tax acts as an antigen recognized by cytotoxic T-lymphocytes (CTLs). Therefore, host CTLs can eliminate HTLV-1 infected cells [13], resulting in a decrease in these cells after long-term latent infection [13]. Tax expression can also be attenuated by partial loss of provirus, nonsense mutations, and 5\u0026prime; long terminal repeat (LTR) promoter region methylation [14\u0026ndash;17]. Furthermore, HBZ downregulates Tax, which activates the transcription of the viral genes via the 5\u0026prime;LTR [18]. The decrease in Tax expression might be a potential mechanism via which HTLV-1 infected cells escape the host immune system, allowing the infection to persist throughout life [19]. HBZ, expressed constitutively in all ATL cells, promotes the proliferation of the infected cells [20]. Single-molecule RNA fluorescence in situ hybridization (FISH) studies showed that 30\u0026ndash;80% of HTLV-1-infected peripheral blood mononuclear cells are \u003cem\u003eHBZ\u003c/em\u003e-positive [21]. \u003cem\u003eHBZ\u003c/em\u003e mRNA is a bifunctional RNA that encodes the protein and acts as an RNA [22]. Several studies have suggested that the \u003cem\u003eHBZ\u003c/em\u003e mRNA might be essential for the proliferation, suppression of apoptosis, and production of cytokines/chemokines in HTLV-1 infected cells [23\u0026ndash;24]. Furthermore, \u003cem\u003eHBZ\u003c/em\u003e mRNA can exist in spliced or unspliced forms. The spliced mRNA suppresses the functions of Tax more effectively than the unspliced form [25]. Transgenic mice expressing HBZ have been shown to develop T-cell lymphoma. Based on these observations, the \u003cem\u003eHBZ\u003c/em\u003e mRNA has been implicated in the carcinogenesis of HTLV-1-infected cells [26]. Thus, although the roles of \u003cem\u003eHBZ\u003c/em\u003e in the pathogenesis of HTLV-1 infection and its associated diseases have been investigated, much remains unclear.\u003c/p\u003e\u003cp\u003eIn this study, we aimed to investigate the roles and functions of HBZ in ATL cell lines using synthetic guide RNAs (sgRNAs). These sgRNAs were designed to introduce mutations in the sequence adjacent to the translation initiation site of the spliced \u003cem\u003eHBZ\u003c/em\u003e mRNA and into \u003cem\u003eHBZ\u003c/em\u003e using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated \u003cem\u003eHBZ\u003c/em\u003e gene knockout resulted in an increase in \u003cem\u003eTax\u003c/em\u003e expression and a decrease in cell proliferation. Therefore, the functions of HBZ in HTLV-1 infected cells and its potential as a therapeutic target can be studied using the CRISPR/Cas9 system with an efficient sgRNA.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eCell lines\u003c/p\u003e\n\u003cp\u003eFor this study, we used three ATL cell lines: MT-1, TL-Om1, and ST-1. MT-1 was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB 1209 Lot No. 12172007, Osaka, Japan) [27]. TL-Om1\u0026nbsp;and ST-1 were kindly gifted by Kazuo Sugamura [28] and Hiroo Hasegawa (Department of Laboratory Medicine, Nagasaki University School of Medicine), respectively [29]. MT-1 was grown at 37°C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum with 5\u0026nbsp;μg/mL penicillin, 5\u0026nbsp;μg/mL streptomycin, and 10\u0026nbsp;μg/mL neomycin.\u0026nbsp;The ST-1 and TL-Om1 cell lines were grown at 37°C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e in RPMI-1640 medium (Nacalai\u0026nbsp;Tesque, Kyoto, Japan) containing 10% fetal bovine serum and 10 μg/mL recombinant human IL2 (PeproTech, Cranbury, USA) with 5 μg/mL penicillin, 5 μg/mL streptomycin, and 10 μg/mL neomycin.\u003c/p\u003e\n\u003cp\u003ePreparation and transfection of sgRNA\u003c/p\u003e\n\u003cp\u003eThe sgRNAs were designed using the CRISPR/Cas9 guide RNA design software CRISPRdirect (https://crispr.dbcls.jp/) to search for candidate sgRNA sequences within the \u003cem\u003eHBZ\u003c/em\u003e sequence (Accession no. AB219938). We designed five different sgRNAs by selecting a region with minimal homology to the human genomic DNA based on the idea that\u0026nbsp;a frameshift mutation can efficiently inactivate\u003cem\u003e\u0026nbsp;HBZ\u003c/em\u003e if a deletion can be introduced in a region close to the N-terminus of the \u003cem\u003eHBZ\u003c/em\u003e. One of these sgRNAs, sgRNA 171, was predicted to cleave the translation start site of \u003cem\u003eHBZ\u003c/em\u003e. The remaining sgRNAs (sgRNA 255, sgRNA 258, sgRNA 395, and sgRNA 405) were presumed to cleave downstream of the translation start site (Table 1).\u0026nbsp;A protospacer adjacent motif was present in these sgRNA sequences. TrueGuide™ sgRNA N.C. (Thermo Fisher Scientific, Tokyo, Japan) was used as the negative control (NC). Cas9 and sgRNA (Cas9/sgRNA) were transfected into the cell lines using the Invitrogen™ Neon™ Transfection System kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions.\u0026nbsp;Electroporation was performed using a Neon Transfection System with a single pulse at 1,650 V for 20 ms.\u0026nbsp;The cell solution was added to a 24-well plate and incubated at 37°C for 6 days under 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eConfirmation of the mutations\u003c/p\u003e\n\u003cp\u003eThe mutations created by CRISPR/Cas9 were screened using the Genomix Cleavage Detection Kit (Thermo Fisher Scientific). After culturing Cas9/sgRNA-transfected cells for 48 h, the cell pellets were collected by centrifugation at 1,500 rpm for 5 min and lysed using Cell Lysis Buffer (50 µL) containing 4% protein degrader by heated initially at 68°C for 15 min, followed by 95°C for 10 min. Then, 2 μL of this solution was used as a template for PCR. The primers used for sgRNA 171 target region amplification were forward primer—HTLV-8396F: 5′-CAGACTAAGGCTCTGACGTC-3′ and reverse primer—HTLV-8886R: 5′-AGACGTAGAGTTGAGCAAGC-3′. The primers for sgRNA 255 and sgRNA 258 target region amplification were forward primer—HTLV-7171F: 5′-CAAGCACAGCTTCCTCCTCC-3′ and reverse primer—HTLV-7518R: 5′-TGAGCCGATAACGCGTCCATC-3′. The primers for sgRNA 395 and sgRNA 405 target region amplification were forward primer—HTLV-6737F: 5′-CAAATCCTCCTTCTCCTGCA-3′, reverse primer—HTLV-7181R: 5′-AGCTGTGCTTGACGGTTTGC-3′. A suitable NC was selected for each of the three sgRNA target regions. The PCR was performed under the following conditions: one cycle of 95°C for 10 min, 40 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 20 s, and extension at 72°C for 30 s. Subsequently, 6 µL of the detection buffer was added to 3 µL of the PCR product and heated at 95°C. The mixture was cooled at 6°C per minute from 85°C to 25°C. After adding 1 µL of the detection enzyme, the mixture was heated at 37°C for 60 min. PCR products, including the mismatch base pair, were recognized by this enzyme, followed by cleavage to form two short bands. The PCR products digested with the detection enzyme were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining. Concurrently, non-digested PCR products were cloned to identify the mutations in the HTLV-1-infected cells. Then, they were subjected to a sequence assay using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Massachusetts, USA) and ABI Prism 3500 Genetic Analyzer (Applied Biosystems ThermoFisher, California, United States), according to the manufacturer’s instructions.\u003c/p\u003e\n\u003cp\u003eCell proliferation assay after CRISPR/Cas9 transfection\u003c/p\u003e\n\u003cp\u003eCells treated with CRISPR/Cas9 were collected and counted every 2 days after CRISPR/Cas9 transfection to assess cell proliferation. After staining the cells using trypan blue (Nacalai Tesque, Kyoto, Japan), viable cells were counted using a hemocytometer (NanoEntek, Seoul, Korea).\u0026nbsp;The accuracy of the cell count of 5 × 10\u003csup\u003e5\u003c/sup\u003e/mL cells were 1.4 × 10\u003csup\u003e4\u003c/sup\u003e/mL standard deviation and 2.78% coefficient of variation.\u003c/p\u003e\n\u003cp\u003eQuantification of\u003cem\u003e\u0026nbsp;HBZ\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;tax/rex\u0026nbsp;\u003c/em\u003emRNA\u003c/p\u003e\n\u003cp\u003eThese cells were collected every 2 days and transfected with Cas9/sgRNA 171. Total RNA was extracted from these cells using the TRIzol® Reagent (Life Technology Japan, Tokyo, Japan), according to the manufacturer’s instructions. The extracted RNA\u0026nbsp;was reverse transcribed into complementary DNA using an M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, USA) with random primers (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. Reverse transcription real-time PCR (RT-qPCR) was performed to detect HTLV-1 \u003cem\u003etax\u003c/em\u003e\u003cem\u003e/rex\u003c/em\u003e and\u003cem\u003eHBZ\u003c/em\u003e mRNA levels.\u0026nbsp;The PCR data was processed and analyzed based on the method described by Hashikura et al. [30]. RT-qPCR was performed in a duplicate manner using Light Cycler 2.0 (Roche Diagnostics, Mannheim, Germany). Relative quantification of HTLV-1 \u003cem\u003etax/rex\u003c/em\u003e and \u003cem\u003eHBZ\u003c/em\u003e mRNA was performed using \u003cem\u003eGAPDH\u003c/em\u003e mRNA as an internal control.\u0026nbsp;\u003cem\u003eHBZ\u003c/em\u003e and \u003cem\u003etax/rex\u0026nbsp;\u003c/em\u003emRNA were tested for significant differences from Cas9/sgRNA N.C. transfection.\u003c/p\u003e\n\u003cp\u003eWestern blot analysis of HBZ and Tax\u003c/p\u003e\n\u003cp\u003eThe cells transfected with Cas9/sgRNA 171 were collected every 2 days and then lysed using an sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.01% bromophenol blue, 10 mM β-β-mercaptoethanol) containing a protease inhibitor mixture (Thermo Fisher Scientific, Waltham, USA). The resultant cell lysates were heated to 98°C for 5 min in the sample buffer and separated on 8% SDS-polyacrylamide gels. Then, they were electroblotted onto polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes\u0026nbsp;(GE Healthcare, Little Chalfont, UK). The primary antibodies used in this study were the mouse anti-Tax antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the rat anti-HBZ monoclonal antibody, mAb 91-1 [31]. As a loading control, mouse anti-α-tubulin antibody (Abcam, Cambridge, MA, USA) was used. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (horse antimouse IgG and goat antirat IgG; Cell Signaling Technology, Inc. Danvers, MA, USA). An enhanced chemiluminescence detection system was used to detect the protein signals (Cytiva, Tokyo, Japan), and they were measured by a luminescent image analyzer FUSION SOLO.7S. EDGE (VILBER, Marne-la-Vallée, France).\u0026nbsp;The expression level of HBZ was measured as a value corrected using the optical density (O.D.) ratio of HBZ and Tubulin.\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eParametric tests (Student t-test) were used to compare the differences in cell proliferation and RNA expression between the Cas9/sgRNA 171-treated cells and the NCs. P-values \u0026lt;0.05 were considered statistically significant. Statistical analyses were performed using SPSS Statistics 20 (IBM, Chicago, IL, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eEvaluation of the mutation efficiency of the sgRNAs using the cleavage assay\u003c/p\u003e\n\u003cp\u003eAmong the five sgRNAs tested, Cas9/sgRNA 171 was the most effective at introducing mutations in \u003cem\u003eHBZ\u003c/em\u003e, as shown by the cleavage assay results (Fig. 1; Supplementary Figure\u0026nbsp;S1). Furthermore, Cas9/sgRNA 171 induced mutations at the methionine-encoding sequence of the HBZ translation start site. Consequently, sgRNA 171 was selected for inactivating\u0026nbsp;\u003cem\u003eHBZ\u0026nbsp;\u003c/em\u003ein\u0026nbsp;the following studies.\u003c/p\u003e\n\u003cp\u003eAnalysis of CRISPR/Cas9-induced mutations in LTR containing \u003cem\u003eHBZ\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter transfecting cells with Cas9/sgRNA 171 and culturing them for 2 days, the mutations introduced in these cells were detected using the Genomix Cleavage Detection Kit. Mutations were observed in the translation initiation site of \u003cem\u003eHBZ\u003c/em\u003e in the Cas9/sgRNA171-transfected cells (Table 2; Supplementary Figure S2).\u0026nbsp;Furthermore, the genomic DNA extracted from the cells were\u0026nbsp;subcloning and sequenced 2 days after the transfection. The results revealed\u0026nbsp;various mutations, such as deletions and insertions in 24/30 clones (80%) in MT-1, 22/30 clones (73%) in TL-Om1, and 21/30 clones (70%) in ST-1 cell lines (Table 2). However,\u0026nbsp;the mutation rate 6 days after transfection was reduced to 5/30 clones (17%) and 11/30 clones (37%) in the MT-1 and TM-Om1 cells, respectively. More than half of the sequences had no mutations\u0026nbsp;(Table 2). Conversely, sequencing results revealed mutations in 19/30 clones (63%) in the ST-1 cells 6 days after transfection, which is comparable to that observed 2 days after transfection\u0026nbsp;(Table 2). To confirm that\u0026nbsp;Cas9/sgRNA 171 does not cleave human genomic DNA, we examined its off-target effects in the four human genome regions with partial homology to the sgRNA 171 sequences (12 of the 20 bases of sgRNA 171 matched).\u0026nbsp;The results showed no\u0026nbsp;insertions and deletions in any of the four regions. As far as we investigated, no recombination with the off-target regions in human genome DNA was observed (Supplementary Figure S3)\u003c/p\u003e\n\u003cp\u003eCell proliferation after CRISPR/Cas9 transfection\u003c/p\u003e\n\u003cp\u003eThe proliferation of CRISPR/Cas9-transfected cells was analyzed by counting cells at 2, 4, and 6 days after the transfection. MT-1 and TL-Om1 transfected with Cas9/sgRNA 171 exhibited suppressed proliferation compared with those transfected with Cas9/sgRNA NC, although the difference was not significant (Fig. 2a, b).\u0026nbsp;The cell proliferation of Cas9/sgRNA 171-transfected ST-1 cells was markedly suppressed compared to those transfected with the NC (Fig. 2c).\u003c/p\u003e\n\u003cp\u003eExpression of \u003cem\u003eHBZ\u003c/em\u003e mRNA and \u003cem\u003etax/rex\u003c/em\u003e mRNA after transfection of Cas9/sgRNA 171\u003c/p\u003e\n\u003cp\u003eThe mRNA levels of \u003cem\u003eHBZ\u003c/em\u003e and \u003cem\u003etax/rex\u003c/em\u003e were investigated using RT-qPCR. The \u003cem\u003eHBZ\u003c/em\u003e mRNA levels in the MT-1 and TL-Om1 cells decreased significantly until 4 days after transfection compared to those transfected with Cas9/sgRNA NC (p \u0026lt; 0.05) (Fig. 3a, b). However, this difference was non-significant 6 days after transfection (Fig. 3a, b). The \u003cem\u003eHBZ\u003c/em\u003e mRNA levels in the Cas9/sgRNA 171-transfected ST-1 cells were significantly decreased compared with those transfected with Cas9/sgRNA NC until 6 days after transfection (Fig. 3c). In contrast, \u003cem\u003etax/rex\u003c/em\u003e mRNA levels in Cas9/sgRNA 171-treated MT-1 cells increased significantly till 6 days after transfection compared with the Cas9/sgRNA NC-transfected cells (Fig. 3d). While the \u003cem\u003etax/rex\u003c/em\u003e mRNA levels in TL-Om1 cells were below the detection limit until 4 days after CRISPR/Cas9 transfection. However, these levels increased significantly 6 days after transfection compared with those transfected with Cas9/sgRNA NC (Fig. 3e).\u0026nbsp;No significant difference was seen in the \u003cem\u003etax/rex\u003c/em\u003e mRNA levels in ST-1 cells transfected with Cas9/sgRNA 171 and Cas9/sgRNA NC (Fig. 3f).\u003c/p\u003e\n\u003cp\u003eExpression of HBZ and Tax protein after transfection of Cas9/sgRNA 171\u003c/p\u003e\n\u003cp\u003eThe protein expression levels of HBZ and Tax in all three cell lines transfected with Cas9/sgRNA 171 were detected using Western blotting (Fig. 4a; Supplementary Figure\u0026nbsp;S4–6). HBZ expression decreased in all cell lines after CRISPR/Cas9 transfection (Fig. 4b–d). While the \u003cem\u003etax\u003c/em\u003e mRNA levels showed an upward trend, its protein levels could not be detected using Western blotting (Fig. 4a).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, CRISPR/Cas9 was used to knock out the \u003cem\u003eHBZ\u003c/em\u003e gene and suppress the expression of the corresponding mRNA and protein. The newly designed sgRNA 171 induced a mutation in the codon at the translation initiation site of spliced-type \u003cem\u003eHBZ\u003c/em\u003e mRNA (Supplementary Figure S2), suppressing the \u003cem\u003eHBZ\u003c/em\u003e mRNA and protein expression in all three cell lines. In ST-1 cells, which contain one copy of the HTLV-1 provirus per cell, cell proliferation was suppressed by \u003cem\u003eHBZ\u003c/em\u003e gene knockout, whereas this inhibition was not observed in the MT-1 and TL-Om1 cell lines, in which multiple HTLV-1 proviruses were integrated per cell. This might be because the suppression of \u003cem\u003eHBZ\u003c/em\u003e expression by CRISPR/Cas9 might upregulate \u003cem\u003etax\u003c/em\u003e expression in the MT-1 and TL-Om1 cell lines, suggesting that \u003cem\u003eHBZ\u003c/em\u003e expression might affect cell proliferation and \u003cem\u003etax\u003c/em\u003e expression in the HTLV-1-infected cell lines.\u003c/p\u003e\u003cp\u003eTwo days after transfection with Cas9/sgRNA 171, the mutation efficiency observed in MT-1, TL-Om1, and ST-1 cells was approximately 70%. Deletions, substitutions, and insertions were observed in 80%, 73%, and 70% of the cells, respectively. The mutation introduction efficiency was comparable to that reported in a previous study in which CRISPR/Cas9 was transfected into activated human T cells. The results of this study were also consistent with ours [32].\u003c/p\u003e\u003cp\u003eUsing DNA sequencing, we examined the off-target effects by analyzing the cleavage of similar DNA sequences. However, no off-target effects were observed with Cas9/sgRNA 171, indicating that this system can efficiently and specifically induce mutations in \u003cem\u003eHBZ\u003c/em\u003e and control its expression. Other studies have shown that CRISPR/Cas9-based gene editing of the LTR region in the provirus of the human immunodeficiency virus (HIV), a retrovirus similar to HTLV-1, has low off-target effects [33]. These results suggest that provirus-specific DNA sequences nonhomologous to the human genome are ideal targets for CRISPR/Cas9 to achieve virus-specific genome editing in cells infected with retroviruses, including HTLV-1 [34].\u003c/p\u003e\u003cp\u003eThe proliferation of ST-1 cells transfected with Cas9/sgRNA 171 was suppressed. However, this suppressive effect was lower in the MT-1 and TL-Om1 cells than the ST-1 cells. Spliced \u003cem\u003eHBZ\u003c/em\u003e mRNA has been reported to suppress cell death and enhance cell proliferation [23, 25]. The proliferation ability of the MT-1 and TL-Om1 cell lines, which do not have genetic mutations in \u003cem\u003eHBZ\u003c/em\u003e, can be attributed to the expression of the spliced \u003cem\u003eHBZ\u003c/em\u003e mRNA in these cells.\u003c/p\u003e\u003cp\u003eThe three cell lines used in this study have been reported to have different numbers of proviruses integrated into the genome of each cell [35\u0026ndash;37]. MT-1 and TL-Om1 have approximately 3 and 1.78 copies of provirus per cell, respectively, whereas ST-1 has one copy per cell. Therefore, the efficiency of Cas9/sgRNA 171 in introducing \u003cem\u003eHBZ\u003c/em\u003e mutations per cell in MT-1 and TL-Om1 cells, which contain multiple proviruses per cell, may be relatively lower than in ST-1. Hence, \u003cem\u003eHBZ\u003c/em\u003e mRNA expression was maintained in MT-1 and TL-Om1 cells, leading to cell proliferation.\u003c/p\u003e\u003cp\u003eIn this study, a significant decrease in \u003cem\u003eHBZ\u003c/em\u003e mRNA was observed in the three cell lines 2 days after the transfection with Cas9/sgRNA 171. This might be due to the suppression of \u003cem\u003eHBZ\u003c/em\u003e transcription. Furthermore, the deletion or insertion of the translation initiation codon sequence of \u003cem\u003eHBZ\u003c/em\u003e by Cas9/sgRNA 171 might result in the generation of premature translation-termination codons due to a frameshift in \u003cem\u003eHBZ\u003c/em\u003e, leading to the expression of mutant \u003cem\u003eHBZ\u003c/em\u003e mRNA. The mRNA quality control mechanism of the host also might eliminate this mutant \u003cem\u003eHBZ\u003c/em\u003e mRNA [38\u0026ndash;40]. Reports have shown that the proproliferative effect of \u003cem\u003eHBZ\u003c/em\u003e on HTLV-1-infected cells depends on the \u003cem\u003eHBZ\u003c/em\u003e mRNA structure [20]. The Cas9/sgRNA 171-induced mutations in \u003cem\u003eHBZ\u003c/em\u003e might also affect its RNA structure and suppress cell proliferation. Therefore, we speculated that HTLV-1-infected cells in which \u003cem\u003eHBZ\u003c/em\u003e was completely knocked out were unable to proliferate and underwent cell death, whereas those in which \u003cem\u003eHBZ\u003c/em\u003e was partially knocked out maintained \u003cem\u003eHBZ\u003c/em\u003e expression and could survive and proliferate. As no mutations were introduced into the \u003cem\u003eHBZ\u003c/em\u003e region of the proviruses in cells containing multiple proviruses, the \u003cem\u003eHBZ\u003c/em\u003e mRNA levels were maintained, allowing the cells to proliferate. Furthermore, in HTLV-1-infected cells in which the \u003cem\u003eHBZ\u003c/em\u003e gene was knocked out using Cas9/sgRNA, the expression of \u003cem\u003etax\u003c/em\u003e mRNA increased slightly, consistent with previous reports showing that \u003cem\u003eHBZ\u003c/em\u003e regulates \u003cem\u003eTax\u003c/em\u003e expression [41].\u003c/p\u003e\u003cp\u003eThis study has several limitations. First, the efficiency of inducing mutations in \u003cem\u003eHBZ\u003c/em\u003e within the HTLV-1-infected cells was low. Although electroporation was selected as the transfection technique in this study, other transfection methods that are more efficient in inducing gene mutations in HTLV-1-infected cell lines should be considered. In the future, the transfection of CRISPR/Cas9 using a viral vector should be investigated to verify whether \u003cem\u003eHBZ\u003c/em\u003e gene mutations can be introduced into various HTLV-1-infected cell lines containing multiple proviruses in the host genome. Second, as the initiation codon for \u003cem\u003eHBZ\u003c/em\u003e, the target of sgRNA 171, is located in the 3\u0026prime;LTR, it is not easy to completely distinguish the 5\u0026prime;LTR from the 3\u0026prime;LTR for analysis and evaluation. Therefore, if mutations are introduced not only in the \u003cem\u003eHBZ\u003c/em\u003e gene but also in the 5\u0026prime;LTR, they might be included in this analysis. Third, we have not yet verified whether \u003cem\u003eHBZ\u003c/em\u003e can be edited by Cas9/sgRNA 171 in HTLV-1-infected cells from patients. \u003cem\u003eHBZ\u003c/em\u003e mRNA and protein have been shown to be involved in the onset of ATL [20,24,42]. Therefore, future studies are required to determine whether Cas9/sgRNA 171 can also control the proliferation of HTLV-1-infected and ATL cells derived from patients.\u003c/p\u003e\u003cp\u003eIn conclusion, we used gene editing technology using CRISPR/Cas9 and a newly designed sgRNA 171 to introduce mutations into \u003cem\u003eHBZ\u003c/em\u003e and suppress the expression of \u003cem\u003eHBZ\u003c/em\u003e mRNA and protein. Our results demonstrated that knocking out \u003cem\u003eHBZ\u003c/em\u003e resulted in the suppression of cell proliferation in HTLV-1-infected cell lines. Furthermore, the target retroviral and proviral gene sequences were specific to the retrovirus-infected cells and did not overlap with any region within the human genome, reducing the off-target effects and achieving successful gene modification using CRISPR/Cas9. Therefore, \u003cem\u003eHBZ\u003c/em\u003e gene editing technology using CRISPR/Cas9 might be a potential therapeutic approach for HTLV-1 infections and ATL.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Ms. Y. Kaseda (Miyazaki University) for technical support and assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.H. and M.I. contributed equally to this work. Conceptualization, Y.H., M.I. and K.U.; methodology, Y.H., M.I. and K.U.; software, Y.H., M.I. and K.U.; validation, Y.H., M.I. and K.U.; formal analysis, Y.H., M.I. and K.U.; investigation, Y.H., M.I., K.U., M.A. and K.K.; resources, Y.H., M.I., H.H. and M.S.; data curation, Y.H. and M.I.; writing—original draft preparation, Y.H., M.I. and K.U.; writing—review and editing, Y.H., M.I. and K.U.; visualization, Y.H. and M.I.; supervision, K.U.; project administration, K.U.; funding acquisition, Y.H., M.I. and K.U. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have no disclosure\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental data are\u0026nbsp;provided within the manuscript or supplementary information files.\u0026nbsp;The sequence datasets analyzed during the current study are available in the DDBJ repository, LC874449, LC874450, LC874451, LC874452, LC874453, LC874454, LC874455, LC874456, LC874457, LC874458, LC874459, LC874460, LC874461, LC874462, LC874463, LC874464, LC874465, LC874466, LC874467, LC874468, LC874469, LC874470, LC874471, LC874472, LC874473, LC874474, LC874475, LC874476, LC874477, LC874478, LC874479, LC874480, LC874481, LC874482, LC874483, LC874484, LC874485, LC874486, LC874487, LC874488, LC874489, LC874490, LC874491, LC874492, LC874493, LC874494, LC874495, LC874496, LC874497, LC874498, LC874499, LC874500, LC874501, LC874502, LC874503, LC874504, LC874505, LC874506, LC874507, LC874508, LC874509, LC874510, LC874511, LC874512, LC874513, LC874514, LC874515, LC874516, LC874517, LC874518, LC874519, LC874520, LC874521, LC874522, LC874523, LC874524, LC874525, LC874526, LC874527, LC874528, LC874529, LC874530, LC874625, LC874626, LC874627, LC874628, LC874629, LC874630, LC874631, LC874632, LC874633, LC874634, LC874635 and LC874636.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by JSPS KAKENHI Grant Number 23K15371 and a Grant for Clinical Research from Miyazaki University Hospital.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePoiesz, B. J. et al. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e\u003cstrong\u003e 77\u003c/strong\u003e, 7415-7419 (1980).\u003c/li\u003e\n\u003cli\u003eHinuma, Y. et al. Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e78\u003c/strong\u003e, 6476-6480 (1981).\u003c/li\u003e\n\u003cli\u003eOsame, M. et al. HTLV-I associated myelopathy, a new clinical entity. \u003cem\u003eLancet\u003c/em\u003e. \u003cstrong\u003e1\u003c/strong\u003e, 1031-1032 (1986).\u003c/li\u003e\n\u003cli\u003eGessain, A et al. Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis. \u003cem\u003eLancet.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 407-410 (1985).\u003c/li\u003e\n\u003cli\u003eTajima, K. et al. Epidemiological features of HTLV-I carriers and incidence of ATL in an ATL-endemic island: a report ofae community-based cooperative study in Tsushima, Japan. \u003cem\u003eInt. J. Cancer\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e40\u003c/strong\u003e, 741-746 (1987).\u003c/li\u003e\n\u003cli\u003eGiam, C. Z. \u0026amp; Semmes, O. J. HTLV-1 infection and adult T-cell leukemia/lymphoma-A tale of two proteins: tax and HBZ. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 161 (2016).\u003c/li\u003e\n\u003cli\u003eMatsuoka, M. \u0026amp; Jeang, K. T. Human T-cell leukemia virus type 1 (HTLV-1) and leukemic transformation: viral infectivity, Tax, HBZ and therapy. \u003cem\u003eOncogene\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1379-1389 (2011).\u003c/li\u003e\n\u003cli\u003eTakahashi, M. et al. HTLV-1 Tax oncoprotein stimulates ROS production and apoptosis in T cells by interacting with USP10. \u003cem\u003eBlood \u003c/em\u003e\u003cstrong\u003e122\u003c/strong\u003e, 715-725 (2013).\u003c/li\u003e\n\u003cli\u003eTattermusch, S. \u0026amp; Bangham, C. R. HTLV-1 infection: what determines the risk of inflammatory disease? \u003cem\u003eTrends Microbiol.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 494-500 (2012).\u003c/li\u003e\n\u003cli\u003eKawata, S., Ariumi, Y. \u0026amp; Shimotohno, K. p21(Waf1/Cip1/Sdi1) prevents apoptosis as well as stimulates growth in cells transformed or immortalized by human T-cell leukemia virus type 1-encoded tax. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 7291-7299 (2003).\u003c/li\u003e\n\u003cli\u003eChlichlia, K. et al. Immediate effects of reversible HTLV-1 tax function: T-cell activation and apoptosis. \u003cem\u003eOncogene\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 269-277 (1995).\u003c/li\u003e\n\u003cli\u003eKitajima, I. et al. Induction of apoptosis in murine clonal osteoblasts expressed by human T-cell leukemia virus type I tax by NF-kappa B and TNF-alpha.\u003cem\u003e J. Bone Miner. Res. \u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, 200-210 (1996).\u003c/li\u003e\n\u003cli\u003eKannagi, M., Ohashi, T., Harashima, N., Hanabuchi, S. \u0026amp; Hasegawa, A. Immunological risks of adult T-cell leukemia at primary HTLV-I infection. \u003cem\u003eTrends Microbiol.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 346-352 (2004).\u003c/li\u003e\n\u003cli\u003eTamiya, S. et al. Two types of defective human T-lymphotropic virus type I provirus in adult T-cell leukemia. \u003cem\u003eBlood \u003c/em\u003e\u003cstrong\u003e88\u003c/strong\u003e, 3065-3073 (1996).\u003c/li\u003e\n\u003cli\u003eFurukawa, Y., Kubota, R., Tara, M., Izumo, S. \u0026amp; Osame, M. Existence of an escape mutant in the HTLV-I tax during the development of adult T-cell leukemia. \u003cem\u003eBlood \u003c/em\u003e\u003cstrong\u003e97\u003c/strong\u003e, 987-993 (2001).\u003c/li\u003e\n\u003cli\u003eFan, J. et al. APOBEC3G generates nonsense mutations in human T-cell leukemia virus type 1 proviral genomes in vivo. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cstrong\u003e84\u003c/strong\u003e, 7278-7287 (2010).\u003c/li\u003e\n\u003cli\u003eTaniguchi, Y. et al. Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms. \u003cem\u003eRetrovirology \u003c/em\u003e\u003cstrong\u003e2\u003c/strong\u003e, 64 (2005).\u003c/li\u003e\n\u003cli\u003eGaudray, G. et al. The complementary strand of the human T-cell leukemia virus type 1 RNA genome encodes a bZIP transcription factor that downregulates viral transcription.\u003cem\u003e J. Virol.\u003c/em\u003e \u003cstrong\u003e76\u003c/strong\u003e, 12813-12822 (2002).\u003c/li\u003e\n\u003cli\u003eYasunaga. J. Strategies of human T-cell leukemia virus type 1 for persistent infection: implications for the leukemogenesis of adult T-cell leukemia-lymphoma. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cstrong\u003e11,\u003c/strong\u003e 979 (2020).\u003c/li\u003e\n\u003cli\u003eSatou, Y., Yasunaga, J., Yoshida, M. \u0026amp; Matsuoka, M. HTLV-I basic leucine zipper factor gene mRNA supports proliferation of adult T cell leukemia cells. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e103\u003c/strong\u003e, 720-725 (2006).\u003c/li\u003e\n\u003cli\u003eMiura, M. et al. Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modeling.\u003cem\u003e PLoS Pathog. \u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, e1008164 (2019).\u003c/li\u003e\n\u003cli\u003eToyoda, K. \u0026amp; Matsuoka, M. Functional and pathogenic roles of retroviral antisense transcripts. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 875211 (2022).\u003c/li\u003e\n\u003cli\u003eMitobe, Y., Yasunaga, J., Furuta, R. \u0026amp; Matsuoka, M. HTLV-1 bZIP factor RNA and protein impart distinct functions on T-cell proliferation and survival. \u003cem\u003eCancer Res. \u003c/em\u003e\u003cstrong\u003e75\u003c/strong\u003e, 4143-4152 (2015).\u003c/li\u003e\n\u003cli\u003eMa, G. et al. Human retroviral antisense mRNAs are retained in the nuclei of infected cells for viral persistence. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, e2014783118 (2021).\u003c/li\u003e\n\u003cli\u003eYoshida, M., Satou, Y., Yasunaga, J., Fujisawa, J. \u0026amp; Matsuoka M. Transcriptional control of the spliced and unspliced human T-cell leukemia virus type 1 bZIP factor (HBZ) gene. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 9359-9368 (2008).\u003c/li\u003e\n\u003cli\u003eSatou, Y. et al. HTLV-1 bZIP factor induces T-cell lymphoma and systemic inflammation in vivo. \u003cem\u003ePLoS Pathog.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e1001274 (2011).\u003c/li\u003e\n\u003cli\u003eMiyoshi, I. et al. A novel T-cell line derived from adult T-cell leukemia. \u003cem\u003eGan \u003c/em\u003e\u003cstrong\u003e71\u003c/strong\u003e, 155-156 (1980).\u003c/li\u003e\n\u003cli\u003eSugamura, K. et al. Cell surface phenotypes and expression of the viral antigens of various human cell lines carrying the human T-cell leukemia virus. \u003cem\u003eInt. J. Cancer \u003c/em\u003e\u003cstrong\u003e34\u003c/strong\u003e, 221-228 (1984).\u003c/li\u003e\n\u003cli\u003eYamada, Y. et al. Interleukin-15 (IL-15) can replace the IL-2 signal in IL-2-dependent adult T-cell leukemia (ATL) cell lines: expression of IL-15 receptor alpha on ATL cells. \u003cem\u003eBlood\u003c/em\u003e\u003cstrong\u003e91\u003c/strong\u003e, 4265-4272(1998).\u003c/li\u003e\n\u003cli\u003eHashikura, Y. et al. The diversity of the structure and genomic integration sites of HTLV-1 provirus in MT-2 cell lines. \u003cem\u003eHum. Cell\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 122-129 (2016). \u003c/li\u003e\n\u003cli\u003eShiohama, Y. et al. Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status. \u003cem\u003eRetrovirology\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 29 (2016).\u003c/li\u003e\n\u003cli\u003eSeki, A. \u0026amp; Rutz, S. Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. \u003cem\u003eJ. Exp. Med.\u003c/em\u003e \u003cstrong\u003e215\u003c/strong\u003e, 985-997 (2018).\u003c/li\u003e\n\u003cli\u003eHu, W. et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 11461-11466 (2014).\u003c/li\u003e\n\u003cli\u003ePanfil, A. R., Green, P. L. \u0026amp; Yoder, K. E. CRISPR genome editing applied to the pathogenic retrovirus HTLV-1. \u003cem\u003eFront. Cell Infect. Microbiol.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 580371 (2020). \u003c/li\u003e\n\u003cli\u003eLevin, M. C. et al. PCR-in situ hybridization detection of human T-cell lymphotropic virus type 1 (HTLV-1) tax proviral DNA in peripheral blood lymphocytes of patients with HTLV-1-associated neurological disease. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, 924-933 (1996). \u003c/li\u003e\n\u003cli\u003eKuramitsu, M. et al. Identification of TL-Om1, an adult T-cell leukemia (ATL) cell line, as a reference material for quantitative PCR for human T-lymphotropic virus 1. \u003cem\u003eJ. Clin. Microbiol.\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 587-596 (2015).\u003c/li\u003e\n\u003cli\u003eHata, T. et al. Multi-clonal expansion of unique human T-lymphotropic virus type-I-infected T cells with high growth potential in response to interleukin-2 in prodromal phase of adult T cell leukemia. \u003cem\u003eLeukemia \u003c/em\u003e\u003cstrong\u003e13\u003c/strong\u003e, 215-221 (1999).\u003c/li\u003e\n\u003cli\u003eIsken, O. \u0026amp; Maquat, L. E. Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. \u003cem\u003eGenes Dev.\u003c/em\u003e\u003cstrong\u003e21\u003c/strong\u003e, 1833-1856 (2007).\u003c/li\u003e\n\u003cli\u003eBaker, K. E. \u0026amp; Parker, R. Nonsense-mediated mRNA decay: terminating erroneous gene expression. \u003cem\u003eCurr. Opin. Cell. Biol.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 293-299 (2004).\u003c/li\u003e\n\u003cli\u003eChang, Y. F., Imam, J. S. \u0026amp; Wilkinson, M. F. The nonsense-mediated decay RNA surveillance pathway. \u003cem\u003eAnnu. Rev. Biochem. \u003c/em\u003e\u003cstrong\u003e76\u003c/strong\u003e, 51-74 (2007).\u003c/li\u003e\n\u003cli\u003eMatsuoka, M. \u0026amp; Mesnard, J. M. HTLV-1 bZIP factor: the key viral gene for pathogenesis. \u003cem\u003eRetrovirology.\u003c/em\u003e\u003cstrong\u003e 17\u003c/strong\u003e, 2 (2020).\u003c/li\u003e\n\u003cli\u003eMiyazaki, M. et al. Preferential selection of human T-cell leukemia virus type 1 provirus lacking the 5\u0026apos; long terminal repeat during oncogenesis. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 5714-5723 (2007).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"human T-cell leukemia virus type-1, HTLV-1 basic zip factor, CRISPR/Cas9, antivirus targeted therapies, synthetic guide RNA","lastPublishedDoi":"10.21203/rs.3.rs-6562052/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6562052/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHerein, we investigated the role of an essential transcription factor in the human T-cell leukemia virus type 1 (HTLV-1) provirus, the HTLV-1 basic zip factor (HBZ), in HTLV-1 infections and adult T-cell leukemia/lymphoma (ATL). We designed five synthetic guide RNAs (sgRNAs) targeting \u003cem\u003eHBZ\u003c/em\u003e and introduced them into ATL and HTLV-1 infected cell lines using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Of all sgRNAs, sgRNA 171 was the most efficient in introducing mutations at the target site as 70\u0026ndash;80% of Cas9/sgRNA 171-transfected host cells contained mutations. Various types of mutations, including deletions, substitutions, insertion, and combinations, were detected in the Cas9/sgRNA 171-treated cells. Based on the predicted peptide sequence, most mutant clones were assumed to inactivate the \u003cem\u003eHBZ\u003c/em\u003e mRNA. The mRNA levels of the \u003cem\u003etransactivator from the X-gene region\u003c/em\u003e (\u003cem\u003etax\u003c/em\u003e) increased after \u003cem\u003eHBZ\u003c/em\u003e editing by Cas9/sgRNA 171. No off-target effects were observed in the four human genome regions partially homologous to the sgRNA 171 target sequence. Furthermore, ST-1 cells transfected with Cas9/sgRNA 171 displayed significantly reduced proliferation. These findings suggest that the \u003cem\u003eHBZ\u003c/em\u003e mRNA might be crucial for the survival of HTLV-1-infected cells, including ATL, providing insights into the molecular pathogenesis of the HTLV-1 provirus.\u003c/p\u003e","manuscriptTitle":"Targeting the gene encoding human T-cell leukemia virus type 1 basic zip factor via CRISPR/Cas9 potentially mitigates viral infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 06:49:39","doi":"10.21203/rs.3.rs-6562052/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-11T08:37:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T13:54:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-13T14:33:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"319497445923929268081096394279461496913","date":"2025-08-29T05:13:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47830322310549140831113963890171903812","date":"2025-08-28T11:23:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-19T04:12:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-12T10:04:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-26T03:14:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-25T05:40:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-30T07:06:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dd1178b2-220f-4ff6-812d-14620d41e9ba","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53703502,"name":"Health sciences/Diseases/Haematological diseases/Haematological cancer/Lymphoma/Non hodgkin lymphoma/T cell lymphoma"},{"id":53703503,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"}],"tags":[],"updatedAt":"2026-02-06T06:24:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 06:49:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6562052","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6562052","identity":"rs-6562052","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}