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Genetic Stability of Human T Lymphotropic Virus Type I despite Antiviral Pressures by CTLs¹

Ryuji Kubota^2,*, Kousuke Hanada, Yoshitaka Furukawa, Kimiyoshi Arimura, Mitsuhiro Osame, Takashi Gojobori and Shuji Izumo^*

^* Center for Chronic Viral Diseases, Division of Blood Transfusion Medicine, and Department of Neurology and Geriatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan; and Center for Information Biology, National Institutes of Genetics, Shizuoka, Japan

	Abstract

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Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurological disease. Patients with HAM/TSP show high proviral load despite increased HTLV-I Tax-specific CTL. It is still unknown whether the CTL efficiently eliminate the virus in vivo and/or whether a naturally occurring variant virus becomes predominant by escaping from the CTL. To address these issues, we sequenced a large number of HTLV-I tax genes from HLA-A*02 HAM/TSP patients and estimated synonymous and nonsynonymous changes of the genes to detect positive selection pressure on the virus. We found the pressures in three of six CTL epitopes in HTLV-I Tax, where amino acid substitutions preferentially occurred. Although some of variant viruses were not recognized by the CTL, no variant viruses accumulated within 3–8 years, indicating genetic stability of HTLV-I tax gene. These results suggest that CTL eliminate the infected cells in vivo and naturally occurring variant viruses do not predominate. As Tax is a regulatory protein which controls viral replication, the amino acid substitutions in Tax may reduce viral fitness for replication. Viral fitness and host immune response may contribute to the viral evolution within the infected individuals. Furthermore, the genetic stability in the epitopes despite the antiviral pressures suggests that the three epitopes can be the candidate targets for HTLV-I vaccine development.

	Introduction

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Human T lymphotropic virus type I (HTLV-I)³ is a retrovirus, which causes two different human diseases in some infected individuals: HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia (1, 2). Adult T cell leukemia is severe leukemia, for which an effective treatment has not yet been established. HAM/TSP is an inflammatory disease in the spinal cord, where CD4⁺ and CD8⁺ T cells infiltrate to the perivascular area (3). The patients show spastic gait and sphincter dysfunction with mild sensory dysfunction (4). They have increased proviral load as compared with HTLV-I carriers, which is a strong predictor for the development of HAM/TSP from the carrier state (5). Furthermore, an increase of proviral load is associated with disease progression (6). These suggest that reducing the proviral load prevents the development and progression of HAM/TSP. However, an effective treatment to reduce the virus has not yet been developed.

HAM/TSP patients have high frequency of circulating CTL specific for HTLV-I Tax and CTL efficiently kill Ag-expressed target cells in an in vitro assay (7, 8, 9). However, the fact that the proviral load is still high despite these vigorous CTL responses may raise the question of whether the CTL really eliminate the virus in vivo. Recently, it was proposed that the killing activity of Tax-specific CTL may be disturbed (10). It has been difficult to show that CTL kill virus-infected cells in vivo; however, calculation of synonymous (without amino acid substitution) and nonsynonymous (with amino acid substitution) changes of virus genes has been developed to show an immunological antiviral pressure in vivo (11). If the rate of nonsynonymous change is greater than that of synonymous change in a region of the virus, this will suggest that an in vivo positive selection pressure occurs on the region. In HTLV-I infection, it is shown that the ratio of nonsynonymous changes to synonymous changes in the tax gene is greater in HTLV-I carriers than in HAM/TSP patients (12). We had previously sequenced the HTLV-I tax gene that codes for an immunodominant and viral regulatory protein, Tax, in a large number of viruses from patients with HAM/TSP. In this study, using the sequence data, we estimated the numbers of nonsynonymous nucleotide substitutions per nonsynonymous sites (dn) and the numbers of synonymous nucleotide substitutions per synonymous sites (ds) in the genes coding the CTL epitopes as well as in the remaining regions and compared those.

Virus-specific CD8⁺ CTL recognize viral peptide on the MHC and play a pivotal role in controlling viral infections (13). To develop a CTL vaccine in viral infections, it is fundamental to know whether virus-specific CTL exist, what the CTL epitopes are, and whether the virus escapes from the host immune system. In HIV infection, naturally occurring mutants escape from CTL and predominate in infected individuals (14, 15, 16). This is a strong obstacle to establish an effective CTL vaccine for HIV infection. In HTLV-I infection, it is unclear whether naturally occurring variant viruses escape from the host immune system and become predominant in the infected individuals. We analyzed longitudinal changes of variant virus proportion in association with variant virus-specific CTL.

	Materials and Methods

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Patients

Three patients with HAM/TSP (nos. 31, 38, 48) who had HLA-A*0201 allele were included (17). They were residing in Kagoshima, an endemic area of HTLV-I in Japan. The patients were diagnosed as HAM/TSP based on the neurological symptoms and seroreactivity to HTLV-I in accordance with the World Health Organization guidelines. These patients had not been treated with any antiretroviral drugs. PBMC were separated by Ficoll gradient centrifugation from heparinized blood repeatedly obtained from the patients and stored in liquid nitrogen until use. The Institutional Ethical Committee of Kagoshima University approved this study and informed consents were obtained from the patients.

Sequence analysis of HTLV-I tax gene

We used three samples from each patient as previously described (17). Cellular immune responses have predominantly been detected in a regulatory protein, HTLV-I Tax, in HTLV-I-infected individuals (7, 8, 9). The T cell epitopes restricted to HLA-A*02 accumulate in the N-terminal portion of the HTLV-I Tax protein (18). We therefore sequenced N-terminal of Tax (amino acid position 1–133). The method was previously described (17). Briefly, 100 ng of DNA extracted from the PBMC was amplified by 35 cycles of PCR. The first PCR products were further amplified by 20 cycles of nested PCR. The amplified products were purified using the QIA quick purification kit (Qiagen). The purified tax gene was subcloned into pCR-Blunt II-TOPO cloning vector (Invitrogen Life Technologies). After linearization by EcoRI digestion, the vector was purified by the QIA quick purification kit. The tax gene was sequenced using the Dye Terminator DNA Sequencing kit (Applied Biosystems) in an automatic sequencer (377 DNA Sequencer; Applied Biosystems). Approximately 50 clones were sequenced in each sample.

Comparison of selective pressures between CTL epitopes and the remaining regions

The CTL epitopes in the HTLV-I Tax were previously reported by epitope mapping (18, 19, 20, 21). The reported CTL epitopes that restricted to HLA-A*02 in aa 1–133 are as follows: Tax 11–19 (aa 11–19; LLFGYPVYV), XN3 (aa 21–35; GDCVQGDWCPISGGL), XN4 (aa 31–45; ISGGLCSARLHRHAL), XN9 (aa 80–95; TQRTSKTLKVLTPPIT), XN11 (aa 101–115; IPPSFLQAMRKYSPF), and XN12 (aa 111–125; KYSPFRNGYMEP). Based on our sequence data of the tax genes, three phylogenetic trees were independently constructed by the maximum likelihood method for each patient (22). The ancestral sequence was inferred at each node in the phylogenetic tree using the maximum parsimony method (23). Then, the numbers of synonymous and nonsynonymous substitutions throughout each phylogenetic tree were estimated for each codon site. The total numbers (count) of synonymous (Cs) and nonsynonymous substitutions (Cn) independently occurring in three patients were summed in each codon site. The total numbers of Cs and Cn were counted in six regions identified as CTL epitopes and the remaining regions in tax genes. Also, we computed the total numbers of synonymous (sS) and nonsynonymous (sN) sites in the regions of compared sequences. To examine selective pressure in the regions, the test of significance between the rate of Cs to sS and the rate of Cn to sN was performed in the regions by the two-tailed ² test (24). Values of p < 0.05 were considered significant.

Detection of positively selected regions of the tax gene

Positive selection pressure to the tax gene was examined by the modified method of Suzuki and Gojobori (25, 26) by three sequence data isolated from three patients. In this method, a phylogenetic tree was reconstructed and the ancestral sequence was inferred as described above. Then, the average number of synonymous (sS) and nonsynonymous (sN) sites and the total number of synonymous (Cs) and nonsynonymous (Cn) substitutions throughout the phylogenetic tree were estimated for each codon site by the Nei-Gojobori method (11). To examine positively selected regions in the tax gene, Cs, Cn, sS, and sN for a window size of five codon sites were calculated by sliding the window on the tax gene. The test of significance between the rate of Cs to sS and the rate of Cn to sN was performed in each window by the two-tailed Fisher’s exact test (22).

Peptides

Substituted amino acids were predicted from the obtained sequence data of the tax gene. The variant epitope peptides of Tax 11–19 and influenza virus M1 peptide (GILGFVFTL) were synthesized using F-moc solid-phase methodology (Kurabo). All the variant epitopes were designated as G4R, when the glycine at position 4 of the Tax 11–19 was substituted to arginine. Influenza virus M1 peptide was used as a control peptide that binds to HLA-A*02 (27). Purity of the peptides was over 90% by HPLC analysis. The synthetic peptides were resolved in 50% DMSO in PBS at 1 mM.

Intracellular cytokine detection by flow cytometry

The assay was conducted by a modified protocol as previously described (17). Briefly, Hmy2.C1R cells transfected with HLA-A*0201 (Hmy-A2) were prepulsed with 1 µM Tax 11–19 or variant epitopes for 1 h and were washed. Cryopreserved PBMC were quickly thawed and washed. A total of 5 x 10⁵ PBMC were cocultivated with the same number of peptide-prepulsed Hmy-A2 cells for 6 h. Brefeldin A (Sigma-Aldrich) was added to the cells at a final concentration of 10 µg/ml at the beginning of the culture to minimize the endogenous expression of HTLV-I protein on the infected cell surface. After culture, cells were harvested, washed, and stained with anti-human CD8 Ab conjugated with PC5 (Beckman Coulter) at 4°C for 20 min. Cells were washed and fixed with 4% paraformaldehyde for 5 min, then washed again. The cells, resuspended in 50 µl of permeabilization buffer containing 0.1% saponin (Sigma-Aldrich), were stained with anti-human IFN- Ab conjugated with FITC (BD Pharmingen) at 4°C for 20 min. Epics-XL flow cytometer and SYSTEM II software were used for fluorescent signal detection and data analysis (Beckman Coulter). Lymphocytes were readily distinguished from Hmy-A2 cells by size and were gated on forward and side scatter image. Ten thousand CD8⁺ cells were further gated and the proportion of IFN-⁺ cells in the CD8⁺ cell population was analyzed. The frequency of peptide-specific CD8⁺ T cells was obtained by subtracting the percentage of IFN-⁺ cells without peptide from that with a peptide. The relative frequency of variant epitope-specific T cells to the frequency of Tax 11–19-specific T cells was given by the following formula: (frequency of variant epitope-specific T cells)/(frequency of Tax 11–19-specific T cells) x 100.

	Results

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Detection of positive selection pressures to tax gene

We sequenced 146 clones of the tax gene from patient 31, 152 clones from patient 38, and 147 clones from patient 48. The phylogenetic tree of all cloned sequences was constructed to examine the phylogenetic relationship of the tax gene isolated from three patients. The sequences isolated from a patient would be clustered if the sequences isolated from a patient evolve in a specific pattern. However, the sequences isolated from each patient were not clustered in the resulting phylogenetic tree, indicating that there are not patient-specific variant viruses in the three patients. We depicted the amino acid replacements in the Tax protein with the previously reported CTL epitopes in HLA-A*02 HAM/TSP patients in Fig. 1. The consensus tax sequence in each patient was the same to the ATK-1 sequence first reported (28), which was referred to as the prototype amino acid sequence in Fig. 1. The number of amino acid replacement at each position was 1.36 ± 1.53 (mean ± SD). Amino acid replacements over 4.42 (mean + 2 SD) were observed at amino acid positions 14, 20, 29, 43, and 54. The replacements at 14, 29, and 43 were within the defined epitopes Tax 11–19, XN3, and XN4, respectively. However, the replacements at 20 and 54 were not within the epitopes. Overall, it is unclear whether amino acid replacements significantly occur in CTL epitope regions in this analysis.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Amino acid replacements in HTLV-I Tax 1–122 in three HAM/TSP patients with HLA-A*02. The consensus tax gene sequence in each patient is the same as the ATK-1 sequence first reported. The prototype amino acid sequence from the ATK-1 is described above the line. The transverse bars indicate all the known CTL epitopes which can bind to HLA-A*02. The numbers above the prototype sequence indicate position number of the Tax protein. The capital letter under the line represents a single occurrence of amino acid replacement found at the position.

View larger version (47K): [in this window] [in a new window]   FIGURE 2. Detection of positive selection pressures on the HTLV-I tax gene. The figure indicates the distribution of the value (1-P) for natural selection. When dn is larger than ds, the value is indicated above the abscissa, whereas in the opposite situation, below the abscissa. Light black columns that are over the dashed line indicate positively selected sites (p < 0.05 by the Fisher’s exact test). The abscissa indicates the amino acid positions. This analysis was performed in a sliding window of five amino acids, and the dn or ds for the sequence is expressed at the middle position of the five amino acids. The transverse bars indicate all the known CTL epitopes which can bind to HLA-A*02.

Because we found that the three epitopes of Tax 11–19, XN4, and XN11 were exposed to antiviral pressures by CTL in the patients with HAM/TSP, we investigated whether some variant epitopes accumulate during the time course of the disease in these epitopes. We summarized the frequencies of variant epitopes of Tax 11–19, XN4, and XN11 in Table II. In the Tax 11–19 epitope, the glycine to arginine change at position 4 was frequently observed in the three patients (1.8% in all the clone sequenced). However, no variant viruses accumulated in the time course. The prototype epitope (LLFGYPVYV) was predominant throughout the time course in all patients. In the XN4 epitope, amino acid replacements randomly occurred with little preferential replacements at positions 13 and 15, and the prototype sequence was predominant. In the XN11 epitope, replacements randomly occurred with small clusters around at positions 1 and 7, and the prototype amino acid sequence was predominant during the entire time course. Consequently, there was no accumulation of any variant viruses that escaped from the immune system in these patients.

We questioned whether no accumulation of variant viruses results from increasing CTL responses to the variant virus. Therefore, we investigated the frequency of variant virus-specific T cells during the time course of the disease. We focused on Tax 11–19 replacements, because the Tax 11–19 peptide was reported to be a strong immunodominant epitope in patients with HAM/TSP, and indeed in this study we detected the positive selection pressure in this region (7, 29). We synthesized several variant epitope peptides of Tax 11–19 according to the sequence data. In each patient, the variant epitopes emerged during the time course was tested to be recognized by CD8⁺ T cells. We performed intracellular IFN- detection, where variant epitope-specific CD8⁺ T cells were detected by their recognition of the epitopes loaded on APCs. We used no peptide-loaded APCs to evaluate background IFN- production, and influenza virus M1 peptide as a peptide control. The background IFN--positive cells in CD8⁺ cells from these patients were <0.49%, and the positive cells for M1 peptide were 1.61–5.41% in patient 31, 0.32–0.68% in patient 38, and 0.35–0.57% in patient 48 (data not shown). As shown in Fig. 3, variant epitope-specific CD8⁺ T cells were detected; Ag-specific T cells in the CD8⁺ T cell population were 10.20% for Tax 11–19 and for F3Y; to a lesser extent, L1P and Y5H were recognized by the T cells, however, G4R was rarely recognized. According to the formula described in Materials and Methods, we calculated relative frequency of variant epitope-specific T cells to Tax 11–19-specific T cells and depicted this in Fig. 4. In patient 31, F3Y peptide was recognized at the same level as Tax 11–19 as 100%, L1P and Y5H were moderately recognized, and G4R was rarely recognized by the CD8⁺ T cells. The relative frequencies of variant virus-specific T cells were considerably stable despite the emergence of variant viruses during the time course. In patient 38, many mutants emerged in 1999, whereas the relative frequencies of variant virus-specific T cells were almost the same. Interestingly, G4R was also rarely recognized by the T cells as in patient 31. Y5H recognition was different between these patients: 40% in patient 31, whereas <10% in patient 38. In patient 48, the relative frequencies fluctuated; recognition of all of the variant viruses was relatively low in 1993, whereas the recognition of all of the variant viruses, except G4RV7A, was relatively high in 2000. Overall, the apparent increase of variant virus-specific T cells was not observed in association with the appearance of any variant viruses in these patients.

View larger version (53K): [in this window] [in a new window]   FIGURE 3. Representative detection of variant epitope-specific CD8+ T cells in patient 31 in March 1999 by flow cytometry. The naturally occurring variant epitopes derived from Tax 11–19 were synthesized and used to detect variant epitope-specific CD8+ T cells by intracellular IFN- staining. The number in the box is the Ag-specific T cell frequency in CD8+ T cell population. The name of the variant epitope is designated by the amino acid replacement, i.e., G4R indicates that glycine at position 4 of the Tax 11–19 is substituted by arginine. NP indicates spontaneous IFN- production with APCs without any peptides.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Longitudinal analysis of variant epitope-specific CD8+ T cells in the patients. The x- and y-axis indicate date and relative frequency of variant epitope-specific CD8+ T cells, respectively. The relative frequency was obtained by dividing the variant epitope-specific T cell frequency in the CD8+ T cell population by the Tax 11–19-specific T cell frequency in the population. The variant epitopes above the boxes are indicated at the time point when the epitopes were detected in each patient.

	Discussion

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The positive selection pressures detected indicate that the CTL exert an effect to eliminate the virus in vivo. This is consistent with the previous report, where mutations in CTL epitope-coding regions occur significantly more frequently in HLA-A2-positive subjects than in HLA-A2-negative subjects, which suggests that HLA-A2-restricted, Tax-specific CTL induce in vivo antiviral pressures (30). The detection of positive selection pressures in Tax 11–19 is consistent with previous reports; Tax 11–19 has been demonstrated as an immunodominant epitope, which induces strong CTL responses both in HLA-A*02 HAM/TSP patients and the carriers (7, 8, 18, 29). In HIV infection, a virus with variant epitope easily escapes from the host immune system and predominates during the time course (14, 15, 16). This phenomenon has made it difficult to establish an effective CTL vaccine. However, in HTLV-I infection, although there were antiviral pressures by CTL in vivo, a virus with variant epitope did not become dominant during the time course. This may be an advantage in establishing an effective CTL vaccine. The three epitopes, where positive selection pressure and no escape variants were observed, could be candidates in designing a CTL vaccine.

The rate of amino acid replacements in HTLV-I Tax is higher than previously considered. In patient 38, the rate in Tax 11–19 ranged from 0% (in February 1997 in Table II) to 20.4% (in July 1999), suggesting that the virus actively replicates in vivo. However, any variant virus did not become dominant over the prototype virus during 3–8 years. The relative frequency of T cells specific for these peptides did not significantly change during the time course as shown in Fig. 4, and the frequencies of T cells specific for G4R (in patient 31, 38, and 48), Y5H (in patient 38), and Y5N (in patient 38) were constantly low. More importantly, the frequency of G4R-specific T cells was <10% and did not increase during the time course in all patients. These results suggest that nonaccumulation of variant viruses is not due to CTL responses. These raise the question of why the variant viruses do not become predominant, especially variants G4R, Y5H, and Y5N, which were rarely recognized by CTL (Fig. 4). Tax protein is a viral regulatory protein, which facilitates viral replication via trans-activator function that up-regulates the numerous promoter genes including its long terminal repeat promoter, IL-2R promoter (31). Niewiesk et al. (30) explored whether naturally occurring variants of HTLV-I Tax impair the transactivation function, and they demonstrated that most of the amino acid substitutions in Tax protein severely reduced its ability to transactivate three promoters: the HTLV-I long terminal repeat, the c-fos promoter, and the IL-2R -chain promoter (30). The replacement of the tax gene that codes for the G4R epitope is frequently observed both in their study and ours. In their study, the G4R replacement strongly reduces transactivator function (30). The reduction of transactivator activity by a replacement is reported not only in the Tax 11–19 epitope but also in XN4 and XN11 (30). Furthermore, artificial random mutagenesis of the tax gene, which introduces amino acid substitutions in Tax protein, abolishes the Tax regulatory functions (32). Therefore, some replacements in the regulatory protein Tax may impair the transactivator function for viral replication, which may lead to nonpredominance of the variant viruses, even if they are rarely recognized by CTL. Recently, reversion of CTL escape-variant SIV to the original virus is reported in newly infected animals in the absence of selective pressure by CTL, which suggests that viral evolution is a result of the equilibrium between viral fitness for replication and viral escape from the immune system (33, 34). Our data may support this hypothesis.

It is reported that the replacement rate is lower in HTLV-I than in HIV in vivo (35). Retroviruses can replicate by two ways within infected individuals: mitotic division of virus-infected cells and infectious spread among cells via reverse transcriptase. Although HIV spread mainly by free viral infection within infected individuals, it has been considered that HTLV-I mainly spread by mitotic replication, because infectivity of HTLV-I is extremely low in vitro and there is no evidence that HTLV-I is transmissible from HTLV-I-infected individuals to another person by blood component-depleted lymphocytes. Furthermore, inverse PCR analysis, which can distinguish each HTLV-I-infected T cell clone, reveals that clonal expansion of several infected cells is a common feature of HTLV-I infection (36). However, in our study, the proportion of mutant virus reached up to 20.4% (Table II, patient 38 on Jul/99). Moreover, viral sequence analysis revealed positive selection pressures, which is generally detected when retroviruses replicate by reverse transcriptase and not by mitosis of the infected cells. Thus, although HTLV-I may mainly spread via mitotic replication, replication via reverse transcriptase can play a role in increasing proviral load at least in some individuals. Quantification of the ratio of mitotic replication vs replication via reverse transcriptase may be important in using reverse transcriptase inhibitors for therapeutic purposes.

We found positive selection pressure around position 54 in the Tax protein (Fig. 2); however, HLA-A*02-restricted CTL epitopes have not been reported in these regions. Although the reason why the pressure was observed in the region is unclear, there is a possibility that positive selection pressure by CTL occurs on these amino acids; another T cell epitope restricted to an HLA allele other than HLA-A*02 may be found in these patients.

In conclusion, Tax-specific CTL act as killer cells, which induce positive selection pressure to HTLV-I in vivo. The naturally occurring variant viruses do not become predominant in the viral population unlike that seen in HIV infection. Therefore, these epitopes may be candidate targets for HTLV-I vaccine development. A search for a viral protein, which includes CTL epitopes and is essential for viral replication, may be important in designing a CTL vaccine for chronic viral infections.

	Acknowledgments

 
We thank Drs. Arlene R. Ng and Moe Moe Aye for the critical reading of the manuscript.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a Grant-in-Aid for Research on Brain Science of the Ministry of Health, Labor and Welfare of Japan, and a Grant-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology of Japan.

² Address correspondence and reprint requests to Dr. Ryuji Kubota, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. E-mail address: kubotar{at}m2.kufm.kagoshima-u.ac.jp

³ Abbreviations used in this paper: HTLV-I, human T lymphotropic virus type I; HAM/TSP, HTLV-I-associated myelopathy/tropical spastic paraparesis; Cn, count of nonsynonymous substitutions; Cs, count of synonymous substitutions; sN, nonsynonymous site; sS, synonymous site.

Received for publication September 26, 2006. Accepted for publication February 14, 2007.

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