ALBERT

Description: Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in ∼10% of infected people. A typical host has between 104 and 105 clones of HTLV-1–infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP (HBZ) factor gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo, allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here, we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a fundamental role in controlling higher order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF-BS introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.

Description: Adult T-cell leukemia (ATL) patients and human T-cell leukemia virus-1 (HTLV-1) infected individuals succumb to opportunistic infections. Cell mediated immunity is impaired, yet the mechanism of this impairment has remained elusive. The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the viral DNA and is constitutively expressed in infected cells and ATL cells. To test the hypothesis that HBZ contributes to HTLV-1–associated immunodeficiency, we challenged transgenic mice that express the HBZ gene in CD4 T cells (HBZ-Tg mice) with herpes simplex virus type 2 or Listeria monocytogenes, and evaluated cellular immunity to these pathogens. HBZ-Tg mice were more vulnerable to both infections than non-Tg mice. The acquired immune response phase was specifically suppressed, indicating that cellular immunity was impaired in HBZ-Tg mice. In particular, production of IFN-γ by CD4 T cells was suppressed in HBZ-Tg mice. HBZ suppressed transcription from the IFN-γ gene promoter in a CD4 T cell–intrinsic manner by inhibiting nuclear factor of activated T cells and the activator protein 1 signaling pathway. This study shows that HBZ inhibits CD4 T-cell responses by directly interfering with the host cell-signaling pathway, resulting in impaired cell-mediated immunity in vivo.

Description: Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is etiologically associated with adult T-cell leukemia. The HTLV-1 bZIP factor (HBZ), which is encoded by the minus strand of the provirus, is involved in both regulation of viral gene transcription and T-cell proliferation. We showed in this report that HBZ interacted with Smad2/3, and enhanced transforming growth factor-β (TGF-β)/Smad transcriptional responses in a p300-dependent manner. The N-terminal LXXLL motif of HBZ was responsible for HBZ-mediated TGF-β signaling activation. In a serial immunoprecipitation assay, HBZ, Smad3, and p300 formed a ternary complex, and the association between Smad3 and p300 was markedly enhanced in the presence of HBZ. In addition, HBZ could overcome the repression of the TGF-β response by Tax. Finally, HBZ expression resulted in enhanced transcription of Pdgfb, Sox4, Ctgf, Foxp3, Runx1, and Tsc22d1 genes and suppression of the Id2 gene; such effects were similar to those by TGF-β. In particular, HBZ induced Foxp3 expression in naive T cells through Smad3-dependent TGF-β signaling. Our results suggest that HBZ, by enhancing TGF-β signaling and Foxp3 expression, enables HTLV-1 to convert infected T cells into regulatory T cells, which is thought to be a critical strategy for virus persistence.

Description: EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a rare yet devastating disorder caused by EBV infection in humans. However, the mechanism of this disease has yet to be elucidated because of a lack of appropriate animal models. Here, we used a human CD34+ cell-transplanted humanized mouse model and reproduced pathologic conditions resembling EBV-HLH in humans. By 10 weeks postinfection, two-thirds of the infected mice died after exhibiting high and persistent viremia, leukocytosis, IFN-γ cytokinenemia, normocytic anemia, and thrombocytopenia. EBV-infected mice also showed systemic organ infiltration by activated CD8+ T cells and prominent hemophagocytosis in BM, spleen, and liver. Notably, the level of EBV load in plasma correlated directly with both the activation frequency of CD8+ T cells and the level of IFN-γ in plasma. Moreover, high levels of EBV-encoded small RNA1 were detected in plasma of infected mice, reflecting what has been observed in patients. These findings suggest that our EBV infection model mirrors virologic, hematologic, and immunopathologic aspects of EBV-HLH. Furthermore, in contrast to CD8+ T cells, we found a significant decrease of natural killer cells, myeloid dendritic cells, and plasmacytoid dendritic cells in the spleens of infected mice, suggesting that the collapse of balanced immunity associates with the progression of EBV-HLH pathogenesis.

Description: The retrovirus human T-cell leukemia virus type 1 (HTLV-1) integrates into the host genome and persists for the lifetime of the host. There are tens of thousands of different infected clones in a HTLV-1 carrier and each clone can be identified by its unique viral integration site. Only about 5% of infected people develop the hematological malignancy, adult T-cell leukemia-lymphoma (ATL). However, it is unclear how a certain infected clone, among various different ones, is selected as a malignant clone. It has been reported that viral integration alters transcripts of the cellular host genes adjacent to the integration site, even generating truncated or virus-host chimeric transcripts. Because each infected clone has a unique viral integration site, each clone possibly has unique virus-host chimeric transcripts, which were not present in the host before infection. Therefore, we hypothesized that the integrated provirus generates virus-host chimeric transcripts that may play a role in the clonal selection of the HTLV-1-infected cell. We previously reported HTLV-1 DNA-capture-seq using biotinylated DNA-probes for the viral genome, to increase the sensitivity and efficiency of viral-sequences detection. In this study, we used HTLV-1 RNA-capture-seq for PBMCs samples from ATL patients to test the hypothesis in a highly sensitive manner. The results showed the presence of chimeric transcripts in 19 out of 30 ATL patients. We next quantified the abundance of chimeric transcripts by droplet digital PCR, and found that the expression levels of chimeric transcripts were similar to those of viral RNAs containing splice junction of HBZ, in 5 of 19 chimeric transcripts positive ATL cases, although the levels varied among different ATL cases. To identify the whole sequences of the chimeric transcripts, we performed Oxford Nanopore sequencing. This approach revealed that the HTLV-1 provirus generates various splicing chimeric transcripts with the host genes in both viral sense and antisense orientations. The transcriptional start site of most of the sense chimeric transcripts was the R region of the 5'- or 3'-long terminal repeats (LTRs) in the proviral sequences, indicating that the chimeric transcripts were generated using the viral promoters because the LTRs work as a promoter for the viral transcripts. Given the structure of the chimeric transcripts with the viral promotors, the expression of the fused host genes could be enhanced by generating the chimeric transcripts. We evaluated the mRNA expression of the fused host genes of the chimeric transcripts by RNA-seq, and the results correlated with those obtained by ddPCR. To clarify the impact of viral integration on the clonal expansion, we analyzed HTLV-1-infected Jurkat cells. The clonality analysis of infected cells by HTLV-1 DNA-capture-seq showed that some infected clones were remarkably expanded for 4-6 months culture. We also confirmed that some of them harbored virus-host chimeric transcripts by HTLV-1 RNA-capture-seq. This study revealed the expression levels and the structures of virus-host chimeric transcripts in ATL patients. We are currently investigating the functional role of chimeric transcripts in the clonal proliferation of infected cells in vitro. Disclosures Uchimaru: Daiichi Sankyo Co., Ltd..: Research Funding. Kimura:Novartis: Honoraria, Research Funding; Ohara Pharmaceutical Co.: Research Funding.

Description: Adult T-cell leukemia (ATL) is a highly aggressive neoplasm of helper T lymphocytes and is etiologically associated with human T-cell leukemia virus type I (HTLV-I). Although HTLV-I encoded Tax protein is thought to play a central role in the leukemogenesis of ATL, ATL cells frequently could not produce Tax protein by the somatic mutations, deletion and loss of 5′-LTR. Moreover, a long-term latent period (almost 60 years in Japan) precedes the onset of ATL, suggesting that multistep tumorigenesis is involved in development of ATL in addition to role of viral protein. Such transformation process is thought to include alterations of host genome: genetic and epigenetic changes. The epigenetic alteration, such as DNA methylation and histone modification, is commonly observed in various cancer cells. Recently, we reported that the aberrant expression of MEL1S gene, which is hypomethylated in ATL cells, confers resistance against transforming growth factor-beta. In this study, we identified 53 aberrantly hypermethylated DNA sequences in ATL cells using methylated CpG island amplification/representational difference analysis (MCA/RDA) method. In addition, we found that the DNA methylation of these regions tends to accumulate with disease progression. Seven genes, which were expressed in normal T-cells, but suppressed in ATL cells, were identified near the hypermethylated regions. Among these silenced genes, Kruppel-like factor 4 (KLF4) gene is a cell cycle regulator and early growth response 3 (EGR3) gene is a critical transcriptional factor for induction of Fas ligand (FasL) expression. Treatment with 5-aza-2′-deoxycytidine resulted in the recovery of their transcription, indicating that their silencing is associated with DNA hypermethylation. To study their role in oncogenesis of ATL, we transfected recombinant adenovirus vectors expressing KLF4 and EGR3 genes into ATL cell lines. Expression of KLF4 induced apoptosis of ATL cells whereas enforced expression of EGR3 induced the expression of FasL gene, resulting in apoptosis. This EGR3-induced apoptosis has been inhibited by stable transfection of cellular and viral FLIP, which suppress the activation of caspase-8 by preventing pro-caspase-8 in Fas-mediated apoptosis pathway. Therefore, suppressed expression of EGR3 enabled ATL cells to escape from activation-induced cell death mediated by Fas-FasL signaling. Our results showed that the DNA hypermethylation plays an important role in the leukemogenesis of ATL by silencing transcriptions of genes that inhibit the abnormal growth of ATL cells.

Description: Human T-cell leukemia virus type I (HTLV-I) causes adult T-cell leukemia (ATL) in about 5% of carriers after a long latent period. HTLV-I is one of complex retrovirus, which encodes accessory genes to control viral replication and proliferation of infected cells. Previous studies reported the pleiotropic actions of tax gene in proliferation of infected cells and leukemogenesis. However, tax gene expression in ATL cells is disrupted by several mechanisms. Our previous study showed that the 5′-LTR of HTLV-I is frequently hypermethylated or deleted in ATL cells, while the 3′-LTR remains unmethylated and intact. These findings suggest the involvement of the 3′-LTR in leukemogenesis. Transcription from the minus strand of HTLV-I has been reported, and the HTLV-I bZIP factor (HBZ) was subsequently found to inhibit Tax-mediated transactivation of viral gene transcription from the 5′-LTR by heterodimerizing with either CREB2, c-Jun or JunB. Based on these previous studies, we hypothesized that HBZ had an important role in ATL cells. We first identified the transcription start site of HBZ gene in the 3′-LTR and found the novel splicing form. The HBZ gene transcription could be detected in all ATL cases and two of three HTLV-I asymptomatic carriers. Suppression of HBZ gene transcription by short hairpin RNA inhibits proliferation of ATL cells. In addition, HBZ gene expression promotes proliferation of a human T-cell line. Transcriptional profiling showed that BTG2, which is known as an antiproliferative molecule, and MX-1, which has an antiviral function, were down regulated. In addition, HBZ up-regulated the transcription of E2F-1 and its target genes. These results suggest that HBZ is associated with the proliferation and survival of HTLV-I infected cells. Furthermore, HBZ mutant analyses suggested that HBZ promotes T-cell proliferation in its RNA form, while HBZ protein suppresses Tax-mediated viral transcription through the 5′-LTR. The studies of microarrays showed that transcriptional changes by HBZ gene could be categorized into two groups: those caused by HBZ RNA and HBZ protein. HBZ protein enhanced the transcription of cellular genes in transfected cells such as GRAP2, an adaptor molecule in the downstream signaling of T cell receptor, which should be important in pathogenesis by HBZ. To analyze the function of HBZ gene in vivo, we generated transgenic (Tg) mice expressing HBZ under the control of the mouse CD4 promoter/enhancer. The percentage of CD4 T cells increased in splenocytes of the Tg mice. In addition, proliferation induced by cross-linking with an immobilized anti-CD3 antibody was augmented in thymocytes of these Tg mice. These data indicate that the HBZ gene promotes proliferation of CD4 T cells in vivo. Interestingly, one of three strains of HBZ mice spontaneously develops dermatitis at about 3–4 months of age. Histological analyses revealed severe dermatitis with massive dermal and epidermal infiltration of lymphocytes. In other strains of transgenic mice, which did not present dermatitis, infiltration of lymphocytes was also observed. In HTLV-I carriers with high provirus loads, infiltration of CD4 T-lymphocytes into the skin has been reported. The spontaneous dermatitis in the HBZ mice resembles to that observed in HTLV-I infected individuals. Taken together, these data suggest that HBZ gene is implicated not only in oncogenesis by HTLV-I, but also in HTLV-I associated diseases.

Description: Human T-cell leukemia virus type I (HTLV-I) is a causative agent of neoplastic disease, adult T-cell leukemia (ATL). Although the encoding viral proteins play an important role in oncogenesis, the role of the HTLV-I proviral integration site remains unsolved. We determined the integration sites of HTLV-I proviruses in ATL cells and HTLV-I–infected cells in asymptomatic carriers. In carrier and ATL cells, HTLV-I provirus was integrated into the transcriptional unit at frequencies of 26.8% (15/56) and 33.9% (20/59), respectively, which were equivalent to the frequency calculated based on random integration (33.2%). In addition, HTLV-I provirus was prone to integration near the transcriptional start sites in leukemic cells (P = .006), and the transcriptional direction of the provirus was in accordance with that of integrated cellular genes in 70% of cases. More importantly, the integration sites in the carrier cells favored the alphoid repetitive sequences (11/56; 20%) whereas in leukemic cells they disfavored these sequences (2/59; 3.4%). Taken together, during natural course from carrier to onset of ATL, HTLV-I–infected cells with integration sites favorable for viral gene transcription are susceptible to malignant transformation due to increased viral gene expression.