Key Points• Humanized mice, IBMIhuNOG, were generated by intra-bone marrow injection of human CD1331 hematopoietic stem cells.• HTLV-1-infected IBMIhuNOG mice recapitulated distinct ATL-like symptoms as well as HTLV-1-specific adaptive immune responses.Human T-cell leukemia virus type 1 (HTLV-1) is causally associated with adult T-cell leukemia (ATL), an aggressive T-cell malignancy with a poor prognosis. To elucidate ATL pathogenesis in vivo, a variety of animal models have been established; however, the mechanisms driving this disorder remain poorly understood due to deficiencies in each of these animal models. Here, we report a novel HTLV-1-infected humanized mouse model generated by intra-bone marrow injection of human CD133 1 stem cells into NOD/Shi-scid/IL-2Rgc null (NOG) mice (IBMI-huNOG mice).Upon infection, the number of CD4 1 human T cells in the periphery increased rapidly, and atypical lymphocytes with lobulated nuclei resembling ATL-specific flower cells were observed 4 to 5 months after infection. Proliferation was seen in both CD25 2 and CD25 1 CD4 T cells with identical proviral integration sites; however, a limited number of CD25 1 -infected T-cell clones eventually dominated, indicating an association between clonal selection of infected T cells and expression of CD25. Additionally, HTLV-1-specific adaptive immune responses were induced in infected mice and might be involved in the control of HTLV-1-infected cells. Thus, the HTLV-1-infected IBMI-huNOG mouse model successfully recapitulated the development of ATL and may serve as an important tool for investigating in vivo mechanisms of ATL leukemogenesis and evaluating anti-ATL drug and vaccine candidates. (Blood. 2014;123(3):346-355)

In this study, we have identified protein kinase A-interacting protein 1 (AKIP1) as a binding partner of NF-B p65 subunit, and AKIP1 enhances the NF-B-mediated gene expression. AKIP1 is a nuclear protein and known to interact with the catalytic subunit of PKA (PKAc). We identified AKIP1 by a yeast two-hybrid screen using the N terminus region of p65 as bait. The interaction between AKIP1 and p65 was confirmed by glutathione S-transferase pull-down assay in vitro and immunoprecipitation-Western blotting assay in vivo. We found that the PKAc was present in the AKIP1⅐p65 complex and enhanced the transcriptional activity of NF-B by phosphorylating p65. In a transient luciferase assay, AKIP1 cotransfection efficiently increased the transcriptional activity of NF-B induced by phorbol 12-myristate 13-acetate (PMA). When AKIP1 was knocked down by RNA interference, the PMA-mediated NF-B-dependent gene expression was abolished, indicating a physiological role of AKIP1. We found that PKAc, which is maintained in an inactive form by binding to IB␣ and NF-B in resting cells, was activated by PMA-induced signaling and could phosphorylate p65. Overexpression of AKIP1 increased the PKAc binding to p65 and enhanced the PKAc-mediated phosphorylation of p65 at Ser-276. Interestingly, this p65 phosphorylation promoted nuclear translocation of p65 and enhanced NF-B transcription. In fact, we observed that AKIP1 colocalized with p65 within the cells and appeared to retain p65 in nucleus. These findings indicate a positive role of AKIP1 in NF-B signaling and suggest a novel mechanism by which AKIP1 augments the transcriptional competence of NF-B.NF-B is an inducible transcription factor for the expression of wide variety of genes involved in immunoinflammatory responses, cell proliferation, and survival, thus playing crucial roles in the pathogenesis of many diseases including cancer, leukemia, and autoimmune diseases (1-4). NF-B exists as either a heterodimer or a homodimer, among which the p65/ p50 is the most ubiquitous heterodimer. In resting cells, NF-B dimers are sequestered in the cytoplasm through association with inhibitory proteins IBs (5). Upon treatment with NF-B inducers such as phorbol 12-myristate 13-acetate (PMA) 2 or pro-inflammatory cytokines, IB is phosphorylated and degraded through the ubiquitin/proteasome pathway, which eventually leads to the nuclear translocation of NF-B and binding to the B site of target genes (6, 7).It has been established that the phosphorylation of p65 is important for the transcriptional activity of NF-B (8 -12). The phosphorylation of p65 by the PKA catalytic subunit dramatically enhances NF-B transcriptional activity by recruiting histone acetyltransferase CBP/p300 (13-15). PKA, existing predominantly in the cytoplasm as an inactive tetramer holoenzyme in resting cells, is composed of two catalytic subunits and a homodimer of two regulatory subunits that can dissociate upon activation by cAMP (16 -20). In resting cells, PKAc is involved in the IB⅐NF-B complex present in the cytoplasm, a...

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo.

The life cycle of human papillomaviruses (HPVs) is tightly coupled to the differentiation program of their host epithelial cells. HPV E4 gene expression is first observed in the parabasal layers of squamous epithelia, suggesting that the E4 gene product contributes to the mechanism of differentiation-dependent virus replication, although its biological function remains unclear. We analyzed the effect of HPV type 18 E4 on cell proliferation and found that E4 expression induced cell cycle arrest at the G 2 /M boundary. The functional region of E4 necessary for the growth arrest activity was located in the central portion of the molecule, and this activity was independent of the E4-mediated collapse of cytokeratin intermediate filament structures.

Graphical Abstract Highlights d A method for comprehensive analysis of HTLV-1 proviruses in infected individuals d The method provides viral sequence, integration site, and degree of cell expansion d Defective proviruses are present in asymptomatic carriers and HAM/TSP, as well as ATL d Infected cells with defective proviruses proliferate more than those with intact ones Correspondence y-satou@kumamoto-u.ac.jp In Brief Katsuya et al. demonstrate that HTLV-1 DNA-capture-seq provides comprehensive information, including the entire viral sequence, integration site, and clonal abundance of infected cells.Infected clones with defective-type proviruses are present in disease states and in asymptomatic carriers, and they proliferate more than full-length proviruses. SUMMARYThe retrovirus human T-cell leukemia virus type 1 (HTLV-1) integrates into the host DNA, achieves persistent infection, and induces human diseases.Here, we demonstrate that viral DNA-capture sequencing (DNA-capture-seq) is useful to characterize HTLV-1 proviruses in naturally virus-infected individuals, providing comprehensive information about the proviral structure and the viral integration site. We analyzed peripheral blood from 98 naturally HTLV-1-infected individuals and found that defective proviruses were present not only in patients with leukemia, but also in those with other clinical entities. We further demonstrated that clones with defective-type proviruses exhibited a higher degree of clonal abundance than those with full-length proviruses. The frequency of defective-type proviruses in HTLV-1-infected humanized mice was lower than that in infected individuals, indicating that defective proviruses were rare at the initial phase of infection but preferentially selected during persistent infection. These results demonstrate the robustness of viral DNA-captureseq for HTLV-1 infection and suggest potential applications for other virus-associated cancers in humans.