Takeo Ohsugi

Viral interference with host mRNA surveillance, the nonsense-mediated mRNA decay (NMD) pathway, through a new function of HTLV-1 Rex: implications for retroviral replication.

Nonsense-mediated mRNA decay (NMD) is an essential and conserved cellular mRNA quality control mechanism. RNA signals to express viral genes from overlapping open reading frames potentially initiate NMD, nevertheless it is not clear whether viral RNAs are sensitive to NMD or if viruses have evolved mechanisms to evade NMD. Here we demonstrate that the genomic and full-length mRNAs of Human-T-cell Leukemia Virus type-I (HTLV-1), a retrovirus responsible for Adult T-cell Leukemia (ATL), are sensitive to NMD. They exhibit accelerated turnover in NMD-activated cells, while siRNA-mediated knockdown of NMD-master-regulator, UPF1, promotes enhanced stability of them. These effects on RNA stability were recapitulated by a reporter construct encoding the HTLV-1 translational frameshift signal of gag-pol. In agreement with the RNA stability, viral protein expression from the integrated provirus was inversely correlated with cellular NMD activity. We further demonstrated that the viral RNA-binding protein, Rex, approves the stability of viral RNA by inhibiting NMD. Significantly, Rex establishes a general block to NMD, as both NMD-responsive reporter transcripts and natural host-encoded NMD substrates were stabilized in the presence of Rex. Thus, we suggest that Rex not only stabilizes viral transcripts, but also perturbs cellular mRNA metabolism and host cell homeostasis via inhibition of NMD.

The antiretroviral potency of APOBEC1 deaminase from small animal species

Although the role of the APOBEC3-dependent retroelement restriction system as an intrinsic immune defense against human immunodeficiency virus type1 (HIV-1) infection is becoming clear, only the rat ortholog of mammalian APOBEC1s (A1) thus far has been shown to possess antiviral activity. Here, we cloned A1 cDNAs from small animal species, and showed that similar to rat A1, both wild-type and Δvif HIV-1 infection was inhibited by mouse and hamster A1 (4- to 10-fold), whereas human A1 had negligible effects. Moreover, rabbit A1 significantly reduced the infectivity of both HIV-1 virions (>300-fold), as well as that of SIVmac, SIVagm, FIV and murine leukemia virus. Immunoblot analysis showed that A1s were efficiently incorporated into the HIV-1 virion, and their packaging is mediated through an interaction with the nucleocapsid Gag domain. Interestingly, there was a clear accumulation of particular C-T changes in the genomic RNAs of HIV-1 produced in their presence, with few G-A changes in the proviral DNA. Together, these data reveal that A1 may function as a defense mechanism, regulating retroelements in a wide range of mammalian species.

Biscoclaurine alkaloid cepharanthine inhibits the growth of primary effusion lymphoma in vitro and in vivo and induces apoptosis via suppression of the NF-κB pathway

Primary effusion lymphoma (PEL) is a unique and recently identified non‐Hodgkins lymphoma that was originally identified in patients with AIDS. PEL is caused by the Kaposi sarcoma‐associated herpes virus (KSHV/HHV‐8) and shows a peculiar presentation involving liquid growth in the serous body cavity and a poor prognosis. As the nuclear factor (NF)‐κB pathway is activated in PEL and plays a central role in oncogenesis, we examined the effect of a biscoclaurine alkaloid, cepharanthine (CEP) on PEL derived cell lines (BCBL‐1, TY‐1 and RM‐P1), in vitro and in vivo. An methylthiotetrazole assay revealed that the cell proliferation of PEL cell lines was significantly suppressed by the addition of CEP (1–10 μg/ml). CEP also inhibited NF‐κB activation and induced apoptotic cell death in PEL cell lines. We established a PEL animal model by intraperitoneal injection of BCBL‐1, which led to the development of ascites and diffuse infiltration of organs, without obvious solid lymphoma formation, which resembles the diffuse nature of human PEL. Intraperitoneal administration of CEP inhibited ascites formation and diffuse infiltration of BCBL‐1 without significant systemic toxicity in this model. These results indicate that NF‐κB could be an ideal molecular target for treating PEL and that CEP is quite useful as a unique therapeutic agent for PEL.

Low CD4/CD8 T-Cell Ratio Associated with Inflammatory Arthropathy in Human T-Cell Leukemia Virus Type I Tax Transgenic Mice

Background Human T-cell leukemia virus type I (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATL) as well as inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A transgenic mouse that expresses HTLV-1 Tax also develops T-cell leukemia/lymphoma and an inflammatory arthropathy that resembles rheumatoid arthritis. The aim of this study was to identify the primary T-cell subsets involved in the development of arthropathy in Tax transgenic mice. Principal Findings By 24 months of age, Tax transgenic mice developed severe arthropathy with a cumulative incidence of 22.8%. The pathological findings of arthropathy in Tax transgenic mice were similar to those seen in human rheumatoid arthritis or mouse models of rheumatoid arthritis, with synovial proliferation and a positive rheumatoid factor. Before the onset of spontaneous arthropathy, young and old Tax transgenic mice were not sensitive to collagen and did not develop arthritis after immunization with type II collagen. The arthropathic Tax transgenic mice showed a significantly decreased proportion of splenic CD4+ T cells, whereas the proportion of splenic CD8+ T cells was increased. Regulatory T cells (CD4+CD25+Foxp3+) were significantly decreased and CD8+ T cells that expressed the chemokine receptor CCR4 (CD8+CCR4+) were significantly increased in arthropathic Tax transgenic mice. The expression of tax mRNA was strong in the spleen and joints of arthropathic mice, with a 40-fold increase compared with healthy transgenic mice. Conclusions Our findings reveal that Tax transgenic mice develop rheumatoid-like arthritis with proliferating synovial cells in the joints; however, the proportion of different splenic T-cell subsets in these mice was completely different from other commonly used animal models of rheumatoid arthritis. The crucial T-cell subsets in arthropathic Tax transgenic mice appear to resemble those in HAM/TSP patients rather than those in rheumatoid arthritis patients.

A transgenic mouse model of human T cell leukemia virus type 1-associated diseases.

Human T cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia/lymphoma (ATLL) and several inflammatory diseases. Tax, the protein encoded by HTLV-1, may be responsible for the development of the diseases caused by this virus. To investigate the pathogenic role of Tax, several transgenic mouse strains expressing Tax have been developed in recent years. These mice develop various tumors including large granular lymphocytic leukemia, as well as inflammatory diseases such as arthritis. These results suggest that Tax expression alone is sufficient to cause both malignant neoplastic diseases and inflammatory diseases. However, until recently, there were no tax transgenic mice that develop T cell leukemia and lymphoma resembling ATLL. The first successful induction of leukemia in T cells was pre-T cell leukemia generated in transgenic mice in which a mouse lymphocyte-specific protein tyrosine kinase p56lck (lck)-proximal promoter was used to express the tax gene in immature T cells. Subsequently, transgenic mice were established in which the lck-distal promoter was used to express Tax in mature T cells; these mice developed mature T cell leukemia and lymphoma that more closely resembled ATLL than did earlier mouse models.

Natural infection of murine norovirus in conventional and specific pathogen-free laboratory mice

Noroviruses cause most cases of acute viral gastroenteritis worldwide. The lack of a cell culture infection model for human norovirus necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict norovirus inactivation. Murine norovirus (MNV) may be used to construct a small animal model for studying the biology and pathogenesis of noroviruses because MNV is the only norovirus that replicates in cell culture and a small animal model. However, recent studies have shown that natural MNV infection is widespread in laboratory mouse colonies. We investigated MNV infection in both conventional and specific pathogen-free (SPF) genetically modified mice from Japan and the US, and commercial mice from several animal breeders in Japan, using serological and molecular techniques. MNV antibodies were detected in 67.3% of conventional mice and 39.1% of SPF mice from Japan and 62.5% of conventional mice from the US. MNV antibodies were also found in 20% of commercial SPF C57BL/6 mice from one of three breeders. Partial gene amplification of fecal isolates from infected animals showed that the isolates were homologous to reported MNV sequences. These results suggest that both conventional and SPF laboratory mice, including commercial mice, are widely infected with MNV, which might require considerable attention as an animal model of human disease.

Rapid tumor death model for evaluation of new therapeutic agents for adult T-cell leukemia.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell neoplasm. The health of ATL patients rapidly deteriorates resulting in death; however, the induction of death in a small animal model due to tumor has not yet been reported. SCID mice, 5 weeks old, younger than those previously used, which were inoculated with ATL cells, eliminated NK cell activity and showed rapid tumor formation resulting in death. Age is the crucial factor influencing tumor formation and death in the SCID mice model for cancer.

Invasion of histiocytic sarcoma into the spinal cord of HTLV-1 tax transgenic mice with HTLV-1-associated myelopathy/tropical spastic paraparesis-like disease

Human T-cell leukemia virus type 1 (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATLL) as well as inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Transgenic (Tg) mice expressing HTLV-1 Tax also develop T-cell leukemia/lymphoma and an inflammatory arthropathy that resembles rheumatoid arthritis. We found that 8 of 297 Tax-Tg mice developed HAM/TSP-like disease with symmetrical paraparesis of the hind limbs, but these symptoms were absent in non-Tg littermates and in other mice strains at our animal facilities. We could perform detailed evaluations for five of these mice. These evaluations showed that the disease was not inflammatory, unlike that in HAM/TSP patients, but instead involved the invasion of histiocytic sarcoma cells into the lumbar spinal cord from the bone marrow where they had undergone extensive proliferation.

p53 dysfunction precedes the activation of nuclear factor-κB during disease progression in mice expressing Tax, a human T-cell leukemia virus type 1 oncoprotein

Transgenic (Tg) mice expressing Tax, a human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, develop mature T-cell leukemia/lymphoma. The leukemic cells in Tg mice expressing Tax show p53 dysfunction and nuclear factor-κB (NF-κB) activation, similar to that seen in adult T-cell leukemia/lymphoma (ATLL) cells from patients infected with HTLV-1. However, it is unclear when these effects occur in HTLV-1 carriers during the development of ATLL. Here, we examined p53 function and NF-κB activity before the onset of leukemia in Tax-expressing Tg (Tax-Tg) mice between 4 and 25 months of age. At 4-10 months of age, 71% of mice showed p53 inactivation, without evidence for NF-κB activation, even though tax expression was consistent from 4 to 25 months of age. The decline in p53 function resulted from decreased p53 accumulation after DNA damage. From 11 months of age onward, 75% of mice showed p53 dysfunction and 37.5% showed constitutive NF-κB activation with the components of p50 and RelB. An NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), reduced NF-κB activity (i.e. p50/RelB) but did not restore p53 function. In vivo, treatment with DHMEQ until 24 months of age prevented the onset of T-cell leukemia in Tax-Tg mice. These results suggest that the Tax-induced decline in p53 function, which is independent of NF-κB activation in the early stage, might be the first stage in the onset of ATLL. NF-κB activity is involved in the later stages of ATLL onset.

Current Topics in Prevention of Human T-Cell Leukemia Virus Type I Infection: NF-κ B Inhibitors and APOBEC3

Human T-cell leukemia virus type I (HTLV-I) is the first human retrovirus and causes adult T-cell leukemia/lymphoma (ATL). Constitutive activation of nuclear factor-κ B (NF-κ B) in the leukemic cells is essential for their growth and survival. Thus, NF-κ B inhibitors have been attracting attention as a potential strategy to treat ATL. Recently, the field of retrovirus research has been stimulated by the discovery of an innate host defense factor, APOBEC3, against the retroviruses. HTLV-I is relatively resistant to the antiviral effects of APOBEC3. To clarify the resistance of HTLV-I against APOBEC3 might contribute to the design of effective therapeutic approaches.