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Human T-cell leukemia virus type 1 (HTLV-1) infects target cells primarily through cell-to-cell routes. Here, we provide evidence that cellular protein M-Sec plays a critical role in this process. When purified and briefly cultured, CD4+ T cells of HTLV-1 carriers, but not of HTLV-1- individuals, expressed M-Sec. The viral protein Tax was revealed to mediate M-Sec induction. Knockdown or pharmacological inhibition of M-Sec reduced viral infection in multiple co-culture conditions. Furthermore, M-Sec knockdown reduced the number of proviral copies in the tissues of a mouse model of HTLV-1 infection. Phenotypically, M-Sec knockdown or inhibition reduced not only plasma membrane protrusions and migratory activity of cells, but also large clusters of Gag, a viral structural protein required for the formation of viral particles. Taken together, these results suggest that M-Sec induced by Tax mediates an efficient cell-to-cell viral infection, which is likely due to enhanced membrane protrusions, cell migration, and the clustering of Gag., Author summary In the present study, we identified the cellular protein M-Sec as a host factor necessary for de novo infection of human T-cell leukemia virus type 1 (HTLV-1), the causative retrovirus of an aggressive blood cancer known as adult T-cell leukemia/lymphoma. The inhibition or knockdown of M-Sec in infected cells resulted in a reduced viral infection in several culture models and a mouse model. We recently demonstrated a similar role of M-Sec in macrophages infected with another human retrovirus HIV-1, but it has been generally thought that M-Sec is not related to HTLV-1 infection because of the lack of its expression in CD4+ T cells, the major target of HTLV-1. In this study, we revealed that CD4+ T cells of HTLV-1 asymptomatic carriers, but not those of HTLV-1- individuals, expressed M-Sec, and that the viral protein Tax mediated the induction of M-Sec. Thus, M-Sec is a new and useful tool for further understanding the process of HTLV-1 transmission.

Background HIV-1 promotes the formation of tunneling nanotubes (TNTs) that connect distant cells, aiding cell-to-cell viral transmission between macrophages. Our recent study suggests that the cellular protein M-Sec plays a role in these processes. However, the timing, mechanism, and to what extent M-Sec contributes to HIV-1 transmission is not fully understood, and the lack of a cell line model that mimics macrophages has hindered in-depth analysis. Results We found that HIV-1 increased the number, length and thickness of TNTs in a manner dependent on its pathogenic protein Nef and M-Sec in U87 cells, as observed in macrophages. In addition, we found that M-Sec was required not only for TNT formation but also motility of U87 cells, both of which are beneficial for viral transmission. In fact, M-Sec knockdown in U87 cells led to a significantly delayed viral production in both cellular and extracellular fractions. This inhibition was observed for wild-type virus, but not for a mutant virus lacking Nef, which is known to promote not only TNT formation but also migration of infected macrophages. Conclusions By taking advantage of useful features of U87 cells, we provided evidence that M-Sec mediates a rapid and efficient cell–cell transmission of HIV-1 at an early phase of infection by enhancing both TNT formation and cell motility.

Additional file 1. Supplemental Fig. S1–Fig. S16.

Objectives Infiltration of macrophages through the tyrosine kinase receptor CSF1R is a poor prognosis factor in various solid tumors. Indeed, these tumors produce CSF1R ligand, macrophage colony‐stimulating factor (M‐CSF) or interleukin‐34 (IL‐34). However, the significance of these cytokines, particularly, the newly discovered IL‐34 in haematological malignancies, is not fully understood. We therefore analysed the role of IL‐34 in diffuse large B‐cell lymphoma (DLBCL), the most common subtype of malignant lymphoma. Methods We analysed formalin‐fixed paraffin‐embedded lymphoma tissues of 135 DLBCL patients for the expression of IL‐34 and the number of macrophages, and the survival of these patients. The expression of IL‐34 in DLBCL cell lines and the activity of IL‐34 to induce the migration of monocytic cells were also characterised. Results Several lymphoma tissues showed a clear IL‐34 signal, and such signal was detectable in 36% of patients. DLBCL cell lines also expressed IL‐34. Interestingly, the percentage of IL‐34+ patients in the activated B‐cell subtype was significantly higher than that in the germinal centre B‐cell subtype. More interestingly, IL‐34+ patients showed shorter survival periods and higher number of macrophages in lymphoma tissues. The recruitment of monocytes is likely the first step for the higher macrophage density in the IL‐34+ lymphoma tissues. Indeed, IL‐34 induced the migration of monocytic cells. Conclusion Our results raise the possibility that IL‐34 in lymphoma tissues of DLBCL patients recruits monocytes, leading to the higher number of macrophages in the tissues and poor prognosis of patients. IL‐34 may be an additional therapeutic target of DLBCL.

The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important role of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.

The interaction between HIV-1 Nef and the Src kinase Hck in macrophages has been shown to accelerate the progression to AIDS. We previously showed that Nef disturbed the N-glycosylation/trafficking of Fms, a cytokine receptor essential for maintaining macrophages in an anti-inflammatory state, in an Hck-dependent manner. Here, we show the underlying molecular mechanism of this effect. Using various Hck isoforms and their mutants and Golgi-targeting Hck mutants, we confirmed that Hck activation at the Golgi causes the Nef-induced Fms N-glycosylation defect. Importantly, we found that both the co-expression of Nef and Hck and the expression of a Golgi-targeted active Hck mutant caused alterations in the distribution of GM130, a Golgi protein that was shown to be required for efficient protein glycosylation. Moreover, the activation of Hck at the Golgi caused strong serine phosphorylation of the GM130-interacting Golgi structural protein GRASP65, which is known to induce Golgi cisternal unstacking. Using pharmacological inhibitors, we also found that the activation of Hck at the Golgi followed by the activation of the MAP kinase ERK-GRASP65 cascade is involved in the Fms N-glycosylation defect. These results suggest that Nef perturbs the structure and signaling of the Golgi by activating Hck at the Golgi, and thereby, induces the N-glycosylation/trafficking defect of Fms, which is in line with the idea that Src family kinases are crucial Golgi regulators.

Nef assembles a multi-kinase complex triggering MHC-I down-regulation. We identify an inhibitor that blocks MHC-I down-regulation, identifying a temporally regulated switch in Nef action from directing MHC-I endocytosis to blocking cell surface delivery. These findings challenge current dogma and reveal a regulated immune evasion program., HIV-1 Nef triggers down-regulation of cell-surface MHC-I by assembling a Src family kinase (SFK)-ZAP-70/Syk-PI3K cascade. Here, we report that chemical disruption of the Nef-SFK interaction with the small molecule inhibitor 2c blocks assembly of the multi-kinase complex and represses HIV-1–mediated MHC-I down-regulation in primary CD4+ T-cells. 2c did not interfere with the PACS-2–dependent trafficking of Nef required for the Nef-SFK interaction or the AP-1 and PACS-1–dependent sequestering of internalized MHC-I, suggesting the inhibitor specifically interfered with the Nef-SFK interaction required for triggering MHC-I down-regulation. Transport studies revealed Nef directs a highly regulated program to down-regulate MHC-I in primary CD4+ T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days postinfection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action.

Macrophage colony-stimulating factor (M-CSF) regulates the production, survival and function of macrophages through Fms, the receptor tyrosine kinase. Recently, interleukin-34 (IL-34), which shares no sequence homology with M-CSF, was identified as an alternative Fms ligand. Here, we provide the first evidence that these ligands indeed resemble but are not necessarily identical in biological activity and signal activation. In culture systems tested, IL-34 and M-CSF showed an equivalent ability to support cell growth or survival. However, they were different in the ability to induce the production of chemokines such as MCP-1 and eotaxin-2 in primary macrophages, the morphological change in TF-1-fms cells and the migration of J774A.1 cells. Importantly, IL-34 induced a stronger but transient tyrosine phosphorylation of Fms and downstream molecules, and rapidly downregulated Fms. Even in the comparison of active domains, these ligands showed no sequence homology including the position of cysteines. Interestingly, an anti-Fms monoclonal antibody (Mab) blocked both IL-34-Fms and M-CSF-Fms binding, but another MAb blocked only M-CSF-Fms binding. These results suggested that IL-34 and M-CSF differed in their structure and Fms domains that they bound, which caused different bioactivities and signal activation kinetics/strength. Our findings indicate that macrophage phenotype and function are differentially regulated even at the level of the single receptor, Fms.

HIV-1 Nef accelerates the progression to AIDS by binding with and activating a Src kinase Hck, but underlying molecular basis is not understood. We revealed that Nef disturbed N-glycosylation/trafficking of a cytokine receptor Fms in an Hck-dependent manner, a possible trigger to worsen uncontrolled immune system. Here, we provide direct evidence that dys-regulated activation of Hck pre-localized to the Golgi apparatus causes this Fms maturation arrest. A striking change in Hck induced by Nef other than activation was its skewed localization to the Golgi due to predominant Golgi-localization of Nef. Studies with different Nef alleles and their mutants showed a clear correlation among higher Nef-Hck affinity, stronger Hck activation, severe Golgi-localization of Hck and severe Fms maturation arrest. Studies with a newly discovered Nef-Hck binding blocker 2c more clearly showed that skewed Golgi-localization of active Hck was indeed the cause of Fms maturation arrest. 2c blocked Nef-induced skewed Golgi-localization of an active form of Hck (Hck-P2A) and Fms maturation arrest by Nef/Hck-P2A, but showed no inhibition on Hck-P2A kinase activity. Our finding establishes an intriguing link between the pathogenesis of Nef and a newly emerging concept that the Golgi-localized Src kinases regulate the Golgi function.

Xtr is present exclusively in early embryonic and germline cells. We have previously shown that loss-of-function of the Xtr in embryos causes arrest of karyokinesis progression. Since Xtr contains plural tudor domains, which are known to associate with target proteins directly, we examined Xtr-interacting proteins by immunoprecipitation with an anti-Xtr monoclonal antibody and detected a few RNA-binding proteins such as FRGY2, a component of messenger ribonucleoprotein (mRNP) particle. The coexistence of Xtr with FRGY2 by constituting an mRNP particle was further confirmed by gel filtration assay. Search of mRNAs in the immunoprecipitate with Xtr suggested that the Xtr-associated molecules included several mRNAs, of which translational products were known to play crucial roles in karyokinesis progression (RCC1, XRHAMM, and so on) and in germ cell development (XDead end). Immunohistochemical observation clearly showed the co-localization of Xtr with FRGY2 also in germ plasm, in which XDead end mRNA has been shown to be localized specifically. Taken together, we proposed the possible role of Xtr in translational activation of the maternal mRNAs repressed in mRNP particle.

Fibrocytes (fibroblastic leukocytes) are recently identified as unique hematopoietic cells with features of both macrophages and fibroblasts. Fibrocytes are known to contribute to the remodeling or fibrosis of various injured tissues. However, their role in viral infection is not fully understood. In this study, we show that differentiated fibrocytes are phenotypically distinguishable from macrophages but can be infected with HIV-1. Importantly, fibrocytes exhibited persistently infected cell-like phenotypes, the degree of which was more apparent than macrophages. The infected fibrocytes produced replication-competent HIV-1, but expressed HIV-1 mRNA at low levels and strongly resisted HIV-1–induced cell death, which enabled them to support an extremely long-term HIV-1 production at low but steady levels. More importantly, our results suggested that fibrocytes were susceptible to HIV-1 regardless of their differentiation state, in contrast to the fact that monocytes become susceptible to HIV-1 after the differentiation into macrophages. Our findings indicate that fibrocytes are the previously unreported HIV-1 host cells, and they suggest the importance of considering fibrocytes as one of the long-lived persistently infected cells for curing HIV-1.

Tunneling nanotubes (TNTs), the long membrane extensions connecting distant cells, have emerged as a novel form of cell-to-cell communication. However, it is not fully understood how and to what extent TNTs contribute to intercellular spread of pathogens including HIV-1. In this study, we show that HIV-1 promotes TNT formation per se via its protein Nef and a cellular protein M-Sec, which appears to mediate approximately half of viral spread among monocyte-derived macrophages (MDMs). A small compound that inhibits M-Sec–induced TNT formation reduced HIV-1 production by almost half in MDMs. Such inhibition was not observed with Nef-deficient mutant HIV-1 that fails to promote TNT formation and replicates less efficiently than the wild-type HIV-1 in MDMs. The TNT inhibitor–sensitive/Nef-promoting viral production was also observed in a T cell line ectopically expressing M-Sec, but not in another M-Sec− T cell line. Our results suggest the importance of TNTs in HIV-1 spread among MDMs and might answer the long-standing question how Nef promotes HIV-1 production in a cell type–specific manner.

Background Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) infection. However, there are no therapies to prevent ATL development in high-risk asymptomatic carriers. To develop a therapy targeting HTLV-1-infected cells that are known to express CCR4 frequently, we tested whether truncated Pseudomonas exotoxin (PE38) fused to a CCR4 ligand, CCL17/thymus and activation-regulated chemokine (TARC), selectively eliminates such cells. Results Our data show that TARC–PE38 efficiently killed HTLV-1-infected cell lines. It also shrank HTLV-1-associated solid tumors in an infected-cell-engrafted mouse model. In HTLV-1-positive humanized mice, TARC–PE38 markedly inhibited the proliferation of HTLV-1-infected human CD4+CD25+ or CD4+CD25+CCR4+ cells and reduced the proviral loads (PVLs) in peripheral blood mononuclear cells (PBMCs). Importantly, TARC–PE38 significantly reduced the PVLs in PBMCs obtained from asymptomatic carriers. We show that the cytotoxicity of TARC–PE38 is mediated by the expression of the proprotein convertase, furin. The expression of furin was enhanced in HTLV-1-infected cells and correlated positively with PVLs in HTLV-1-infected individuals, suggesting that infected cells are more susceptible to TARC–PE38 than normal cells. Conclusions TARC–PE38 robustly controls HTLV-1 infection by eliminating infected cells in both a CCR4- and furin-dependent manner, indicating the excellent therapeutic potential of TARC–PE38. Electronic supplementary material The online version of this article (doi:10.1186/s12977-015-0199-8) contains supplementary material, which is available to authorized users.

To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies.

Figure S2. Phenotype of MT-2 cells. Expression of the indicated molecules on MT-2 cells was analyzed by flow cytometry. This analysis showed that MT-2 cells are CD45â CD3â CD4+CD8â . This phenotype of cells was analyzed similarly in samples from humanized mice inoculated with MT-2 cells and data obtained are shown in Additional file 3: Table S1.

Figure S4. Independence of furin expression from induced expression of HTLV-1 Tax. Although expression of Tax was definitely induced by adding Cd2+ to JPX-9 cells (lower panel), expression of furin was not increased as compared with that before adding it (upper panel).

Table S2. Characteristics of HTLV-1-infected individuals from which blood samples were collected to test a correlation between PVLs and expression of furin.

Figure S3. Therapeutic effects of TARC–PE38 on CD4+CCR4+ cells in HTLV-1-infected humanized mice. To assess the therapeutic effect of TARC–PE38 on HTLV-1-infected cells, peripheral blood was obtained from all the mice. The PBMCs were isolated and the frequency of human CD4+CCR4+ cells was determined with flow cytometry. The data are representative of each group as follows: none, uninfected untreated mice; MT-2 + PBS, HTLV-1-infected mice treated with PBS; MT-2 + TARC–PE38, HTLV-1-infected mice treated with TARC–PE38. The number indicated in the upper right quadrant is the frequency of human CD4+CCR4+ cells (%).

Figure S1. Viability of MT-2 cells treated with MMC. After MT-2 cells were treated with 50 Îźg/ml MMC, they were cultured in vitro and the number of live cells was counted daily with the standard Trypan-blue exclusion method to check the viability of MT-2 cells. At three days post-MMC treatment most of the cells were dead.

Table S1. Detection of MT-2 cells in humanized mice at 2Â weeks post-inoculation with non- or MMC-treated MT-2 cells. Frequencies of MT-2 cells and HTLV-1 PVLs were tested with flow cytometry and quantitative PCR, respectively, in splenocytes and PBMCs obtained from humanized mice at 2Â weeks post-inoculation with non- or MMC-treated MT-2 cells. In either mouse group MT-2 cells were not detected by flow cytometry (see Additional file 2: Figure S2 also). While the provirus was detected in splenocytes alone obtained from mice inoculated with non-treated MT-2 cells, it was not at all in splenocytes and PBMCs obtained from mice inoculated with MMC-treated MT-2 cells.

Embryonic patterning and germ-cell specification in mice are regulative and depend on zygotic gene activities. However, there are mouse homologues of Drosophila maternal effect genes, including vasa and tudor , that function in posterior and germ-cell determination. We report here that a targeted mutation in Tudor domain containing 1 / mouse tudor repeat 1 ( Tdrd1 / Mtr-1 ), a tudor -related gene in mice, leads to male sterility because of postnatal spermatogenic defects. TDRD1/MTR-1 predominantly localizes to nuage/germinal granules, an evolutionarily conserved structure in the germ line, and its intracellular localization is downstream of mouse vasa homologue / DEAD box polypeptide 4 ( Mvh / Ddx4 ), similar to Drosophila vasa - tudor . Tdrd1/Mtr-1 mutants lack, and Mvh / Ddx4 mutants show, strong reduction of intermitochondrial cement, a form of nuage in both male and female germ cells, whereas chromatoid bodies, another specialized form of nuage in spermatogenic cells, are observed in Tdrd1/Mtr-1 mutants. Hence, intermitochondrial cement is not a direct prerequisite for oocyte development and fertility in mice, indicating differing requirements for nuage and/or its components between male and female germ cells. The result also proposes that chromatoid bodies likely have an origin independent of or additional to intermitochondrial cement. The analogy between Mvh - Tdrd1 in mouse spermatogenic cells and vasa - tudor in Drosophila oocytes suggests that this molecular pathway retains an essential role(s) that functions in divergent species and in different stages/sexes of the germ line.

We compared the secondary spermatogonia and the primary spermatocytes of Xenopus for the proteins in their microsomal fractions and identified a newly synthesized protein (94 kDa) and three other proteins (99, 85, and 72 kDa) which increased their amount after entering the meiotic phase. These four proteins were used as antigens to produce polyclonal antibody which was found to react with the four proteins as well as two other proteins (208 and 60 kDa). Immunoscreening of Xenopus testis cDNA library with this polyclonal antibody yielded two cDNA clones (Xmegs and Xtr) encoding novel proteins. Xmegs mRNA was specifically expressed in the spermatogenic cells from the mid-pachytene stage to completion of two meiotic divisions. The putative Xmegs protein contained 19 tandem repeats of 26 amino acid residues rich in proline as well as potential phosphorylation sites (i.e., serine and threonine residues). Around this repetitive area, we found five PEST sequences known as a proteolytic signal to target protein for degradation. The presence of PEST sequences was believed to allow protein levels to closely parallel mRNA abundance. These results suggested the possible role of this novel protein in the regulation of two meiotic divisions specific to the spermatogenesis in a phosphorylation- and/or dephosphorylation-dependent manner. On the other hand, Xtr mRNA was expressed in both spermatogenic and oogenic cells except for round spermatids and the later stage cells. This mRNA was also expressed in the early stage embryos and its amount was kept constant from the St. I oocyte to the gastrula stage and decreased thereafter. The putative Xtr protein contained four complete and one partial tudor-like domains that were discovered in Drosophila tudor protein which plays an important role in PGC differentiation and abdominal segmentation. The characteristic expression profile of Xtr and the protein structure similar to the Drosophila tudor protein suggested its possible role in the progression of meiosis and PGC differentiation.

Previous studies indicated that the acquisition of egg fertilizability during transit through the pars recta portion of the oviduct in Bufo japonicus is accompanied by hydrolytic conversion of the vitelline coat 40- to 52-kDa components to 39-kDa components induced by a 66-kDa serine protease, “oviductin.” In this study, we cloned a 3028-bp cDNA that contained an open reading frame encoding 974 amino acids with a calculated molecular mass of 107.6 kDa, including two protease domains and three repeats of CUB domains. Sequence analysis indicated that the catalytically active 66-kDa protein comprised an N-terminally located oviductin protease and two CUB domains. The oviductin gene was transcribed as a part of 6-kb mRNA that was expressed specifically in the cells lining the bottom of epithelial folds in the oviductal pars recta, and this expression was highly accelerated when the pars recta fragments were cultured in the presence of hCG. Western blot analyses using antibodies against a protease domain revealed that the catalytically inactive 102-kDa proteins in the pars recta granules yield 66-kDa catalytically active and 82- and 59-kDa inactive molecules. We propose that the oviductin translated as 107.6-kDa precursors are processed both N- and C-terminally to give rise to a 66-kDa active form comprising a serine protease and two CUB domains.

Xtr in the fertilized eggs of Xenopus has been demonstrated to be a member of a messenger ribonucleoprotein (mRNP) complex that plays a crucial role in karyokinesis during cleavage. Since the Xtr is also present both in oocytes and spermatocytes and its amount increases immediately after spematogenic cells enter into the meiotic phase, this protein was also predicted to act during meiotic progression. Taking advantage of Xenopus oocytes' large size to microinject anti-Xtr antibody into them for inhibition of Xtr function, we examined the role of Xtr in meiotic progression of oocytes. Microinjection of anti-Xtr antibody into immature oocytes followed by reinitiation of oocyte maturation did not affect germinal vesicle break down and the oscillation of Cdc2/cyclin B activity during meiotic progression but caused abnormal spindle formation and chromosomal alignment at meiotic metaphase I and II. Immunoprecipitation of Xtr showed the association of Xtr with FRGY2 and mRNAs such as RCC1 and XL-INCENP mRNAs, which are involved in the progression of karyokinesis. When anti-Xtr antibody was injected into oocytes, translation of XL-INCENP mRNA, which is known to be repressed in immature oocytes and induced after reinitiation of oocyte maturation, was inhibited even if the oocytes were treated with progesterone. A similar translational regulation was observed in oocytes injected with a reporter mRNA, which was composed of an enhanced green fluorescent protein open reading frame followed by the 3' untranslational region (3'UTR) of XL-INCENP mRNA. These results indicate that Xtr regulates the translation of XL-INCENP mRNA through its 3'UTR during meiotic progression of oocyte.

IFN-inducible IFITM proteins (IFITM1, 2, and 3) inhibit the replication of various viruses including HIV-1 through poorly understood mechanisms. Here, we further analyzed characteristics of these newly identified HIV-1 restriction factors. Firstly, in contrast to other anti-HIV-1 proteins, such as tetherin and APOBEC3G, IFITMs were resistant to a down-regulation of surface expression or degradation by HIV-1 proteins. Secondly, the enforced expression of IFITMs reduced the production of HIV-1 viruses from cells transfected with proviral plasmids containing whole viral sequences. Although their inhibitory activities were modest when compared to that of tetherin, IFITMs, but not tetherin, directly reduced the expression of HIV-1 proteins including Gag, Vif and Nef. Of importance, however, IFITMs had no inhibitory effect when these viral proteins were expressed by codon-optimized cDNAs that bypassed the viral-specific expression machinery. Indeed, our results supported the idea that IFITMs interfere with viral protein expression mediated by double-stranded viral RNAs, such as RRE and TAR. Finally, the S-palmitoylation of IFITMs, which is crucial for their anti-influenza virus activity, was not required for their anti-HIV-1 activity, indicating that IFITMs restrict these viruses at different steps. These characteristics lead to a better understanding of the mechanism by which IFITMs restrict HIV-1 and other viruses.

Xtr is present exclusively in early embryonic and germline cells. We have previously shown that loss-of-function of the Xtr in embryos causes arrest of karyokinesis progression. Since Xtr contains plural tudor domains, which are known to associate with target proteins directly, we examined Xtr-interacting proteins by immunoprecipitation with an anti-Xtr monoclonal antibody and detected a few RNA-binding proteins such as FRGY2, a component of messenger ribonucleoprotein (mRNP) particle. The coexistence of Xtr with FRGY2 by constituting an mRNP particle was further confirmed by gel filtration assay. Search of mRNAs in the immunoprecipitate with Xtr suggested that the Xtr-associated molecules included several mRNAs, of which translational products were known to play crucial roles in karyokinesis progression (RCC1, XRHAMM, and so on) and in germ cell development (XDead end). Immunohistochemical observation clearly showed the co-localization of Xtr with FRGY2 also in germ plasm, in which XDead end mRNA has been shown to be localized specifically. Taken together, we proposed the possible role of Xtr in translational activation of the maternal mRNAs repressed in mRNP particle.

Cholangiocarcinoma (CCA) is a major cause of cancer deaths in northeast Thailand. It is aggressive, highly metastatic, and responds poorly to traditional chemotherapy. We demonstrated the potential for Cepharanthine (CEP), a biscoclaurine alkaloid extracted from Stephania cepharantha, to treat CCA. CEP significantly inhibited growth of human CCA cell lines in a dose- and time-dependent manner, regardless of the histologic type of tumor origin. Increasing cell apoptosis via caspase-3 and capase-9 activation was demonstrated in CEP-treated cells. We found that CEP controlled the growth of CCA cells through nuclear factor-kappa B (NF-κB) inactivation by inhibiting nuclear translocation. CEP treatment effectively reduced tumor size in CCA-inoculated mice without serious side effects. CEP also increased cell apoptosis in primary histocultures of CCA patients’ tissues; this was demonstrated by immunohistochemistry using TUNEL staining. Our results suggest that CEP possesses therapeutic potential against human CCA. (Cancer Sci 2010)

Primary effusion lymphoma (PEL) is a unique and recently identified non-Hodgkin's 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)-kappaB 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 microg/ml). CEP also inhibited NF-kappaB 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-kappaB could be an ideal molecular target for treating PEL and that CEP is quite useful as a unique therapeutic agent for PEL.

Nef is a multifunctional pathogenetic protein of HIV-1, the interaction of which with Hck, a Src tyrosine kinase highly expressed in macrophages, has been shown to be responsible for the development of AIDS. However, how the Nef-Hck interaction leads to the functional aberration of macrophages is poorly understood. We recently showed that Nef markedly inhibited the activity of macrophage colony-stimulating factor (M-CSF), a primary cytokine for macrophages. Here, we show that the inhibitory effect of Nef is due to the Hck-dependent down-regulation of the cell surface expression of M-CSF receptor Fms. In the presence of Hck, Nef induced the accumulation of an immature under–N-glycosylated Fms at the Golgi, thereby down-regulating Fms. The activation of Hck by the direct interaction with Nef was indispensable for the down-regulation. Unexpectedly, the accumulation of the active Hck at the Golgi where Nef prelocalized was likely to be another critical determinant of the function of Nef, because the expression of the constitutive-active forms of Hck alone did not fully down-regulate Fms. These results suggest that Nef perturbs the intracellular maturation and the trafficking of nascent Fms, through a unique mechanism that required both the activation of Hck and the aberrant spatial regulation of the active Hck.

Stem cells in various somatic tissues including hematopoietic stem cells can be identified by the "side population (SP)" phenotype based on the efflux of Hoechst33342. Knockout and enforced expression experiments show that the expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype. In this study, we showed that erythroblasts also express a large amount of Bcrp1/ABCG2. The level of expression was increased with maturation, but did not relate to the cell-cycle status. Despite the high expression level of Bcrp1/ABCG2, erythroblasts did not show the "side population" phenotype. Furthermore, a Bcrp1/ABCG2 inhibitor, verapamil, had little effect on the Hoechst33342 staining pattern of erythroblasts. However protoporphyrin IX fluorescence was significantly higher in the presence of verapamil, suggesting that the ABCG2 functions as a transmembrane transporter in erythroblasts. These results indicate that dissociation between Bcrp1/ABCG2 expression and dye efflux function exists in erythroblasts and in stem cells, and that the function of ABCG2 in erythroblasts differs from that in stem cells.

We have previously shown that the transcriptional product of the novel gene, Xenopus tudor repeat (Xtr), occurred exclusively in germline cells and early embryonic cells and that the putative Xtr contained plural tudor domains which are thought to play a role in the protein-protein interactions. To understand the role of Xtr, we produced an antibody against a polypeptide containing Xtr tudor domains as an antigen and investigated the distribution and the function of the Xtr. Immunoprecipitation/Western blot and immunohistochemical analyses indicated a similar occurrence of the Xtr to the mRNA except for a slightly different profile of its amount during spermatogenesis. In spite of a large amount of Xtr mRNA at late-secondary spermatogonial stage, the amount of Xtr was kept at a low level until this stage and increased after entering into the meiotic phase. Depletion of the Xtr function in the activated eggs by injection of the anti-Xtr antibody caused the inhibition both of microtubule assembly around nucleus and of karyokinesis progression after prophase, but not of the oscillation of H1 kinase activity. These results suggest that the karyokinesis of at least early embryonic cells are regulated by unique mechanisms in which the Xtr is involved.

Primary effusion lymphoma (PEL) is a unique recently identified non-Hodgkin’s lymphoma which was originally identified in AIDS patients. PEL is characteristically infected by the Kaposi sarcoma-associated herpesvirus (KSHV) and shows a peculiar presentation involving liquid growth in the serous body cavity with generally poor prognosis. As the NK-κB pathway is activated in PEL and plays a central role in oncogenesis, we examined the effect of biscoclaurine alkaloid, Cepharanthine, on PEL derived cell lines (BCBL-1, TY-1, and RM-P1) in vitro and in vivo. MTT assay revealed the cell proliferation of PEL cell lines was significantly suppressed by the addition of Cepahranthine (1–10μg/ml). Cepharanthine also inhibited NK-κB acitivation and induced apoptotic cell death in PEL cell lines. We established PEL animal model by intraperitoneal injection of BCBL-1 into the nobel immunodeficient NOD/Scid/Jak3null mice. Intraperitoneal injection led to the development of ascites and diffuse infiltration of organs, without obvious solid lymphoma formation, resembling the diffuse nature of human PEL. Intraperitoneal administration of Cepharanthine inhibited the ascites formation and diffuse infiltration of BCBL-1 without significant systemic toxicity in this model. These results indicate that NK-κB could be an ideal molecular target of PEL and Cepharanthine could a unique therapeutic agent for PEL.

Nef, an accessory protein of HIV−1, has been shown to be critical for both high viral loads and progression to AIDS. We recently demonstrated that Nef inhibited the bioactivities of M-CSF, a monocytes/macrophages-specific cytokine (Blood105: 3230–3237, 2005). The inhibitory effect of Nef is a likely explanation for dysregulated functions in HIV−1−infected macrophages. Here, we examined the specificity and molecular mechanism of the newly-characterized function of Nef, to clarify its relevance to the pathogenetic activity of Nef. By analyzing cytokine-dependent cell lines expressing the conditionally active Nef, we found that Nef had a profound inhibitory effect on M-CSF signal, but not on IL-4- and GM-CSF signals. We also found that Nef down-regulated the surface expression of M-CSF receptor (M-CSFR) but not of GM-CSF receptors. The M-CSFR down-regulation by Nef was reproducible in 293 cells when co-transfected with Hck, a member of Src kinases. Interestingly, the down-regulation of cell surface M-CSFR in the presence of Nef and Hck correlated with the concomitant increase of the immature form of M-CSFR. These results suggested that Nef perturbed M-CSFR maturation through Hck and thereby selectively inhibited M-CSF signal.