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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) utilizes a highly complex splice site regulation system, taking advantage of host proteins, to express its own viral protein in an orderly way. We show here that one of the host proteins, high mobility group A protein 1a (HMGA1a), is involved in splice site regulation of 3' splice site 2 (A2) and 5'splice site 3 (D3) of HIV-1 genomic RNA. shRNA knockdown of HMGA1 in HeLa cells resulting in a decrease of HMGA1 showed a significant decrease of Vpr mRNA. RNA electrophoretic mobility shift assays showed HMGA1a specifically binds to a sequence adjacently upstream D3. In vitro splicing using heterologous pre-mRNA with A2 and D3, showed HMGA1a induced a splicing intermediate which decreased when an RNA decoy of the HMGA1a binding site was added. RT-PCR of in vitro splicing products revealed that HMGA1a induced an incomplete splicing product resulting from usage of A2 but inhibition of D3, which is reminiscent of the splicing pattern necessary for Vpr mRNA formation. HMGA1a interacted with hnRNPA1 shown by coimmunoprecipitation and supershifted U1 snRNP in an RNA electrophoretic mobility shift assay. We conclude that HMGA1a anchors U1 snRNP to inhibit D3 function, and that HMGA1a inhibits hnRNPA1 function on exon splicing silencer of Vpr (ESSV) to activate A2 function. We show here for the first time that HMGA1a is involved in specific splice site regulation of HIV-1.
Biochemical and Biophysical Research Communications 02/2011; 406(4):512-7. · 2.48 Impact Factor
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ABSTRACT: OX40 protein is highly expressed on activated CD4-positive T cells that are susceptible for human immunodeficiency virus type 1 (HIV-1) infection. To target and kill HIV-1-infected OX40(+) T cells, we used a recombinant vesicular stomatitis virus (rVSV) lacking its envelope glycoprotein (ΔG) and instead expressing HIV-1 receptors CD4/CXCR4 and OX40 ligand (OX40L). Expression of OX40L as well as HIV-1 receptors on the VSV particles led to specific infection of OX40(+) T cells, including primary cells, either acutely or chronically infected with X4 HIV-1. Consequently, the rVSV rapidly eliminated these infected cells and caused a marked reduction of HIV-1 viral load in culture. Inclusion of the OX40L gene in the VSV recombinant led to significantly better infection and HIV-1 elimination compared with an rVSVΔG expressing only HIV-1 receptors. A novel rVSVΔG encoding both HIV-1 receptors and OX40L has a potentially greater therapeutic value than an rVSVΔG expressing only HIV-1 receptors.
Human immunology 01/2011; 72(4):295-304. · 2.55 Impact Factor
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Tsutomu Murakami,
Sei Kumakura,
Toru Yamazaki,
Reiko Tanaka,
Makiko Hamatake, Kazu Okuma,
Wei Huang,
Jonathan Toma,
Jun Komano,
Mikiro Yanaka,
Yuetsu Tanaka,
Naoki Yamamoto
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ABSTRACT: The previously reported CXCR4 antagonist KRH-1636 was a potent and selective inhibitor of CXCR4-using (X4) human immunodeficiency virus type 1 (HIV-1) but could not be further developed as an anti-HIV-1 agent because of its poor oral bioavailability. Newly developed KRH-3955 is a KRH-1636 derivative that is bioavailable when administered orally with much more potent anti-HIV-1 activity than AMD3100 and KRH-1636. The compound very potently inhibits the replication of X4 HIV-1, including clinical isolates in activated peripheral blood mononuclear cells from different donors. It is also active against recombinant X4 HIV-1 containing resistance mutations in reverse transcriptase and protease and envelope with enfuvirtide resistance mutations. KRH-3955 inhibits both SDF-1alpha binding to CXCR4 and Ca(2+) signaling through the receptor. KRH-3955 inhibits the binding of anti-CXCR4 monoclonal antibodies that recognize the first, second, or third extracellular loop of CXCR4. The compound shows an oral bioavailability of 25.6% in rats, and its oral administration blocks X4 HIV-1 replication in the human peripheral blood lymphocyte-severe combined immunodeficiency mouse system. Thus, KRH-3955 is a new promising agent for HIV-1 infection and AIDS.
Antimicrobial Agents and Chemotherapy 06/2009; 53(7):2940-8. · 4.84 Impact Factor
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ABSTRACT: We have previously demonstrated that normal human T cells either long-term repeatedly stimulated or freshly activated in vitro in the presence of TGF-beta express the cell surface T-cell costimulating molecule OX40 ligand (OX40L). To further elucidate the kinetics of OX40L expression by human T cells, we have examined whether cell proliferation was required for the expression of OX40L. Thus, normal fresh peripheral blood mononuclear cells were stimulated with immobilized anti-CD3 antibody in the presence of the DNA synthesis-blocking agents such as mitomycin C, 5-fluorouracil, or X-ray irradiation. Flow cytometric analyses demonstrated that a significant frequency of these DNA-damaged activated primary CD4+ and CD8+ T cells became OX40L+ as early as 1 hour after treatment. The OX40L induction on the DNA-damaged activated T cells was inhibited by treatment with either RNA or protein synthesis inhibitors, actinomycin D, or cycloheximide, respectively. Induced OX40L on T cells was functional because it bound recombinant OX40. These data indicate that human primary T cells are programmed to rapidly express functional OX40L molecules after stimulation under DNA-damaging conditions, demonstrating that the induction of OX40L by T cells is independent of cell proliferation. The clinical implications of these new findings are discussed.
Human Immunology 09/2008; 69(9):533-42. · 2.84 Impact Factor
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ABSTRACT: Dendritic cell (DC)-based immunotherapy has been utilized for the treatment of not only a number of human malignancies but also a select group of infectious diseases. Conventional techniques for the generation and maturation of DCs require 7 days of in vitro culture, which prompted us to seek alternative methods that would hasten the generation of functional human myeloid DCs in vitro. Following the use of a number of cytokines/growth factors, we found that in vitro culture of purified human monocytes, in media containing interleukin (IL)-4, together with interferon (IFN)-beta for 24 hrs, followed by the addition of non-specific antigenic stimuli, such as keyhole limpet hemocyanin (KLH), lipopolysaccharide (LPS), or inactivated human immunodeficiency virus (HIV)-1 induced the monocytes to differentiated by 3 days into mature DCs (4B-DCs). These 4B-DCs expressed high levels of CD83 and CD11c, as well as markers of immune activation, including CD80 and CD86, human leukocyte antigen (HLA) class I and II, and CD14, but not CD1a. Anti-CD14 blocking antibody interfered with generation of 4B-DCs by LPS, but not by KLH or HIV-1. Interestingly, 4B-DCs, but not conventional DCs generated using macrophage-colony stimulating factor and IL-4 (G4-DCs), expressed OX40 and OX40L. 4B-DCs showed phagocytic activity, and spontaneously produced IL-12 and tumor necrosis factor (TNF)-alpha, but not IL-10. 4B-DCs promoted proliferation of allogeneic naïve CD4(+) T cells, producing IFN-(lambda) at lower levels than those stimulated with G4-DCs. 4B-DCs were more potent stimulators of allogeneic bulk CD8(+) T cells producing IFN-(lambda) than G4-DCs. These data indicate that 4B-DCs are unique and may provide a relatively more rapid alternative tool for potential clinical use, as compared with conventional G4-DCs.
Experimental Biology and Medicine 07/2008; 233(6):721-31. · 2.64 Impact Factor
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ABSTRACT: CXCR4-tropic (X4) human immunodeficiency virus type 1 (HIV-1) does not efficiently infect and replicate in severe combined immunodeficiency (SCID) mice reconstituted with human peripheral blood mononuclear cells, termed "hu-PBL-SCID mice," due to, at least in part, relatively low levels of expression of the CXCR4 coreceptor. To overcome this limitation, interleukin (IL)-4-transgenic hu-PBL-SCID mice were derived that spontaneously synthesized human IL-4, which has been shown to enhance CXCR4 expression and promote X4 virus infection in vitro. Experiments reported here show that (1) synthesis of human IL-4 in vivo augmented CXCR4 expression on human CD4(+) lymphocytes and importantly led to productive infection of not only X4 HIV-1(NL4-3) but also multidrug-resistant primary clinical isolates and that (2) the in vivo infection could be significantly blocked by the administration of a CXCR4 antagonist. Altogether, IL-4-transgenic hu-PBL-SCID mice provide a useful model for X4 HIV-1 study and testing/screening of anti-X4 viral drugs.
The Journal of Infectious Diseases 02/2008; 197(1):134-41. · 6.41 Impact Factor
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ABSTRACT: Interaction between OX40 expressed on activated T cells and its ligand (OX40L) on antigen presenting cells (APC) provides a co-stimulatory signal for T cells to promote acquired immunity. In the present study, we have examined various culture conditions for optimum OX40L expression on T cells stimulated with immobilized anti-CD3/CD28 monoclonal antibodies (mAbs). Although the day 3 primed T cells expressed minimal OX40L, after repeated stimulations both the CD4+ and CD8+ T cells became OX40L positive as determined by flow cytometry. Interleukin (IL)-12 interfered with the OX40L expression. Among activated T cells, a higher frequency of CD8+ T cells expressed OX40L than CD4+ T cells. By blocking OX40L-OX40 interaction by an anti-OX40 mAb, the number of OX40L+ T cells significantly increased. Screening of various cytokines showed that transforming growth factor (TGF)-beta1 was capable of induction of OX40L on the activated T cells within 3 days. The OX40L expressed on T cells was functional, as they bound soluble OX40 and stimulated human immunodeficiency virus-1 (HIV-1) production from cell lines chronically infected with HIV-1 and expressing OX40. Altogether the present study findings indicate that functional OX40L is inducible on human activated CD4+ and CD8+ T cells, and that the expression is enhanced by TGF-beta1.
Human Immunology 08/2007; 68(7):563-71. · 2.84 Impact Factor
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ABSTRACT: Monocytes express on the cell surface several kinds of chemokine receptors that facilitate chemotaxis followed by differentiation in target tissues. In the present study, we found that a large number of monocytes from peripheral blood mononuclear cells (PBMCs) tightly adhered to plastic cell culture plates precoated with a monoclonal antibody (mAb, clone T312) specific for human CCR5 but not an isotype control after overnight incubation. Soluble T312 did not induce such adhesion, indicating that cross-linking of CCR5 is required for the enhanced adhesion of monocytes. The adhesion was blocked by a PI3-K inhibitor and an anti-CD18 blocking mAb. Following the cross-linking of CCR5, monocytes synthesized high levels of M-CSF, RANTES, MIP-1 alpha, and MIP-1 beta associated with a readily detectable down modulation of CD14, CD4, CCR5, and CXCR4 expression. The T312-enriched monocytes differentiated into dendritic cells (DCs) in the presence of interleukin-4 alone. After maturation with beta-interferon, the T312-induced DCs stimulated proliferation of allogeneic naïve CD4(+) T cells accompanied by the synthesis of high levels of gamma-interferon in vitro. Furthermore, the T312-induced DCs were capable of stimulating antigen-specific human T- and B-cell immune responses in our hu-PBL-SCID mouse system. Finally, screening of other anti-chemokine receptor mAbs showed that select clones of mAbs against CXCR4 and CCR3 were also capable of facilitating enrichment of monocytes similar to T312. These results show that cross-linking of chemokine receptors on monocytes by appropriate mAbs leads to activation and differentiation of monocytes and that the method described herein provides an alternate simple strategy for adherence-based isolation of monocytes and generation of functional DCs.
Experimental Biology and Medicine 05/2006; 231(4):431-43. · 2.64 Impact Factor
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ABSTRACT: We have constructed VSV recombinants lacking the viral glycoprotein gene and instead expressing rhesus macaque SIV receptors CD4 and CCR5 with or without the receptor DC-SIGN. The recombinant expressing CD4 and CCR5 specifically infected SIV envelope protein-expressing cells. Incorporation of DC-SIGN into the particles required deletion of the cytoplasmic domain. Inclusion of DC-SIGN in the particles definitely enhanced infection, indicating that the enhancement by coexpression of DC-SIGN with CD4 and CCR5 does not require internalization of the virus into cells. The recombinants also specifically infected, killed, and propagated in CEMx174 cells that were first infected with an SIV expressing EGFP. If cells were superinfected with either of the recombinants after the primary SIV infection, the numbers of SIV-infected cells and titers of infectious SIV in the cultures were significantly reduced. Such antivirals can now be tested in the SIV/non-human primate model for AIDS to determine their therapeutic value in vivo.
Virology 04/2006; 346(1):86-97. · 3.35 Impact Factor
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ABSTRACT: To develop a high-titer surrogate virus for human T-cell leukemia virus type 1 (HTLV-1), we generated recombinant vesicular stomatitis viruses (VSVs) in which the gene encoding the single transmembrane glycoprotein (G) was deleted. Genes encoding HTLV-1 envelope glycoproteins (HTEnv) or HTEnvG hybrid proteins were then inserted into either of two different sites in the VSV genome. The viruses also encoded a green fluorescent protein. With this surrogate virus, we found that a soluble protein, osteoprotegerin (OPG), or an OPG/Fc chimeric protein inhibited the infection of various cell lines. Our experiments indicate that this inhibition resulted from binding of heparan sulfate by OPG.
Journal of Virology 09/2003; 77(15):8562-9. · 5.40 Impact Factor
