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1.  HIV-1 Vpr Induces Interferon-Stimulated Genes in Human Monocyte-Derived Macrophages 
PLoS ONE  2014;9(8):e106418.
Macrophages act as reservoirs of human immunodeficiency virus type 1 (HIV-1) and play an important role in its transmission to other cells. HIV-1 Vpr is a multi-functional protein involved in HIV-1 replication and pathogenesis; however, its exact role in HIV-1-infected human macrophages remains poorly understood. In this study, we used a microarray approach to explore the effects of HIV-1 Vpr on the transcriptional profile of human monocyte-derived macrophages (MDMs). More than 500 genes, mainly those involved in the innate immune response, the type I interferon pathway, cytokine production, and signal transduction, were differentially regulated (fold change >2.0) after infection with a recombinant adenovirus expressing HIV-1 Vpr protein. The differential expression profiles of select interferon-stimulated genes (ISGs) and genes involved in the innate immune response, including STAT1, IRF7, MX1, MX2, ISG15, ISG20, IFIT1, IFIT2, IFIT3, IFI27, IFI44L, APOBEC3A, DDX58 (RIG-I), TNFSF10 (TRAIL), and RSAD2 (viperin) were confirmed by real-time quantitative PCR and were consistent with the microarray data. In addition, at the post-translational level, HIV-1 Vpr induced the phosphorylation of STAT1 at tyrosine 701 in human MDMs. These results demonstrate that HIV-1 Vpr leads to the induction of ISGs and expand the current understanding of the function of Vpr and its role in HIV-1 immune pathogenesis.
PMCID: PMC4149569  PMID: 25170834
3.  Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus 
Bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) make up a unique retrovirus family. Both viruses induce chronic lymphoproliferative diseases with BLV affecting the B-cell lineage and HTLV-1 affecting the T-cell lineage. The pathologies of BLV- and HTLV-induced infections are notably similar, with an absence of chronic viraemia and a long latency period. These viruses encode at least two regulatory proteins, namely, Tax and Rex, in the pX region located between the env gene and the 3′ long terminal repeat. The Tax protein is a key contributor to the oncogenic potential of the virus, and is also the key protein involved in viral replication. However, BLV infection is not sufficient for leukemogenesis, and additional events such as gene mutations must take place. In this review, we first summarize the similarities between the two viruses in terms of genomic organization, virology, and pathology. We then describe the current knowledge of the BLV model, which may also be relevant for the understanding of leukemogenesis caused by HTLV-1. In addition, we address our improved understanding of Tax functions through the newly identified BLV Tax mutants, which have a substitution between amino acids 240 and 265.
PMCID: PMC3820957  PMID: 24265629
BLV; HTLV-1; EBL; B-cell lymphoma; Tax; leukemogensis; transactivation; apoptosis
4.  Estimation of bovine leukemia virus (BLV) proviral load harbored by lymphocyte subpopulations in BLV-infected cattle at the subclinical stage of enzootic bovine leucosis using BLV-CoCoMo-qPCR 
Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. BLV infection may remain clinically silent at the aleukemic (AL) stage, cause persistent lymphocytosis (PL), or, more rarely, B cell lymphoma. BLV has been identified in B cells, CD2+ T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, γ/δ T cells, monocytes, and granulocytes in infected cattle that do not have tumors, although the most consistently infected cell is the CD5+ B cell. The mechanism by which BLV causes uncontrolled CD5+ B cell proliferation is unknown. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method, BLV-CoCoMo-qPCR, which enabled us to demonstrate that the proviral load correlates not only with BLV infection, as assessed by syncytium formation, but also with BLV disease progression. The present study reports the distribution of BLV provirus in peripheral blood mononuclear cell subpopulations isolated from BLV-infected cows at the subclinical stage of EBL as examined by cell sorting and BLV-CoCoMo-qPCR.
Phenotypic characterization of five BLV-infected but clinically normal cattle with a proviral load of > 100 copies per 1 × 105 cells identified a high percentage of CD5+ IgM+ cells (but not CD5- IgM+ B cells, CD4+ T cells, or CD8+T cells). These lymphocyte subpopulations were purified from three out of five cattle by cell sorting or using magnetic beads, and the BLV proviral load was estimated using BLV-CoCoMo-qPCR. The CD5+ IgM+ B cell population in all animals harbored a higher BLV proviral load than the other cell populations. The copy number of proviruses infecting CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells (per 1 ml of blood) was 1/34 to 1/4, 1/22 to 1/3, and 1/31 to 1/3, respectively, compared with that in CD5+ IgM+ B cells. Moreover, the BLV provirus remained integrated into the genomic DNA of CD5+ IgM+ B cells, CD5- IgM+ B cells, CD4+ T cells, and CD8+ T cells, even in BLV-infected cattle with a proviral load of <100 copies per 105 cells.
The results of the recent study showed that, although CD5+ IgM+ B cells were the main cell type targeted in BLV-infected but clinically normal cattle, CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells were infected to a greater extent than previously thought.
PMCID: PMC3648496  PMID: 23641811
Bovine leukemia virus (BLV); Proviral load; BLV-CoCoMo-qPCR; CD5+IgM+ B cell; Cell sorting; Flow cytometry
5.  The pH-Sensitive Fusogenic 3-Methyl-Glutarylated Hyperbranched Poly(Glycidol)-Conjugated Liposome Induces Antigen-Specific Cellular and Humoral Immunity 
We examined the ability of a novel liposome, surface modified by 3-methyl-glutarylated hyperbranched poly(glycidol) (MGlu-HPG), to enhance antigen-specific immunity in vitro and in vivo and to function as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) encapsulated in MGlu-HPG-modified liposomes more effectively than free OVA or OVA encapsulated in unmodified liposomes. Immunization of mice with OVA-containing MGlu-HPG-modified liposomes induced antigen-specific splenocyte proliferation and production of gamma interferon (IFN-γ) more strongly than did immunization with free OVA or OVA encapsulated in unmodified liposomes. The immune responses induced by OVA encapsulated in MGlu-HPG-modified liposomes were significantly suppressed by addition of anti-major histocompatibility complex (MHC) class I and class II monoclonal antibodies, indicating the involvement of antigen presentation via MHC class I and II. Furthermore, delayed-type hypersensitivity responses and OVA-specific antibodies were induced more effectively in mice immunized with OVA encapsulated by MGlu-HPG-modified liposomes than with unencapsulated OVA or OVA encapsulated in unmodified liposomes. These results suggested that MGlu-HPG-modified liposomes effectively induced both cell-mediated and humoral immune responses. Collectively, this study is the first to demonstrate the induction of both cell-mediated and humoral immune responses in vivo by MGlu-HPG-modified liposomes.
PMCID: PMC3428382  PMID: 22815149
6.  Positively charged cholesterol–recombinant human gelatins foster the cellular uptake of proteins and murine immune reactions 
Recombinant human gelatins with defined molecular weights were modified with cholesterol to make them amphiphilic in nature. We investigated the feasibility of these modified human gelatins acting as a carrier of antigenic proteins for inducing cellular immunity. The aim of this study was to synthesize novel and effective compounds for vaccine delivery in vivo.
Two types of cholesterol-modified gelatin micelles, anionic cholesterol-modified gelatin (aCMG) and cationic-cholesterol modified gelatin (cCMG), were synthesized using different cholesterol derivatives such as the cholesterol-isocyanate (Ch-I) for aCMG and amino-modified cholesterol for cCMG. One was anionic and the other cationic, and therefore they differed in terms of their zeta potential. The aCMG and cCMG were characterized for their size, zeta potential, and in their ability to form micelles. Cytotoxicity was also evaluated. The modified human gelatins were then investigated as a carrier of antigenic proteins for inducing cellular immunity both in vitro in DC 2.4 cells, a murine dendritic cell line, as well as in vivo. The mechanism of entry of the polymeric micelles into the cells was also evaluated.
It was found that only cCMG successfully complexed with the model antigenic protein, fluorescein-isothiocyanate ovalbumin (OVA) and efficiently delivered and processed proteins in DC 2.4 cells. It was hypothesized that cCMG enter the cells predominantly by a caveolae-mediated pathway that required tyrosine kinase receptors on the cell surface. Animal testing using mice showed that the cationic cholesterol-modified gelatin complexed with OVA produced significantly high antibody titers against OVA: 2580-fold higher than in mice immunized with free OVA.
Conclusively, cCMG has shown to be very effective in stimulating an immune response due to its high efficiency, stability, and negligible cytotoxicity.
PMCID: PMC3471541  PMID: 23091385
recombinant human gelatin; cholesterol; micelle; protein delivery; caveolae pathway; receptor-mediated endocytosis
7.  BLV-CoCoMo-qPCR: a useful tool for evaluating bovine leukemia virus infection status 
Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in BLV-infected animals. Indeed, the assay was highly effective in detecting BLV in cattle from a range of international locations. This assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression. In this study, we compared the sensitivity of our BLV-CoCoMo-qPCR method for detecting BLV proviruses with the sensitivities of two real-time PCR systems, and also determined the differences of proviral load with serotests.
BLV-CoCoMo-qPCR was found to be highly sensitive when compared with the real-time PCR-based TaqMan MGB assay developed by Lew et al. and the commercial TaKaRa cycleave PCR system. The BLV copy number determined by BLV-CoCoMo-qPCR was only partially correlated with the positive rate for anti-BLV antibody as determined by the enzyme-linked immunosorbent assay, passive hemagglutination reaction, or agar gel immunodiffusion. This result indicates that, although serotests are widely used for the diagnosis of BLV infection, it is difficult to detect BLV infection with confidence by using serological tests alone. Two cattle were experimentally infected with BLV. The kinetics of the provirus did not precisely correlate with the change in anti-BLV antibody production. Moreover, both reactions were different in cattle that carried different bovine leukocyte antigen (BoLA)-DRB3 genotypes.
Our results suggest that the quantitative measurement of proviral load by BLV-CoCoMo-qPCR is useful tool for evaluating the progression of BLV-induced disease. BLV-CoCoMo-qPCR allows us to monitor the spread of BLV infection in different viewpoint compared with classical serotest.
PMCID: PMC3489618  PMID: 22995575
Bovine leukemia virus; Real-time PCR; Proviral load; Serological test; Experimental infection
10.  BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm 
Retrovirology  2010;7:91.
Bovine leukemia virus (BLV) is closely related to human T-cell leukemia virus (HTLV) and is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly extended course that often involves persistent lymphocytosis and culminates in B-cell lymphomas. BLV provirus remains integrated in cellular genomes, even in the absence of detectable BLV antibodies. Therefore, to understand the mechanism of BLV-induced leukemogenesis and carry out the selection of BLV-infected animals, a detailed evaluation of changes in proviral load throughout the course of disease in BLV-infected cattle is required. The aim of this study was to develop a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in clinical animals.
Degenerate primers were designed from 52 individual BLV long terminal repeat (LTR) sequences identified from 356 BLV sequences in GenBank using the CoCoMo algorithm, which has been developed specifically for the detection of multiple virus species. Among 72 primer sets from 49 candidate primers, the most specific primer set was selected for detection of BLV LTR by melting curve analysis after real-time PCR amplification. An internal BLV TaqMan probe was used to enhance the specificity and sensitivity of the assay, and a parallel amplification of a single-copy host gene (the bovine leukocyte antigen DRA gene) was used to normalize genomic DNA. The assay is highly specific, sensitive, quantitative and reproducible, and was able to detect BLV in a number of samples that were negative using the previously developed nested PCR assay. The assay was also highly effective in detecting BLV in cattle from a range of international locations. Finally, this assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression.
Using our newly developed BLV-CoCoMo-qPCR assay, we were able to detect a wide range of mutated BLV viruses. CoCoMo algorithm may be a useful tool to design degenerate primers for quantification of proviral load for other retroviruses including HTLV and human immunodeficiency virus type 1.
PMCID: PMC2988707  PMID: 21044304
11.  The human immunodeficiency virus type 1 Vpr protein and its carboxy-terminally truncated form induce apoptosis in tumor cells 
The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces apoptosis after cell cycle arrest at the G2 phase in primate cells. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation and results in apoptosis without G2 arrest. Here, we investigated whether this property of Vpr and C81 could be exploited for use as a potential anticancer agent. First, we demonstrated that C81 induced G1 arrest and apoptosis in all tumor cells tested. In contrast, Vpr resulted in G2 arrest and apoptosis in HeLa and 293 T cells. Vpr also suppressed the damaged-DNA-specific binding protein 1 (DDB1) in HepG2 cells, thereby inducing apoptosis without G2 arrest. G2 arrest was restored when DDB1 was overexpressed in cells that also expressed Vpr. Surprisingly, C81 induced G2 arrest when DDB1 was overexpressed in HepG2 cells, but not in HeLa or 293 T cells. Thus, the induction of Vpr- and C81-mediated cell cycle arrest appears to depend on the cell type, whereas apoptosis was observed in all tumor cells tested. Overall, Vpr and C81 have potential as novel therapeutic agents for treatment of cancer.
PMCID: PMC2735735  PMID: 19674438
12.  A Mutant Form of the Tax Protein of Bovine Leukemia Virus (BLV), with Enhanced Transactivation Activity, Increases Expression and Propagation of BLV In Vitro but Not In Vivo 
Journal of Virology  2003;77(3):1894-1903.
In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.
PMCID: PMC140974  PMID: 12525624

Results 1-12 (12)