A new indicator cell line established to monitor bovine foamy virus infection

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from −7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.     

牛泡沫病毒BFV3026细胞感染性的确立及包装细胞系的建立

分别将牛泡沫病毒BFV3026接种于胎牛肺细咆(FBL）、牛肺细咆系(BL12)、新生牛肾细胞(NBK)、兔肺细胞(RL)、人乳腺癌上皮细咆(MCF)、293T、HeLa、CV-1、CHO等9种细胞,通过对它们及其传代细胞的病变观察-RT—PCR检测,确立BFV3026对这9种细胞的感染。并以BFV3026原病毒DNA为模板,通过PCR构建以pcDNA3．1(-)为载体的gag—pol、env真核表达质粒共转染BL12细胞,经G418持续筛选,获得8个Neo^R细胞克隆RT—PCR及包装实验证实：其中7个细胞克隆能有效行使包装辅助功能。     

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the B...     

Engineering Viral Promoters for Gene Transfer to Human Neuroblasts

1. The strength and activity of several viral promoters in human neuroblasts were evaluated in vitro. 2. Several luciferase reporter gene contructs under the control of different viral promoters (HIV-1 LTR, HTLV-I LTR, MMTV LTR, RSV LTR, CMV, SV40), in the presence or in the absence of the viral SV40 enhancer, were transfected into two well-established human neural cell lines, including one derived from human embryonic olfactory cells (B4) and one derived from an adrenal neuroblastoma (SH-SY-5Y). The epithelial cell line HeLa was used as a control.3. The enzymatic activity of luciferase was evaluated after normalization with an internal control. The results indicated that in the context of the reporter gene constructs, the CMV promoter alone was, overall, the most active in any tested cell line. However, addition of the SV40 enhancer to the CMV promoter abolished luciferase activity in SH-SY-5Y cells while significantly increasing luciferase expression in the CNS derived B4 fetal neuroblasts.4. The results suggest that gene therapeutic vectors aimed to promote enzymatic activity through gene transfer into undifferentiated human neural cells are feasible. However, since differences in promoter activity in neuroectodermal-derived cells are very relevant, gene construct variants should be considered to optimize the system.     

Subcellular localization analysis of bovine foamy virus Borf1 protein

The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.     

Subcellular Localization Analysis of Bovine Foamy Virus Borfl Protein

The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.     

JDV与三种牛反转录病毒相互关系的初步研究

为研究JDV与其它三种牛反转录病毒BIV、BLV、BFV的相互作用关系,将以JDV、BIV、BLV、BFV的LTR为启动子,以Luc为报告基因的质粒和以上病毒反式激活因子的表达质粒共转染BLl2细胞系,通过瞬时表达分析试验证明了JDV和BIV的LTR和Tat之间亲缘关系很近,能够相互激活;JDV Tat可以反式激活BLVLTR,BLVTax不能激活JDVLTR;JDVLTR上存在BFVTas的应答元件;BLV、BFV和BIV的LTR和反式激活因子问不存在相互激活。     

A quantitative assay for measuring of bovine immunodeficiency virus using a luciferase-based indicator cell line

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay. BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.     

Growth inhibition of cancer cells by co-transfection of diphtheria toxin A-chain gene plasmid with bovine leukemia virus-tax expression vector

We constructed a plasmid containing bovine leukemia virus (BLV)-tax gene driven by SR alpha promoter, designated as pME-BLVtax, to activate the promoter of the long terminal repeat (LTR) of BLV in various tumor cells. Activation of the promoter of BLV-LTR by pME-BLVtax was confirmed by luciferase assay. When the cells, such as COS-1, C8, and KU-1, were transfected with a plasmid pBLV-LUC1, which contained the luciferase gene under the control of BLV-LTR, and pME-BLVtax, luciferase was expressed in these cells, whereas no luciferase gene expression was observed when only pBLV-LUC1 was introduced into the cells. Activation of the BLV-LTR promoter was regulated by pME-BLVtax and 0.5 microg of pME-BLVtax was sufficient for the expression of the gene under the control of BLV-LTR. Furthermore, pME-BLVtax was used to direct the cell expression of the gene for diphtheria toxin A-chain under the control of BLV-LTR (pLTR-DT) to various tumor cell lines, KU-1, C8, COS-1, BL2M3, and HeLa cells. The transfection was carried out with cationic liposomes. In this experiment, co-transfection of pLTR-DT with pME-BLVtax exerted selective growth inhibitory effects on the tumor cell lines. Moreover, three co-introductions of pLTR-DT with pME-BLVtax into the cell lines resulted in significant inhibition of the cell growth. This result suggests that the delivery of the pLTR-DT and pME-BLVtax genes into tumor cells by the use of cationic liposomes may be potentially useful as a novel approach for the treatment of tumor cells.     

HIV LTR-dependent expression of Bax selectively induces apoptosis in Tat-positive cells

HIV integrates into the host cell genome where it persists for the life of the cell. One approach to reducing viral burden is to selectively eliminate cells containing integrated provirus early following infection. We have used the HIV LTR promoter to selectively express transgenes in human cells positive for the HIV transactivator protein Tat. Transient transfection of Jurkat cells, or Jurkat cells stably expressing Tat (Jurkat-Tat), with a LTR construct containing luciferase reporter gene resulted in a 37-fold increase in gene expression when Tat was present. We have demonstrated that when pro-apoptotic Bax was used as the transgene, cytotoxicity was seen only in the Jurkat-Tat cells. Annexin-V staining indicated that Bax induced cell death by apoptosis. In mixed populations of Jurkat and Jurkat-Tat cells, the LTR-Bax construct was selectively cytotoxic to the Tat-positive cells. These results suggest that Bax under the control of the HIV LTR can be used to destroy cells harbouring HIV without affecting uninfected cells.     

Quantitative analysis of luciferase activity of viral and hybrid promoters in bovine preimplantation embryos

This study was conducted to investigate quantitatively the luciferase activity of gene constructs with viral and hybrid enhancers and promoters in bovine preimplantation embryos by using firefly luciferase reporter genes. In Experiment I, to examine the stability of the luciferase, bioluminescence intensity of bovine embryos injected with the luciferase gene driven by the SV40 early promoter and enhancer (SVEluc) was measured with a luminometer at 2 days after microinjection. The results indicated that the bioluminescence could be analysed at any time within 30 min because the luciferase activity was constant during the measurement period from 5 to 30 min. In Experiment II, the luciferase expression of fertilized oocytes injected with four gene constructs (TKEluc, TK6WEluc, SVEluc, and Miwluc) was analysed by using a photon imaging system at 2 or 6 days following microinjection. The results from Experiment II indicated that the reporter gene governed by the Miw promoter (RSV LTR and chicken β-actin promoter) was expressed more intensively in bovine morulae and blastocysts than three other gene constructs. In Experiment III, the effect of SV40 enhancer was investigated when fused downstream to the luciferase cDNA of the Miwluc vector. The results showed that SV40 enhancer further activated the luciferase activity of the Miw promoter in bovine preimplantation embryos. It was concluded, therefore, that the Miw promoter together with the SV40 enhancer would confer the strongest expression of the firefly luciferase reporter gene among the gene constructs tested in preimplantation bovine embryos. Mol. Reprod. Dev. 49:368–373, 1998. © 1998 Wiley-Liss, Inc.     

多聚TAR-CoreRNA诱饵对人免疫缺陷病毒1型Tat蛋白活性的抑制作用

为了抑制Ｔａｔ蛋白的反式激活作用,在细胞内大量表达外源ＴＡＲＲＮＡ使其与Ｔａｔ蛋白结合,从而竞争性抑制其与ＨＩＶ－１ＬＴＲ的ＴＡＲＲＮＡ元件结合．构建了以ＨＩＶ－１ＬＴＲＹＬ１５８（－１５８～＋１８０）为启动子,分别含有４,８和１５个拷贝的ＴＡＲ－ＣｏｒｅＲＮＡ诱饵（ｄｅｃｏｙ）表达质粒;以荧光酶基因为报告基因,检测了瞬时共转染体系中含不同拷贝数的ＴＡＲ－ＣｏｒｅＤＮＡ转录产物对Ｔａｔ蛋白反式激活作用的影响．结果证明,ＴＡＲ－ＣｏｒｅＲＮＡ诱饵对Ｔａｔ蛋白活性具有很强的抑制作用,其抑制程度与ＴＡＲ－ＣｏｒｅＤＮＡ串联体的拷贝数有关．