Enhancement of gene transactivation activity of androgen receptor by hepatitis B virus X protein

Hepatitis B virus (HBV) X protein (HBx) is a regulatory protein that is required for efficient replication of HBV in its natural host. In this report, we demonstrate by co-immunoprecipitation experiments that HBx can physically bind to the androgen receptor (AR), which is a nuclear hormone receptor that is expressed in many different tissues including the liver. This observation is further supported by confocal microscopy, which reveals that HBx can alter the subcellular localization of the AR both in the presence and in the absence of dihydrotestosterone (DHT). Further studies indicate that HBx can enhance the gene transactivation activity of AR by enhancing its DNA binding activity in a DHT-dependent manner. However, HBx does not remain associated with AR on the DNA. As AR can regulate the expression of a number of cellular genes, our results raise the possibility that HBV pathogenesis may be mediated in part via the interaction between HBx and AR.     

Deletion Analysis of Both the Long Terminal Repeat and the Internal Promoters of the Human Foamy Virus

Deletion analyses of the long terminal repeat (LTR) and internal promoters (IP) of human foamy virus (HFV) showed that a negative acting element resides in the U5 region of the 5′ LTR reducing reporter gene expression tenfold. The basal activity of the IP was higher than that obtained with LTR promoter constructs and strongly elevated in permissive BHK-21 cells whereas semi-permissive COS-7 cells showed low basal activity. Since the basal activity of the IP is critical for initiating HFV gene expression by providing Bel 1 transactivator early after infection, the basal activity of the IP may be the crucial factor that contributes to whether cells are permissive for HFV infection or not. Deletion mutagenesis allowed to define the minimal IP region. A region strongly transactivated by Bel 1 extends from −136 to +58 relative to the cap site of the IP. The major Bel 1 response element of the IP required for transactivation is located upstream of the cap site between −136 and −88 relative to the internal cap site. A DNA fragment reported to be protected by recombinant Bel 1 was deleted with marginal reduction of Bel 1 transactivation. HFV gene expression directed by the IP and LTR promoters is thus multiply regulated by positive and negative acting response elements in cis and their binding partners in trans. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the DNA-binding properties and the transactivation activity of Schistosoma mansoni nuclear receptor fushi tarazu-factor 1alpha (SmFTZ-F1alpha)

A FTZ-F1-related orphan nuclear receptor SmFTZ-F1alpha was previously identified from Schistosoma mansoni. The deduced SmFTZ-F1alpha protein contains a highly conserved DNA binding domain (DBD, C domain), a less conserved ligand binding domain (LBD, E domain) and three highly variable regions, the N-terminal A/B domain (108 aa), a large hinge region (D domain, 1027 aa) and an F domain (220 aa). Herein, we characterize the DNA binding properties and the transactivation activity of SmFTZ-F1alpha. In in vitro assays, SmFTZ-F1alpha bound as a monomer to a response element (FF1RE: TCAAGGTCA) recognized by mammalian steroidogenic factor 1 (SF-1), and to related sequences (p14: TTAAGGTCA and SmFF1a-2: CGAAGGTCA) derived from known schistosome gene promoters. Competition assays with p14 oligonucleotides containing a single mutation at each nucleotide position defined the optimum DNA sequence required for SmFTZ-F1alpha binding. The optimal consensus sequence for SmFTZ-F1alpha binding is TN(A/G)AGGTC(A/G) (N: any base). This sequence is similar but not identical to the SF-1 response element (SFRE) consensus sequence [(T/C)CAAGG(T/C)C(A/G)]. By performing yeast one-hybrid assays, the ability of SmFTZ-F1alpha to bind productively to a p14-derived 9-base pair sequence was demonstrated in vivo. The ability of the full-length SmFTZ-F1alpha to transactivate reporter gene expression was shown to be A/B domain-dependent in a yeast system. In addition, the hinge region contained an unexpected activation function (AF) domain, termed AF-3, while no transactivation activity was detected within the E/F domain. This AF-3 region (from aa 982 to aa 1110) revealed a strong autonomous transactivation activity, which was masked when it was present in the full-length SmFTZ-F1alpha. Taken together, our results suggest that SmFTZ-F1alpha possesses the characteristic DNA binding specificity of FTZ-F1 subfamily members and the capacity to transactivate a reporter gene.     

HIV-1 Vpr transactivates LTR-directed expression through sequences present within -278 to -176 and increases virus replication in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vpr, a 14-kDa virion-associated protein, plays an important role in the viral life cycle. Using a panel of truncated HIV-1 LTR-CAT constructs and Vpr expression plasmid, we have identified sequences from nucleotide -278 to -176 in LTR as Vpr-mediated transactivation domain. This region includes the glucocorticoid response element (GRE) in HIV-1 LTR. Transactivation by Vpr was noted with the HIV-1 LTR reporter constructs containing CAT or luciferase. A similar effect was also observed with a construct in which the GRE motif was linked to CAT. Studies involving Vpr mutants identified that helical domains I and III, and amino acid residues at G75 and C76, are responsible for GRE-mediated LTR transactivation. The transactivation function of Vpr is independent of its cell cycle arrest activity. Further, viral replication studies indicated that Vpr-mediated increase in viral replication is directly correlated with the ability of Vpr to transactivate HIV-1 LTR. The results presented here demonstrate that Vpr activates HIV-1 LTR through the host GR pathway and suggest that an intact GRE in the LTR is critical for Vpr activity.     

Establishment of cell lines using a doxycycline-inducible gene expression system to regulate expression of hepatitis B virus X protein

The hepatitis B virus (HBV) X gene plays an important role in HBV-associated pathogenesis, especially hepatocarcinogenesis. Establishment of a stable and regulable HBx expression system will allow study of the function of this gene. Here, we describe the development of a doxycycline-inducible recombinant plasmid (pBPSTR3-FlagX) with the full-length HBV X gene and all components of the tetracycline-on (“Tet-on”) gene expression system. This vector exhibited dose-dependent doxycycline-dependent induction of the Flag-HBx protein in HepG2 and Hep3B cells. We also observed dose-dependent doxycycline transactivation of HBx in HepG2 cells. After transfecting HepG2 cells with the pBPSTR3-FlagX plasmid, we isolated five puromycin-resistant cell clones with stable HBx expression, two of which exhibited stable and tight control of HBx expression by doxycycline. This new system has great potential for functional studies of the HBV X gene.     

β萘黄酮对大鼠谷氨酰半胱氨酸合成酶催化亚单位基因表达的影响

目的 通过了解外界刺激因子β萘黄酮(β-NF)对大鼠谷氨酰半胱氨酸合成酶催化亚单位(GCLC)基因转录的影响,研究大鼠γ-谷氨酰半胱氨酸合成酶(γ-GCS)基因转录的调节机制.方法 利用大鼠GCLC基因调控序列驱动的GCLC-PGL3-enhancer-Luciferase报道载体(GCLC-Luc)转染大鼠支气管上皮细胞(RTE)和肝癌细胞(H4ⅡE),分DMSO空白对照组和不同浓度的各刺激因子组,比较各组荧光素酶值的差异以筛选影响GCLC基因转录调控的刺激因子,并比较筛选到的刺激因子β-NF对GCLC-Luc在RTE和H4ⅡE细胞内表达影响的异同.2种转染后的细胞均分为β-NF实验组(10μmol/L)和DMSO空白对照组,以荧光定量PCR检测各组的细胞内源性γ-GCS转录水平变化.分别将构建的r系列缺失报道载体、GCLC-Luc和激活蛋白1(AP-1)、NF-κB定点突变报道载体转染2种细胞,分β-NF实验组(10 μmol/L)与DMSO空白对照组,比较各组荧光素酶值的变化,分析β-NF作用的调节位点和转录调控作用相关元件.结果 1、10、100μmol/L的β-NF均强烈抑制RTE中GCLC基因表达,荧光素酶值显著低于DMSO空白对照组(16 135±1456、2752±218、1579±294比25 971±1662,均P＜0.01).在H4ⅡE中,1、10、100 μmol/L的β-NF则促进其表达,荧光素酶值显著高于DMSO空白对照组(5686±441、13 601±746、13978±164比3645±367,均P＜0.01).荧光定量PCR显示10 μmol/L的β-NF作用下,RTE内源性γ-GCS的mRNA表达水平是DMSO空白对照组的0.73倍,在H4ⅡE细胞中则是1.98倍.GCLC-Luc、r系列缺失报道载体转染2种细胞后,RTE中各载体β-NF实验组荧光素酶值均低于DMSO空白对照组(P＜0.01),而H4ⅡE中则高于DMSO空白对照组(P＜0.05).β-NF作用的调节位点定位于转录起始位点上游-390～+2 bp范围内.用针对该区域的AP-1、NF-κB定点突变报道载体转染细胞后,转染GCLC-Luc载体的β-NF实验组RTE荧光素酶值较DMSO空白对照组下降(91.50±0.32)%,与之相比,转染AP-1、NF-κB定点突变报道载体的细胞下降幅度并没有减少(P＞0.05).在H4ⅡE,β-NF作用下转染GCLC-Luc载体的细胞荧光素酶值上升幅度则高于转染AP-1定点突变报道载体的细胞[(3.81±0.19)倍比(2.08±0.19)倍,P＜0.05],而与转染NF-κB定点突变报道载体的细胞荧光素酶值上升幅度[(4.1±1.01)倍]相比差异无统计学意义(P＞0.05).结论 在大鼠RTE和H4ⅡE,β-NF影响GCLC基因的表达具有细胞特异性.在大鼠H4ⅡE,β-NF通过激活抗氧化应答元件(ARE)类似的AP-1调控GCLC基因表达.     

Hepatitis B virus X protein enhances activation of nuclear factor κB through interaction with valosin-containing protein

Hepatitis B virus X protein (HBx protein) is a multifunctional regulatory protein. The transactivation of nuclear factor kappa B (NF-κB) by HBx protein has been shown to be of importance in the pathogenesis of HBV-related diseases. However, the mechanism involved remains largely unclear. In this study, a CytoTrap yeast two-hybrid system was employed to screen binding partners of the HBx protein; 29 cellular proteins, including valosin-containing protein (VCP), were identified. The interaction between HBx protein and VCP was further confirmed in vitro and in vivo using a glutathione S-transferase pull-down assay and co-immunoprecipitation, respectively. It was also shown that this interaction is mediated by amino acid residues 51–120 of the HBx protein. In Huh-7 hepatoma cells, HBx protein enhanced the VCP-mediated activation of NF-κB. Our findings provide new insights into the molecular mechanisms that lead to the activation of NF-κB by HBx protein.