Non-identical patterns of proviral insertions around host transcription units in lymphomas induced by different strains of murine leukemia virus

In a small sample of 57 retrovirus integration sites (RISs) isolated from 23 end-stage lymphomas induced in NMRI mice by the B-lymphotropic Akv wt or an enhancer mutant hereof, Akv1-99, we identified 14 novel RISs and defined 9 novel CISs (common insertion sites). Moreover, when comparing with RISs from tumors induced by the T-lymphomagenic SL3-3, we observed that SL3-3 targets RefSeq promoter regions with a significantly higher frequency than Akv/Akv1-99 and in an orientation-dependent way. Altogether, our results strongly emphasize the importance of host genetic background and virus type for retroviral insertion mutagenesis screens and suggest that different types of MLV may favor specific genomic regions and orientations in order exert optimal effect on target gene expression during lymphoma induction and development.     

Activation of the glucocorticoid receptor releases unstimulated PBMCs from an early block in HIV-1 replication

Infection of resting peripheral mononuclear blood cells (PBMCs) with HIV-1 is not productive due to a block prior to integration of the provirus into the host genome. Here we show that a unique restriction is determined by the status of the glucocorticoid receptor (GR). Proviral integration increases after addition of a GR ligand. The ligand dependent effect is confined to an early time period after infection and requires GR and the GR binding viral protein Vpr. Endogenous GR and transiently expressed Vpr are localized in the cytoplasm in unstimulated PMCs and comigrate into the nucleus upon ligand addition. Thus, the predominant cytoplasmic localization of GR seems to be a specific obstacle for HIV replication. Accordingly, efficient proviral integration in a cell line with a constitutive cytoplasmic GR requires addition of a GR ligand. The data suggest that steroids can overcome the restriction on HIV provirus formation and thereby increase the reservoir of virus producing cells.     

从未经培养的HIV-1阳性外周血克隆及系统树分析HIV-1中国株E亚型gp120基因

目的:研究中国HIV-1 E亚型代表株的主要结构基因及其功能。方法:选择3份根据HIV-1 的外膜基因(C2-V3)的序列分析判定为E亚型的阳性血样,采用克隆和系统树分析,通过套式PCR得到全长的gp120 基因片段,并插入到pFastBacl载体中,以双脱氧末端终止法测定全部的DNA 序列。结果:来自广东和广西的样品的gp120 基因归于E亚型,E亚型中国株内的遗传距离为2.56% ～5.87% ,与16 株国际标准株比较,遗传距离为3.60% ～27.98% 。所有克隆到的gp120 基因都具有完整的阅读框架,无大的缺失和插入。结论:HIV-1 E亚型进入中国的时间不长;克隆到的E亚型代表株的gp120 基因具有完整的结构和功能,适合用来构建E亚型的亚单位疫苗表达载体     

L-chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro

The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. l-Chicoric acid (l-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), l-CA inhibits integration at concentrations from 500 nM to 10 microM but also inhibits entry at concentrations above 1 microM. Using recombinant HIV IN, steady-state kinetic analyses with l-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of l-CA, was successively washed. Inhibition of IN diminished, demonstrating that l-CA was reversibly bound to the protein. These data demonstrate that l-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, l-CA likely interacts with amino acids other than those which bind substrate.     

睫状神经营养因子及神经突起素上调莫洛尼小鼠白血病病毒前病毒插入位点1表达促进受损神经元样细胞突起再生

目的 探讨莫洛尼小鼠白血病病毒前病毒插入位点1(Pim-1)基因在体外受损神经元中的表达变化，以及神经营养因子调节Pim-1表达进而促进受损神经元突起再生的相关分子基础。方法 用反式视黄酸将Neuro-2a（N-2a）细胞诱导成为神经元样N-2a（N-2a-N）细胞，用去铁胺亚磺酸盐(DFO)抑制N-2a细胞增殖，用丙烯酰胺（ACR）损伤N-2a-N细胞突起。N-2a-N细胞分为正常对照组、损伤组、睫状神经营养因子（CNTF）及神经突起素（Nrn1）组,每组4个样本。免疫荧光细胞化学法检测N-2a-N细胞表型及Pim-1蛋白表达；用Real-time PCR和Western blotting检测Pim-1在各组的表达变化。用Western blotting检测调节Pim-1活性的相关分子，细胞存活、凋亡相关分子及轴突再生相关分子的表达变化。结果 细胞免疫荧光显示，N-2a-N细胞表达神经元标志分子β Ⅲ-微管蛋白（β-Ⅲ tubulin）和neurofilament-200，并表达Pim-1。50 μmol DFO有效抑制N-2a细胞的增殖。应用 -1 mmol/L-的ACR成功建立N-2a-N细胞突起损伤模型。N-2a-N细胞损伤后，Pim-1基因表达呈现先降低后升高，再降低的趋势。与损伤组相比，CNTF组和Nrn1组最长神经突起所占比例明显增加，细胞内信号转导分子细胞外调节蛋白激酶1/2（ERK1/2）、磷酸化细胞外调节蛋白激酶1/2（p-ERK1/2）、信号转导及转录激活因子3（STAT3）、磷酸化信号转导及转录激活因子3（p-STAT3）及Pim-1表达上调，凋亡相关分子cleaved Caspase-3及Bax表达下调，抗凋亡相关分子Bcl-2表达上调，神经突起生长相关分子生长相关蛋白43（GAP-43）表达上调.结论 修复受损N-2a-N细胞需要过表达Pim-1基因。神经营养因子CNTF、Nrn1可激活受损N-2a-N细胞ERK1/2及STAT3信号通路，进而上调Pim-1及GAP-43表达，并促进细胞突起再生。     

Leukemogenesis of Adult T-Cell Leukemia

Adult T-cell leukemia (ATL) is one of the most aggressive hematologic malignancies and is caused by human T-cell leukemia virus type I (HTLV-I). Tax, encoded by the HTLV-I pX region, has been recognized by its pleiotropic actions as a critical accessory protein playing a central role in leukemogenesis. However, fresh ATL cells frequently lose Tax protein expression via several mechanisms, such as genetic and epigenetic changes in the provirus. Furthermore, there is a long latency period before the onset of ATL, indicating the multistep mechanisms of leukemogenesis. Therefore, additional factors, including other viral proteins, genetic and epigenetic changes of the host genome, and alterations in the gene expression and immune systems of the host cells, may be implicated in ATL leukemogenesis. This review summarizes recent advances in the understanding of ATL leukemogenesis.     

Integrase-independent HIV-1 infection is augmented under conditions of DNA damage and produces a viral reservoir

HIV-1 possesses a viral protein, integrase (IN), which is necessary for its efficient integration in target cells. However, it has been reported that an IN-defective HIV strain is still capable of integration. Here, we assessed the ability of wild type (WT) HIV-1 to establish infection in the presence of IN inhibitors. We observed a low, yet clear infection of inhibitor-incubated cells infected with WT HIV which was identical to cells infected with IN-deficient HIV, D64A. Furthermore, the IN-independent integration could be enhanced by the pretreatment of cells with DNA-damaging agents suggesting that integration is mediated by a DNA repair system. Moreover, significantly faster viral replication kinetics with augmented viral DNA integration was observed after infection in irradiated cells treated with IN inhibitor compared to nonirradiated cells. Altogether, our results suggest that HIV DNA has integration potential in the presence of an IN inhibitor and may serve as a virus reservoir.