Expression of sequence variants of endogenous retrovirus RGH in particle form in multiple sclerosis
Expression of sequence variants of endogenous retrovirus RGH in particle form in multiple sclerosis
复制标题
DOI：
10.1016/s0140-6736(05)60075-x
复制
发表时间：
1998-09-26
期刊：
影响因子：
168.9
通讯作者：
Moller-Larsen, A
中科院分区：
文献类型：
--
作者：
Christensen, T;Sorensen, PD;Moller-Larsen, A
我们观察了在短期和长期培养的外周血单个核细胞从几个多发性硬化症患者的C型逆转录病毒样颗粒的生产。逆转录病毒颗粒具有逆转录酶（RT）活性，并与人T细胞白血病病毒-1共享一些抗原决定簇，但在抗原水平上与已知的逆转录病毒不同。1为了鉴定逆转录病毒，我们对从细胞培养上清液中纯化的裂解的逆转录病毒颗粒进行了RT-PCR。RT活性、EM中逆转录病毒颗粒的存在以及共定位在梯度中的RNA含量。2我们使用mRNA Direct Kit（Dynal，Norway）从RT阳性梯度组分中分离RNA模板。每个RNA样品用DNAse（Life Technologies）处理，并且我们用GeneBank RNA PCR试剂盒（Perkin Elmer）对处理的样品进行RT-PCR。控制RNA模板的完整性，包括特异性引发的cDNA合成与下游引物（负cDNA合成与上游引物），和重复的每个反应没有RT。我们分析了PCR产物的琼脂糖凝胶电泳，克隆，和DEL双脱氧测序。从最初的gag引物，我们开发了gag和env区的特异性引物5 'ATTTTATTACCCAATCTGCTCCAAACAT 3'/5 'AGGTGAGTTGAACAGTCTGATT TTTA 3'; 5 'CGTTTACATATCACTCCCTTCCTAGTCTCTGT 3'/5 'GCATTAACCTTGACTATGTCTTTAGCT CCAG 3'; 5 'GATCCTCCCCACTGGGTTCACCATT 3'/5 'GGAAGTATTGGAGGGTGCCCTGCC 3'。我们在4个多发性硬化细胞系中鉴定了几个与人内源性逆转录病毒RGH-2同源的gag和env片段。RGH属于C型样RTVL-H/HERV-H（含His dRNA PBS的人内源性逆转录病毒）家族，与人肿瘤逆转录病毒人T细胞白血病病毒1和2以及ERV-9（内源性逆转录病毒）相关。已描述了两个RGH克隆：缺乏gag的截短克隆（RGH-1）和全长克隆（RGH-2）。据报道，RGH样序列存在于约100个拷贝/单倍体基因组中。3 RGH颗粒本身先前未报告。我们还测试了多发性硬化细胞系颗粒RNA，其通过我们的标准程序用ERV-9-1相关的巢式引物在设定的ST 1 -1/2和RT-PCR条件下纯化。为了评估颗粒形式的RGH序列是否与体内多发性硬化症相关，我们对多发性硬化症患者或自身免疫性疾病患者以及健康对照的无细胞、过滤、超浓缩血浆临床样本进行了盲法RT-PCR（表）。颗粒水平的RGH序列表达对多发性硬化症具有特异性，所有活动性多发性硬化症患者在采样时均为RGH阳性。这一发现表明，在许多患者中，多发性硬化症与含有RGH序列的逆转录病毒颗粒进入血液有关，可能与疾病活动有关。在细胞系上清液的病毒颗粒和多发性硬化患者血浆的颗粒组分中发现RGH-2序列变体。几个RGH序列分离自个体
We observed production of type C retrovirus-like particles during short-term and long-term cultivation of peripheral blood mononuclear cells from several patients with multiple sclerosis. The retrovirus particles have reverse-transcriptase (RT) activity and share a few antigenic determinants with human T-cell leukaemia virus-1, but are distinct from the known retroviruses at the antigenic level. 1 To identify the retrovirus we did RT-PCR on lysed retroviral particles purified from cell culture supernatants by ultracentrifugation. RT activity, presence of retroviral particles in EM, and RNA content colocated in the gradients. 2 We used the mRNA Direct Kit (Dynal, Norway) to isolate the RNA templates from RT-positive gradient fractions. Each RNA sample was treated with DNAse (Life Technologies) and we did RT-PCR with the GeneAmp RNA PCR Kit (Perkin Elmer) on the treated samples. Controls for the integrity of RNA templates included specifically primed cDNA synthesis with the downstream primer (negative cDNA synthesis with the upstream primer), and duplicates of each reaction without RT. We analysed PCR products by agarose gel electrophoresis, cloning, and DEL dideoxy-sequencing. From initial gag primers we developed specific primers for the gag and env regions 5'ATTTTATTACCCAATCTGCTCCAAACAT3'/5'AGGTGAGTTGAACAGTCTGATT TTTA3'; 5'CGTTTACATATCACTCCCTTCCTAGTCTCTGT3'/5'GCATTAACCTTGACTATGTCTTTAGCT CCAG3'; 5'GATCCTCCCCACTGGGTTCACCATT3'/5'GGAAGTATTGGAGGGTGCCCTGCC3'. We identified several gag and env fragments with homology to the human endogenous retrovirus RGH-2 in four muliple-sclerosis cell lines. RGH belongs to the type-C-like RTVL-H/HERV-H (human endogenous retrovirus with His dRNA PBS) family which is related to the human oncoretroviruses human T-cell-leukaemia viruses 1 and 2, and to ERV-9 (endogenous retrovirus). Two RGH clones have been described: a truncated clone (RGH-1) lacking gag, and a full-length clone (RGH-2). RGH-like sequences were reported to be present in about 100 copies/haploid genome. 3 RGH particles as such have not been reported previously. We also tested multiple sclerosis cell-line particle RNAs, purified by our standard procedure with ERV-9-1-related nested primer with set ST1-1/2 and RT-PCR conditions. 4 The results were negative.To assess whether RGH sequences in particulate form could be associated with multiple sclerosis in vivo, we did blinded RT-PCR on clinical samples of cell-free, filtered, ultracentrifuged plasma from patients with multiple sclerosis or patients with autoimmune diseases, and from healthy controls (table). Expression of RGH sequences at particle level was specific for multiple sclerosis and all patients with active multiple sclerosis at the time of sampling were RGH positive. This finding shows in many patients, multiple sclerosis is associated with production into the blood of retroviral particles containing RGH sequences, possibly related to disease activity. RGH-2 sequence variants were found in the virus particles from cell-line supernatants and in the particulate fractions of plasma from patients with multiple sclerosis. Several RGH sequences were isolated from individual