Analysis of replication and transcription mechanisms in a newly discovered circular, single-stranded DNA virus (TTV)

分析新发现的环状单链 DNA 病毒 (TTV) 的复制和转录机制

基本信息

批准号：
13470067
负责人：
OKAMOTO Hiroaki
金额：
$ 6.85万
依托单位：
Jichi Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470067/
关键词：
TTV circular single-stranded DNA virus replicative intermediate mRNA liver tandem dimer transfection RFLP tandem dimer transfection 培養細胞転写調節機構 Transfection 好中球

项目摘要

TTV is a novel virus with the circular, single-stranded DNA genome that we isolated from the serum of a patient with posttransfusion acute hepatitis of unknown etiology in 1997. Its replication mechanism and transcription profile remained unknown. Therefore, in the present study, we examined the presence of double-stranded DNA as a replicative intermediate and mRNA in various tissues in infected individuals. Circular double-stranded TTV DNA in the replicative intermediate form as well as TTV mRNA were detected not only in liver tissues but also in bone marrow cells, lung tissues, spleen, thyroid gland and pancreas. In addition, it was found that TTV is distributed in various leukocyte subpopulations at distinct levels, with the highest viral load in granulocytes. We tried to find established cell lines supporting efficient replication of TTV, but it remains unsuccessful. Then, we constructed plasmids harboring a tandem dimer of the full-length TTV genome [3,853 nucleotides (nt)] or a monomer and introduced them separately into cultured hepatoma cells (HepG2 or Huh7). TTV mRNAs of 3.0, 1.2, or 1.0 kilobases (kb) were detectable in cells transfected with tandem dimers, but not in cells transfected with monomers. Sequence analysis of TTV mRNAs revealed that three distinct species of TTV rnRNAs possessed in common 5' and 3' termini as well as splicing of 91 nt, and that shorter mRNAs of 1.2 kb and 1.0 kb possessed another splicing of 1662 nt and 1855 nt, respectively. In the additional studies, it was revealed that TATA-box and its upstream 120-nt sequence consisting of GC-rich stem & loop structures have enhancer and promoter activities in vitro. However, production of viral particles in culture after transfection of tandem dimers is still unsuccessful, which indicates necessity of further continuous investigation.

TTV是一种新型病毒，具有圆形的单链DNA基因组，我们从1997年患有转移后急性急性肝炎的患者的血清中分离出来。其复制机制和转录谱尚不清楚。因此，在本研究中，我们检查了受感染个体的各种组织中双链DNA作为复制中间体和mRNA的存在。不仅在肝组织中，而且在骨髓细胞，肺组织，脾脏，甲状腺，甲状腺和胰腺中还检测到复制性中间形式和TTV mRNA中的圆形双链TTV DNA。此外，发现TTV以不同水平的各种白细胞亚群中分布，粒细胞中的病毒负荷最高。我们试图找到支持TTV有效复制的既定细胞系，但仍然没有成功。然后，我们构建了具有全长TTV基因组[3,853个核苷酸（NT）]或单体的串联二聚体的质粒，并将它们分别引入培养的肝癌细胞（HEPG2或HUH7）中。在用串联二聚体转染的细胞中可以检测到3.0、1.2或1.0千酶（Kb）的TTV mRNA，而在用单体转染的细胞中则不能检测到。 TTV mRNA的序列分析表明，在共同的5'和3'末端中拥有的三种不同的TTV rNRNA物种以及91 nt的剪接，并且分别具有1.2 kb和1.0 kb的较短mRNA，另外1662 NT和1855 NT的拼接为1662 NT和1855 NT。在其他研究中，揭示了TATA-Box及其上游的120-NT序列由富含GC的茎和环结构组成，其体外具有增强子和启动子活性。然而，转染串联二聚体后培养中的病毒颗粒的产生仍然没有成功，这表明有必要进一步进行连续研究。