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

• 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

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，具有环状单链DNA基因组，我们于1997年从一名病因不明的输血后急性肝炎患者的血清中分离到。它的复制机制和转录谱仍不清楚。因此，在本研究中，我们研究了双链DNA作为复制中间体和mRNA在感染个体的各种组织中的存在。在肝组织、骨髓细胞、肺组织、脾脏、甲状腺和胰腺中均检测到呈复制中间体形式的环状双链TTV DNA和TTV mRNA。此外，还发现TTV以不同的水平分布在各种白细胞亚群中，其中粒细胞中的病毒载量最高。我们试图找到支持TTV有效复制的细胞系，但仍然不成功。然后，我们构建了含有全长TTV基因组的串联二聚体[3，853个核苷酸（nt）]或单体的质粒，并将它们分别引入培养的肝癌细胞（HepG 2或Huh 7）中。在用串联二聚体转染的细胞中可检测到3.0、1.2或1.0 kb的TTV mRNA，但在用单体转染的细胞中检测不到。TTV mRNAs的序列分析表明，3种不同的TTV mRNAs具有共同的5'和3'末端，并具有91 nt的剪接，而1.2kb和1.0kb的短片段分别具有1662 nt和1855 nt的剪接。在进一步的研究中，发现TATA-box及其上游120-nt序列由富含GC的茎环结构组成，在体外具有增强子和启动子活性。然而，串联二聚体转染后在培养物中产生病毒颗粒仍然是不成功的，这表明有必要进行进一步的持续研究。

项目成果

Yokoyama, H.: "Pathological changes of renal epithelial cells in mice transgenic for the TT virus ORF1 gene"Journal of General Virology. 83. 141-150 (2002)

Yokoyama, H.：“TT病毒ORF1基因转基因小鼠肾上皮细胞的病理变化”普通病毒学杂志。

Kato S, et al.: "Helicobacter pylon and TT virus prevalence in Japanese children."J Gastroenterol. 38. 1126-1130 (2003)

Kato S 等人：“日本儿童中幽门螺杆菌和 TT 病毒的流行情况。”J Gastroenterol。

Muljono D.H.: "Molecular epidemiology of TT virus(TTV)and characterization of two novel genotypes in Indonesia"Archives of Virology. 146. 1249-1266 (2001)

Muljono D.H.：“TT 病毒（TTV）的分子流行病学和印度尼西亚两种新基因型的特征”病毒学档案。

Yokoyama H, et al.: "Pathological changes of renal epithelial cells in mice transgenic for the TT virus ORF 1 gene."J Gen Virol. 83. 141-150 (2002)

Yokoyama H 等人：“TT 病毒 ORF 1 基因转基因小鼠肾上皮细胞的病理变化。”J Gen Virol。

Takahashi M.: "TT virus is distributed in various leukocyte subpopulations at distinct levels, with the highest viral load in granulocytes"Biochemical and Biophysical Research Communications. 290. 242-248 (2002)

Takahashi M.：“TT 病毒以不同的水平分布在各种白细胞亚群中，其中粒细胞中的病毒载量最高”《生物化学和生物物理研究通讯》。