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

TTV是我们于1997年从一例原因不明的输血后急性肝炎患者的血清中分离到的一种具有环状单链DNA基因组的新型病毒。其复制机制和转录谱尚不清楚。因此，在本研究中，我们检测了作为复制中间体的双链DNA的存在，以及在感染个体的不同组织中的mRNA。不仅在肝组织中检测到复制中间体的环状双链TTV DNA，而且在骨髓细胞、肺组织、脾、甲状腺和胰腺中也检测到TTV mRNA。此外，还发现TTV在不同的白细胞亚群中有不同程度的分布，其中粒细胞的病毒载量最高。我们试图寻找支持TTV有效复制的已建立的细胞系，但仍未成功。然后，我们构建了含有全长TTV基因组[3,853个核苷酸(NT)]的串联二聚体或单体的质粒，并将它们分别导入培养的肝癌细胞(HepG2或HuH7)。在串联二聚体细胞中可检测到3.0、1.2或1.0千碱基(Kb)的TTV mRNAs，而在单体细胞中检测不到。序列分析表明，3种不同的TTV rnRNAs在共同的5‘和3’端有91nT的剪接，1.2kb和1.0kb的较短的mRNAs分别有1662nT和1855nT的剪接。进一步的研究表明，TATA-BOX及其上游120-NT序列由富含GC的茎和环结构组成，在体外具有增强子和启动子活性。然而，串联二聚体转染后的培养物仍未产生病毒颗粒，这表明有必要进行进一步的继续研究。

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.