| Research Abstract |
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.
|
