10. Virology Division

Research on hepatitis C virus (HCV) and human T-cell leukemia virus (HTLV-I), the causative agents of hepatocellular carcinoma and adult T-cell leukemia, respectively, are the focus of the Virology Division. The principal concerns are elucidation of the molecular carcinogenic mechanisms of these viruses, and the development of measures to prevent cancers caused by HCV and HTLV-1.

Establishment of an HCV-proliferating System in Human Cell Lines

HCV replication systems were developed using two cultured human cell lines: MT-2C, an HTLV-I-infected cloned cell line, which is more susceptible than the original MT-2 cell line to HCV infection and can support HCV replication, and PH5CH, a non-neoplastic human hepatocyte line.^(114,115) Semiquantitative analysis of HCV RNA by PCR revealed that HCV multiplied during the period of culture following inoculation.^(114,115) Negative-stranded HCV RNA was also detected as an intermediate in HCV replicating cells by two different methods with strand specificity.⁽¹¹⁵⁾ Sequence analysis of the HCV hypervariable region 1 (HVR1) revealed that certain HCV species gained predominance during the culture period after viral inoculation.^(114,116) Persistent HCV infection in MT-2C cells could be reproducibly achieved by reducing the temperature from 37^ĄC to 32^ĄC. Under these culture conditions, HCV RNA was detectable in the cells for more than 6 months postinoculation, and viral transmission via a cell-free mode could be successfully repeated at least 4 times as opposed to only twice at 37^ĄC.⁽¹¹⁶⁾ This HCV-infected culture system is anticipated to be useful for various biological studies, including investigations into the mechanisms of HCV replication and multiplication.

Genetic Analysis of the 3'Terminus of the HCV Genome

A novel 98-nucleotide sequence, which was discovered downstream of the poly(U) stretch that was previously considered to be the 3' end structure of the HCV genome, appeared to be the bona fide 3' terminus of the HCV genome, and was highly conserved among individuals and even between the two most genetically distinct HCV types, 1b and 2a.⁽¹¹⁷⁾

Comparative Analysis of HCV Species from a Patient with Hepatocellular Carcinoma

Genetic variability of HCV was assessed by determining the nucleotide sequence corresponding to the HVR1 and HVR2 of the putative envelope protein (E2/NS1) in positive- and negative-stranded HCV RNA from the cancerous and surrounding non-cancerous liver tissue, peripheral blood mononuclear cells and serum of a patient with hepatocellular carcinoma (HCC). The results revealed that some viral isolates with the same HVR1 sequence were able to replicate in both cancerous and non-cancerous liver tissue, whereas others replicated in HCC tissue only.⁽¹¹⁸⁾

Functional Analysis of HCV Proteins

In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the HCV polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, an in vitro NS3 protease assay system consisting of three purified viral elements (a recombinant NS3 protease which is expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein, synthetic NS4A fragments, and a synthetic peptide substrate which mimics the NS5A/5B junction) was developed.^(119,120) This assay system revealed that the coefficient for proteolytic efficiency of NS3 protease was increased approximately 40 fold by the addition of NS4A, and that amino acid residues 22 to 31 in NS4A constituted the core sequence for the effector activity.⁽¹¹⁹⁾ This assay system was also used to screen for inhibitors of protease NS3-4A.⁽¹²⁰⁾

Regulation of Cell-cycle Regulatory Genes in HTLV-I- infected T-cells

To understand how the growth of HTLV-I-infected T-cells is deregulated, the level of expression of cell-cycle regulatory genes in HTLV-I-infected and non-infected T-cell lines was examined and the following results were obtained: (1) HTLV-I-infected T-cell lines preferentially expressed cyclin D2, whereas cyclin D3 was the major D-type cyclin in HTLV-I-negative T-cell lines; (2) HTLV-I-infected T-cell lines expressed strikingly low levels of p18^Ink4; (3) HTLV-I-infected T-cell lines expressed high levels of p21^{Waf1/Cip1/Sdi1}.⁽¹²¹⁾ These features were also found in T-cells immortalized with Tax1, as was previously established.⁽¹²¹⁾ During the analysis using various T-cell lines, it was found that the Cdk6 protein level rapidly decreased after treatment with the tyrosine kinase inhibitor herbimycin A.⁽¹²²⁾ This decrease is fairly specific, because the level of other Cdks did not change after treatment with herbimycin A. This finding suggests that herbimycin A-sensitive protein tyrosine kinase is involved in regulating the Cdk6 protein level.

List of papers from this division

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