Virology Division

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 mechanisms of carcinogenesis induced by these viruses, and the development of measures to prevent cancers caused by these viruses.
Analysis of the Cell Tropism of HCV by Using in vitro HCV-infected Human Lymphocytes and Hepatocytes

The infective potencies of sev en sera obtained from HCV-positive blood donors were examined in the recently established two HCV replication systems using MT-2, an HTLV-I-infected human T-cell line, and PH5CH, a non-neoplastic human hepatocyte line immortalized with simian virus 40 large T antigen. The results showed that these sera had different infectivities for MT-2 and PH5CH cells, and suggested that the infective potency of each serum depends on the type of target cells.⁽¹⁵⁰⁾ Sequence analysis of the hypervariable region 1 (HVR1) of HCV populations recovered from both MT-2 and PH5CH cells at 8 days postinoculation revealed that the shift to limited HVR1 populations from the quasi-species of HVR1 populations in both cells usually occurred within 8 days after virus inoculation, and that the predominant HVR1 populations in MT-2 and PH5CH cells appeared to be different in two of four sera, suggesting that HCV exhibits cell tropism.⁽¹⁵⁰⁾ The nucleotide sequence of the whole HCV genome, designated HCV-JS, was determined by standardization of the nucleotide sequences of cDNA clones obtained from HCV-infected cloned MT-2C cells, which support viral replica tion more persistently than parental MT-2 cells, at 12 days after inoculation.⁽¹⁵¹⁾Molecular evolutional analyses comparing the sequences of the HCV clones obtained from HCV-infected MT-2C cells and the original inoculum suggest that limited HCV populations are able to replicate in MT-2C cells.⁽¹⁵¹⁾ In the course of this HCV study, it was found that replication of hepatitis G virus (HGV), which has recently been identified, was also sup ported in both MT-2C and PH5CH cells.⁽¹⁵²⁾ Sequence analysis of the 5'-noncoding region of HGV RNA revealed the quasi-species nature of HGV.⁽¹⁵²⁾
Quantitative Analysis of HCV in Extrahepatic Organs

To determine whether HCV is present in extrahepatic organs, quantitative analysis of the HCV genome in surgical specimens from three patients with gynecological cancer was performed, and relatively high HCV genome titers were found in the lymph nodes irrespective of various titers in peripheral blood mononuclear cells.⁽¹⁵³⁾ This result suggested that lymph nodes may play an important role in the carrier state and the persistence of HCV infection.
Analysis of Cellular Proteins Interacting with the 3'-terminal Structure of the HCV Genome

A 38 kDa and a 57 kDa protein were found in PH5CH cells to bind specifically to the 3'-terminal structure (3惔) of HCV positive-stranded RNA, the initiation site of RNA replication, by a UV-induced cross-linking assay.⁽¹⁵⁴⁾ The 57-kDa protein (p57) recognized a 26 nucleotide sequence which showed similarity to the consensus binding sequence of the polypyrimidine tract-binding protein (PTB), and p57 was immunoprecipitated by anti-PTB antibody. From these results, it was concluded that p57 was identical to PTB, and suggested that the 3X-PTB interaction is involved in the specific initiation of HCV genome replication.
Functional Analysis of HCV Proteins

HCV NS5A is a phosphoprotein located in the perinuclear membrane fraction. To clarify the mechanism of NS5A phosphorylation, phosphorylation of deleted and mutated forms of NS5A was analyzed using a transient protein production system in cultured cells in the presence or absence of NS4A. The N-terminal region of NS5A was important for NS4A-dependent phosphorylation of NS5A.⁽¹⁵⁵⁾ The antigenicity of a second envelope glycoprotein (E2) of HCV constitutively produced in Chinese hamster ovary cells was examined. It was found that the antigenicity of the E2 protein glycosylated with complex type oligosaccharides was greater than that of the high-mannose form of E2 protein.⁽¹⁵⁶⁾
Growth Regulation of T-cells Expressing HTLV-I Tax

To elucidate the role of the Tax1-inducible transcriptional pathway in T-cell transformation, Tax1 mutants with different trans-activating phenotypes were introduced into peripheral blood lymphocytes (PBL) by retroviral vectors. Analysis of these PBLs revealed that activation of the NF-kB pathway was sufficient to promote the growth response to IL2, and that activation of the CREB/ATF and SRF pathway was also required for the clonal expansion of CD4+ T-cells.⁽¹⁵⁷⁾ Aberrant expression and function of p53 in T-cells immortalized by HTLV-I Tax1 were revealed by the findings of elevated levels of conformationally wild-type p53 and impairment in the transactivating function of p53 in the T-cells.⁽¹⁵⁸⁾




	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 mechanisms of carcinogenesis induced by these viruses, and the development of measures to prevent cancers caused by these viruses. Analysis of the Cell Tropism of HCV by Using in vitro HCV-infected Human Lymphocytes and Hepatocytes The infective potencies of sev en sera obtained from HCV-positive blood donors were examined in the recently established two HCV replication systems using MT-2, an HTLV-I-infected human T-cell line, and PH5CH, a non-neoplastic human hepatocyte line immortalized with simian virus 40 large T antigen. The results showed that these sera had different infectivities for MT-2 and PH5CH cells, and suggested that the infective potency of each serum depends on the type of target cells.⁽¹⁵⁰⁾ Sequence analysis of the hypervariable region 1 (HVR1) of HCV populations recovered from both MT-2 and PH5CH cells at 8 days postinoculation revealed that the shift to limited HVR1 populations from the quasi-species of HVR1 populations in both cells usually occurred within 8 days after virus inoculation, and that the predominant HVR1 populations in MT-2 and PH5CH cells appeared to be different in two of four sera, suggesting that HCV exhibits cell tropism.⁽¹⁵⁰⁾ The nucleotide sequence of the whole HCV genome, designated HCV-JS, was determined by standardization of the nucleotide sequences of cDNA clones obtained from HCV-infected cloned MT-2C cells, which support viral replica tion more persistently than parental MT-2 cells, at 12 days after inoculation.⁽¹⁵¹⁾Molecular evolutional analyses comparing the sequences of the HCV clones obtained from HCV-infected MT-2C cells and the original inoculum suggest that limited HCV populations are able to replicate in MT-2C cells.⁽¹⁵¹⁾ In the course of this HCV study, it was found that replication of hepatitis G virus (HGV), which has recently been identified, was also sup ported in both MT-2C and PH5CH cells.⁽¹⁵²⁾ Sequence analysis of the 5'-noncoding region of HGV RNA revealed the quasi-species nature of HGV.⁽¹⁵²⁾ Quantitative Analysis of HCV in Extrahepatic Organs To determine whether HCV is present in extrahepatic organs, quantitative analysis of the HCV genome in surgical specimens from three patients with gynecological cancer was performed, and relatively high HCV genome titers were found in the lymph nodes irrespective of various titers in peripheral blood mononuclear cells.⁽¹⁵³⁾ This result suggested that lymph nodes may play an important role in the carrier state and the persistence of HCV infection. Analysis of Cellular Proteins Interacting with the 3'-terminal Structure of the HCV Genome A 38 kDa and a 57 kDa protein were found in PH5CH cells to bind specifically to the 3'-terminal structure (3惔) of HCV positive-stranded RNA, the initiation site of RNA replication, by a UV-induced cross-linking assay.⁽¹⁵⁴⁾ The 57-kDa protein (p57) recognized a 26 nucleotide sequence which showed similarity to the consensus binding sequence of the polypyrimidine tract-binding protein (PTB), and p57 was immunoprecipitated by anti-PTB antibody. From these results, it was concluded that p57 was identical to PTB, and suggested that the 3X-PTB interaction is involved in the specific initiation of HCV genome replication. Functional Analysis of HCV Proteins HCV NS5A is a phosphoprotein located in the perinuclear membrane fraction. To clarify the mechanism of NS5A phosphorylation, phosphorylation of deleted and mutated forms of NS5A was analyzed using a transient protein production system in cultured cells in the presence or absence of NS4A. The N-terminal region of NS5A was important for NS4A-dependent phosphorylation of NS5A.⁽¹⁵⁵⁾ The antigenicity of a second envelope glycoprotein (E2) of HCV constitutively produced in Chinese hamster ovary cells was examined. It was found that the antigenicity of the E2 protein glycosylated with complex type oligosaccharides was greater than that of the high-mannose form of E2 protein.⁽¹⁵⁶⁾ Growth Regulation of T-cells Expressing HTLV-I Tax To elucidate the role of the Tax1-inducible transcriptional pathway in T-cell transformation, Tax1 mutants with different trans-activating phenotypes were introduced into peripheral blood lymphocytes (PBL) by retroviral vectors. Analysis of these PBLs revealed that activation of the NF-kB pathway was sufficient to promote the growth response to IL2, and that activation of the CREB/ATF and SRF pathway was also required for the clonal expansion of CD4+ T-cells.⁽¹⁵⁷⁾ Aberrant expression and function of p53 in T-cells immortalized by HTLV-I Tax1 were revealed by the findings of elevated levels of conformationally wild-type p53 and impairment in the transactivating function of p53 in the T-cells.⁽¹⁵⁸⁾

10. Virology Division

Analysis of the Cell Tropism of HCV by Using in vitro HCV-infected Human Lymphocytes and Hepatocytes

Quantitative Analysis of HCV in Extrahepatic Organs

Analysis of Cellular Proteins Interacting with the 3'-terminal Structure of the HCV Genome

Functional Analysis of HCV Proteins

Growth Regulation of T-cells Expressing HTLV-I Tax