VIROLOGY DIVISION

10.VIROLOGY DIVISION

    Research in the Virology Division is focused on the molecular mechanisms of oncogenesis by the human papillomavirus (HPV) and hepatitis C virus (HCV). As viral oncogenes perturb the normal cell cycle and its checkpoints and immortalize normal human cells, our study also covers the DNA replication machinery in mammalian cells and stem cell biology.
Immortalization of Human Cells by HPV

    Normal human cells in culture undergo a limited number of divisions and then enter a nondividing state called replicative senescence. Most cancer cells can divide indefinitely by escaping this senescence program. The E6 and E7 genes of the human papillomavirus can cooperatively immortalize normal human epithelial cells originating from the skin and mammary glands. Both inactivation of the RB pathway by E7 and activation of telomerase by E6 are required for immortalization of these cell types. Some cell types such as skin fibroblasts can be immortalized by activation of telomerase alone while other cell types cannot. The underlying mechanisms which can explain these differences remain to be elucidated. Understanding of the immortalization might be applied to cancer therapy by reactivating the senescence program in cancer cells.
Transforming Activity of HPV Genes

    Normal cells have a mechanism of growth inhibition by cell-cell contact, which is called contact inhibition or density arrest. The E6 and E7 genes of HPV attenuate the contact inhibition by different mechanisms. E7 can do so by inactivating the RB pathway, but the mechanism by which E6 does so is not fully understood. So far the PDZ domain-binding motif located at the carboxy terminus of the E6 protein and the ability to bind a ubiquitinligase named E6AP are known to be important for transforming activity of E6. Dlg and Scribble, both of which contain the PDZ domains, are tumor suppressors in Drosophila, and the human orthologues are possible tumor suppressors. The enhanced degradation of these possible tumor suppressors could be the mechanism of transformation by E6.
HCV RNA Replicon

    A HCV RNA replicon (self-replicating RNA molecule) was established from HCV-infected cultured cells with the availability of a long RT-PCR method. The replicon carries a consensus sequence of amino acids and self-cleaving ribozymes in both ends. We have obtained replicon-replicating cells ^(151,¹⁵²⁾. Investigation into the effects of HCV specific RNAi on these cells using dsRNA expression viral vector is currently in process. RNA serves not only as a transcript of a gene but also as an enzyme (ribozyme). Moreover, it became evident that RNAi plays an important physiological role, and its applications are rapidly advancing. In this context, RNA research, including the RNA replicon, should be promising.
Analysis of the Hematopoietic Microenvironment

    In bone marrow, hematopoiesis-supporting cells form a hematopoietic microenvironment called a "niche" and, by producing growth factors, adhesion molecules and matrix proteins, they govern homing, growth, survival and differ-entiation of hematopoietic stem/progenitor cells (HSPCs). Recent reports have suggested that the niche also regulates the proliferation of tumor cells. HSPCs cannot be maintained in vitro without stromal cells. Moreover, direct contact between stromal cells and HSPCs is critical for supporting HSPCs. These findings suggest that bone marrow supportive cells are expected to express unknown membrane molecules that regulate hematopoiesis. By the retrovirus-mediated signal sequence trap (SST) method, secreted and membrane molecules have been isolated from OP9 stromal cells. One of them, SST-4 is a novel type Ia membrane protein that is involved in the hematopoietic supportive capacity of stromal cells. Our approach give one clue to understand the mechanisms by which the hematopoietic microenvironment regulates hematopoiesis and leukemogenesis.
Cell Cycle Regulation of DNA Replication in Mammalian Cells

    Genomic DNA has to be replicated completely and only once during a single cell cycle to maintain genome integrity. Inhibition of re-replication is one of most important aspects in the cell cycle control. The MCM complex, an essential DNA replication initiation factor, is loaded onto chromatin by the origin recognition complex, CDC6 and Cdt1 proteins. In mammalian cells, Cdc2 kinase governs the inhibition of re-replication through prohibiting MCM re-binding to chromatin. Here, mechanisms by which Cdc2 kinase inhibits re-binding of MCM proteins have been analyzed.
    MCM complex and CDC6 proteins were hyperphosphorylated by Cdc2 kinase, which may be one of the mechanisms. In addition, it was found that when Cdc2 kinase is inactivated, Cdt1 re-binds to chromatin. One simple explanation for this might be that Cdc2 kinase enhances interaction between Cdt1 and geminin, a counteractive protein against Cdt1. Indeed, in cells treated with purvalanol A, a very specific Cdk2/Cdc2 inhibitor, the Cdt1-geminin interaction was diminished. It was also found that the Cdc2/cyclinA complex binds to Cdt1 through its Cy motif.




10.VIROLOGY DIVISION



	Research in the Virology Division is focused on the molecular mechanisms of oncogenesis by the human papillomavirus (HPV) and hepatitis C virus (HCV). As viral oncogenes perturb the normal cell cycle and its checkpoints and immortalize normal human cells, our study also covers the DNA replication machinery in mammalian cells and stem cell biology. Immortalization of Human Cells by HPV Normal human cells in culture undergo a limited number of divisions and then enter a nondividing state called replicative senescence. Most cancer cells can divide indefinitely by escaping this senescence program. The E6 and E7 genes of the human papillomavirus can cooperatively immortalize normal human epithelial cells originating from the skin and mammary glands. Both inactivation of the RB pathway by E7 and activation of telomerase by E6 are required for immortalization of these cell types. Some cell types such as skin fibroblasts can be immortalized by activation of telomerase alone while other cell types cannot. The underlying mechanisms which can explain these differences remain to be elucidated. Understanding of the immortalization might be applied to cancer therapy by reactivating the senescence program in cancer cells. Transforming Activity of HPV Genes Normal cells have a mechanism of growth inhibition by cell-cell contact, which is called contact inhibition or density arrest. The E6 and E7 genes of HPV attenuate the contact inhibition by different mechanisms. E7 can do so by inactivating the RB pathway, but the mechanism by which E6 does so is not fully understood. So far the PDZ domain-binding motif located at the carboxy terminus of the E6 protein and the ability to bind a ubiquitinligase named E6AP are known to be important for transforming activity of E6. Dlg and Scribble, both of which contain the PDZ domains, are tumor suppressors in Drosophila, and the human orthologues are possible tumor suppressors. The enhanced degradation of these possible tumor suppressors could be the mechanism of transformation by E6. HCV RNA Replicon A HCV RNA replicon (self-replicating RNA molecule) was established from HCV-infected cultured cells with the availability of a long RT-PCR method. The replicon carries a consensus sequence of amino acids and self-cleaving ribozymes in both ends. We have obtained replicon-replicating cells ^(151,¹⁵²⁾. Investigation into the effects of HCV specific RNAi on these cells using dsRNA expression viral vector is currently in process. RNA serves not only as a transcript of a gene but also as an enzyme (ribozyme). Moreover, it became evident that RNAi plays an important physiological role, and its applications are rapidly advancing. In this context, RNA research, including the RNA replicon, should be promising. Analysis of the Hematopoietic Microenvironment In bone marrow, hematopoiesis-supporting cells form a hematopoietic microenvironment called a "niche" and, by producing growth factors, adhesion molecules and matrix proteins, they govern homing, growth, survival and differ-entiation of hematopoietic stem/progenitor cells (HSPCs). Recent reports have suggested that the niche also regulates the proliferation of tumor cells. HSPCs cannot be maintained in vitro without stromal cells. Moreover, direct contact between stromal cells and HSPCs is critical for supporting HSPCs. These findings suggest that bone marrow supportive cells are expected to express unknown membrane molecules that regulate hematopoiesis. By the retrovirus-mediated signal sequence trap (SST) method, secreted and membrane molecules have been isolated from OP9 stromal cells. One of them, SST-4 is a novel type Ia membrane protein that is involved in the hematopoietic supportive capacity of stromal cells. Our approach give one clue to understand the mechanisms by which the hematopoietic microenvironment regulates hematopoiesis and leukemogenesis. Cell Cycle Regulation of DNA Replication in Mammalian Cells Genomic DNA has to be replicated completely and only once during a single cell cycle to maintain genome integrity. Inhibition of re-replication is one of most important aspects in the cell cycle control. The MCM complex, an essential DNA replication initiation factor, is loaded onto chromatin by the origin recognition complex, CDC6 and Cdt1 proteins. In mammalian cells, Cdc2 kinase governs the inhibition of re-replication through prohibiting MCM re-binding to chromatin. Here, mechanisms by which Cdc2 kinase inhibits re-binding of MCM proteins have been analyzed. MCM complex and CDC6 proteins were hyperphosphorylated by Cdc2 kinase, which may be one of the mechanisms. In addition, it was found that when Cdc2 kinase is inactivated, Cdt1 re-binds to chromatin. One simple explanation for this might be that Cdc2 kinase enhances interaction between Cdt1 and geminin, a counteractive protein against Cdt1. Indeed, in cells treated with purvalanol A, a very specific Cdk2/Cdc2 inhibitor, the Cdt1-geminin interaction was diminished. It was also found that the Cdc2/cyclinA complex binds to Cdt1 through its Cy motif.