Virology and Glycobiology Division

10. Virology and Glycobiology Division

　Research in the Virology and Glycobiology Division is focused on the molecular mechanism(s) of cell sociology, such as the extracellular agent-to-cell and cell-to-cell interactions in carcinogenesis, and infiltration and metastasis of cancer cells. Major concerns are viral carcinogenesis and its prevention, and the roles of glycoconjugates in cancer infiltration and metastasis.
Efficient Detection and Inhibition by Lactoferrin of HCV Infection, and Molecular Cloning of its Extreme 3'-End

　With a set of primers that amplify a 232-base pair sequence unique to the 5'-non-coding region of HCV RNA, as few as 50 copies of negative-strand HCV RNA and as few as 100 copies even in the presence of a 100-fold excess of positive-strand HCV RNA could be specifically detected.⁽¹⁴⁸⁾ Further, to reduce the risk of carry-over contamination during manipulations and to save both time and labor, a novel, practical and highly sensitive single-step RT-PCR method was developed for the detection of HCV RNA in serum and liver biopsy specimens from patients with chronic hepatitis C.⁽¹⁴⁹⁾ Bovine lactoferrin (bLF) was found to effectively prevent HCV infection in human hepatocytes with a different mechanism (interaction of bLF with virus) from that (interaction of bLF with cell) shown in previous studies of infection with other human viruses.⁽¹⁵⁰⁾ Human lactoferrin, but not bovine transferrin, was also demonstrated to have anti-HCV activity. The 3'-end region of the HCV genome is composed of three characteristic elements: a conventional 3'-untranslated region, subsequent poly(U) stretch, and a newly identified 3'-terminal sequence named the 3'X tail.⁽¹⁵¹⁾ The 3'X tail (a specific 98 nucleotide sequence) is a common structure of the HCV genome, and may have important roles in initiation and regulation of genomic replication, and can be easily detected in HCV infected samples by conventional RT-PCR techniques.⁽¹⁵¹⁾
Cell Tropism, Persistent Replication, and a Cytotoxic T Cell Epitope of HCV

　To type HCV populations using 3 MT-2 (a HTLV-I-infected human T-cell line) and 3 PH5CH (a non-neoplastic human hepatocyte line) clones, the hypervariable region 1 (HVR1) was characterized by sequence analysis and HpaII digestion analysis, and could distinguish 3 major HVR1 types (I,II and III). Genomes containing HVR1 types I and II became predominant in MT-2 and PH5CH clones, respectively, during culture.⁽¹⁵²⁾ In search of cell type-specific sequences in regions other than HVR1, 11 amino acids were identified as candidate determinants of cell tropism.⁽¹⁵²⁾ Three clones (PH5CH1, 7 and 8) that supported more persistent HCV replication, with intracellular HCV RNA being detected at least 25 days postinoculation, were subcloned.⁽¹⁵³⁾ HCV produced in these subclones was released into the culture medium, and they contained 10-fold higher levels of HCV RNA than parental cells.⁽¹⁵³⁾ An HLA-B35-restricted cytotoxic T cell (CTL) epitope of HCV was identified using a strategy called reverse immunogenetics.⁽¹⁵⁴⁾ A strong CTL response to this epitope was observed in the acute phase, but not in the recovery phase or in chronic hepatitis C.⁽¹⁵⁴⁾
Expression Cloning and Functional Characterization of a cDNA for Human Ganglioside GM3 Synthase

　Ganglioside GM3 is a major component of glycosphingolipids in the plasma membrane and is widely distributed in vertebrates. A cDNA encoding a protein responsible for GM3 synthesis in humans was cloned.⁽¹⁵⁵⁾ The cDNA was 2,359 bp long with an open reading frame encoding a protein of 362 amino acids. The deduced primary structure shows the characteristic features of the sialyltransferase family, including a type II transmembrane topology, and the sialylmotifs L at the center and S at the C-terminal region. A characteristic amino acid substitution from Asp to His was demonstrated in the sialyl motif L. Despite the ubiquitous distribution of ganglioside GM3 in human tissues, a major 2.4-kb transcript of the gene was found to be expressed in a tissue-specific manner, with predominant expression in brain, skeletal muscle and testis, and very low expression in liver.⁽¹⁵⁵⁾
Functions of an Extracellular Matrix Glycoprotein, Tenascin-C (TN-C), in Hematopoietic Microenvironments

　TN-C is expressed on the surface of stromal cells in hematopoietic or lymphoid organs. The colony-forming capacity (CFC) of bone marrow (BM) cells was considerably lower in TN-C-deficient mice, which develop normally, although BM mononuclear cell count and architecture were not significantly different from those of normal mice.⁽¹⁵⁶⁾ In long-term BM culture (LTBMC) in vitro, hematopoietic cell production, the CFC of the cells produced, and longevity of the cultures were markedly lower in TN-C-deficient mice. The addition of TN-C glycoprotein to LTBMCs of TN-C-deficient mice clearly induced the recovery of hematopoietic cell production and CFC.⁽¹⁵⁶⁾




	10. Virology and Glycobiology Division



		Research in the Virology and Glycobiology Division is focused on the molecular mechanism(s) of cell sociology, such as the extracellular agent-to-cell and cell-to-cell interactions in carcinogenesis, and infiltration and metastasis of cancer cells. Major concerns are viral carcinogenesis and its prevention, and the roles of glycoconjugates in cancer infiltration and metastasis. Efficient Detection and Inhibition by Lactoferrin of HCV Infection, and Molecular Cloning of its Extreme 3'-End 　With a set of primers that amplify a 232-base pair sequence unique to the 5'-non-coding region of HCV RNA, as few as 50 copies of negative-strand HCV RNA and as few as 100 copies even in the presence of a 100-fold excess of positive-strand HCV RNA could be specifically detected.⁽¹⁴⁸⁾ Further, to reduce the risk of carry-over contamination during manipulations and to save both time and labor, a novel, practical and highly sensitive single-step RT-PCR method was developed for the detection of HCV RNA in serum and liver biopsy specimens from patients with chronic hepatitis C.⁽¹⁴⁹⁾ Bovine lactoferrin (bLF) was found to effectively prevent HCV infection in human hepatocytes with a different mechanism (interaction of bLF with virus) from that (interaction of bLF with cell) shown in previous studies of infection with other human viruses.⁽¹⁵⁰⁾ Human lactoferrin, but not bovine transferrin, was also demonstrated to have anti-HCV activity. The 3'-end region of the HCV genome is composed of three characteristic elements: a conventional 3'-untranslated region, subsequent poly(U) stretch, and a newly identified 3'-terminal sequence named the 3'X tail.⁽¹⁵¹⁾ The 3'X tail (a specific 98 nucleotide sequence) is a common structure of the HCV genome, and may have important roles in initiation and regulation of genomic replication, and can be easily detected in HCV infected samples by conventional RT-PCR techniques.⁽¹⁵¹⁾ Cell Tropism, Persistent Replication, and a Cytotoxic T Cell Epitope of HCV 　To type HCV populations using 3 MT-2 (a HTLV-I-infected human T-cell line) and 3 PH5CH (a non-neoplastic human hepatocyte line) clones, the hypervariable region 1 (HVR1) was characterized by sequence analysis and HpaII digestion analysis, and could distinguish 3 major HVR1 types (I,II and III). Genomes containing HVR1 types I and II became predominant in MT-2 and PH5CH clones, respectively, during culture.⁽¹⁵²⁾ In search of cell type-specific sequences in regions other than HVR1, 11 amino acids were identified as candidate determinants of cell tropism.⁽¹⁵²⁾ Three clones (PH5CH1, 7 and 8) that supported more persistent HCV replication, with intracellular HCV RNA being detected at least 25 days postinoculation, were subcloned.⁽¹⁵³⁾ HCV produced in these subclones was released into the culture medium, and they contained 10-fold higher levels of HCV RNA than parental cells.⁽¹⁵³⁾ An HLA-B35-restricted cytotoxic T cell (CTL) epitope of HCV was identified using a strategy called reverse immunogenetics.⁽¹⁵⁴⁾ A strong CTL response to this epitope was observed in the acute phase, but not in the recovery phase or in chronic hepatitis C.⁽¹⁵⁴⁾ Expression Cloning and Functional Characterization of a cDNA for Human Ganglioside GM3 Synthase 　Ganglioside GM3 is a major component of glycosphingolipids in the plasma membrane and is widely distributed in vertebrates. A cDNA encoding a protein responsible for GM3 synthesis in humans was cloned.⁽¹⁵⁵⁾ The cDNA was 2,359 bp long with an open reading frame encoding a protein of 362 amino acids. The deduced primary structure shows the characteristic features of the sialyltransferase family, including a type II transmembrane topology, and the sialylmotifs L at the center and S at the C-terminal region. A characteristic amino acid substitution from Asp to His was demonstrated in the sialyl motif L. Despite the ubiquitous distribution of ganglioside GM3 in human tissues, a major 2.4-kb transcript of the gene was found to be expressed in a tissue-specific manner, with predominant expression in brain, skeletal muscle and testis, and very low expression in liver.⁽¹⁵⁵⁾ Functions of an Extracellular Matrix Glycoprotein, Tenascin-C (TN-C), in Hematopoietic Microenvironments 　TN-C is expressed on the surface of stromal cells in hematopoietic or lymphoid organs. The colony-forming capacity (CFC) of bone marrow (BM) cells was considerably lower in TN-C-deficient mice, which develop normally, although BM mononuclear cell count and architecture were not significantly different from those of normal mice.⁽¹⁵⁶⁾ In long-term BM culture (LTBMC) in vitro, hematopoietic cell production, the CFC of the cells produced, and longevity of the cultures were markedly lower in TN-C-deficient mice. The addition of TN-C glycoprotein to LTBMCs of TN-C-deficient mice clearly induced the recovery of hematopoietic cell production and CFC.⁽¹⁵⁶⁾