We are seeking to understand the molecular mechanisms by which hepatitis C virus (HCV) enters host cells, replicates, escapes the immune system, and induces disease. This department is also currently developing a novel virus vector for gene delivery.
1. Studies on molecular biology of HCV replication and pathogenesis
HCV has infected more than 3% of the world's population, 80% of whom will be persistently infected. Persistent HCV infection often leads to chronic hepatitis, cirrhosis and hepatocellular carcinoma. The incidence of hepatitis C in Japan has decreased signficantly since a screening system for anti-HCV antibodies was introduced in 1999. However, more than two million people in Japan are already infected with HCV. The proportion of patients who achieve a sustained virological response to therapy has been increased by the use of pegylated-IFNα and ribavirin combination therapy. However, half of the patients with HCV genotype 1 fail to mount a response to this combination therapy. Since HCV exhibits quasispecies heterogeneity, it is difficult to determine the mechanisms by which HCV infects the human host by using surrogate systems such as pseudotype and recombinant viruses derived from a single HCV clone. The in vitro replication of genotype 2a HCV (HCVcc) was established recently and surrogate viruses and HCVcc were used to identify several receptor candidates for HCV entry, including hCD81, SR-BI, and Claudins. However, the sera of persistently infected patients contain high levels of neutralization antibodies that recognize these artificial viruses, which suggests that these antibodies do not play a crucial role in the clearance of HCV. Our studies also showed that HCV NS3/4A protease cleaves adaptor molecules involved in the TLR- and RIG-I-dependent signaling pathways, that HCV particles are internalized into cells through endocytosis, and that after uncoating, a viral RNA is translated into a large precursor polyprotein composed of 3,000 amino acids. With regard to the latter viral polyprotein, it is then cleaved by signal peptidase (SP), signal peptide peptidase (SPP) and virus-encoded proteases to generate at least 10 viral proteins. The open reading frame of the polyprotein is flanked at both ends by highly conserved untranslated regions (UTRs), which are required for viral RNA replication. The 5'-UTR harbors an internal ribosome entry site (IRES) that is essential for Cap-independent translation of viral RNA.
Although there has been considerable successful innovation in the drugs used in the clinic to eliminate the virus in patients with HCV, drug-resistant viruses emerge easily. Therefore, the most ideal targets for the development of new chronic hepatitis C therapies that have a broad spectrum and are unlikely to generate breakthrough viruses are host proteins that are indispensable for HCV replication. To search for such molecules, we have taken advantage of the fact that HCV belongs to the family of Flaviviridae, which includes the flavivirus Japanese encephalitis virus (JEV). JEV research is favored by the existence of a robust cell culture system and a small animal model. We are thus investigating the replication and pathogenesis of JEV as a surrogate model of HCV.
2. Development of baculoviral vectors
Viral vectors are essential tools for the studies on replication-deficient viral infectious diseases such as HCV. Furthermore, the development of novel viral vectors is essential for future gene therapy. We are seeking to convert the baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) into a versatile viral vector that will mediate gene delivery both in vitro and in vivo. AcNPV is an insect virus that has a 134-kb double-stranded circular DNA genome. Due to their strong promoters, baculoviruses are commonly used for the large-scale production of recombinant protein in insect cells. Baculoviruses are also capable of entering a variety of mammalian cells and, without replicating the viral genome, facilitating the expression of foreign genes under the control of mammalian promoters. Therefore, baculoviruses are useful viral vectors, not only because they can be used to induce the abundant expression of foreign genes in insect cells, but also because they can efficiently deliver genes to mammalian cells. AcNPV has a number of unique beneficial properties as a viral vector, including a large capacity for foreign gene incorporation, easy manipulation, and replication competence in insect cells combined with incompetence in mammalian cells. Therefore, compared to other viral vectors that are currently in use, AcNPV is much less likely to generate replication-competent revertants that express baculoviral gene products and can induce harmful immune responses against mammalian cells. Furthermore, we have shown that intranasal inoculation with AcNPV induces a strong innate immune response that protects mice from lethal challenges with influenza viruses. In addition, we have demonstrated that AcNPV induces this innate immune response via a TLR9/MyD88-dependent pathway, and that this requires the internalization of viral DNA via membrane fusion with the envelope glycoprotein in the endosome. This finding raises the possibility that AcNPV can be harnessed therapeutically to induce host immune responses against various infectious diseases, especially those that are caused by pathogens that invade from the respiratory tract.
