We are working for the understanding of the molecular mechanisms of entry, replication, immune escape, and pathogenesis of hepatitis C virus (HCV) and the development of 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 worldwide population and 80% of these individuals will develop persistent infection. Persistent HCV infection often leads to chronic hepatitis, cirrhosis and hepatocellular carcinoma. The incidence of hepatitis C has significantly decreased since introduction of screening system for anti-HCV antibodies in 1999, however, more than two million people have already been infected with HCV in Japan. The proportion of patients achieving a sustained virological response has increased by the combination therapy of pegylated-IFNα and ribavirin. However, half of the patients with HCV genotype 1 exhibits no response to the combination therapy. HCV exhibits quasispecies heterogeneity and it is difficult to assess infection mechanisms by using surrogate systems such as pseudotype and recombinant viruses derived from a single HCV clone. In vitro replication of genotype 2a HCV (HCVcc) has recently established and several receptor candidates including hCD81, SR-BI, and Claudins were identified for HCV entry by using the surrogate viruses and HCVcc. However, a high level of neutralization antibodies to the artificial viruses has been detected in sera from persistently infected patients, suggesting that these antibodies do not play a crucial role in the clearance of HCV. Furthermore, HCV NS3/4A protease was shown to cleave adaptor molecules involved in the TLR- and RIG-I-dependent signaling pathways. HCV particles are internalized into cells through endocytosis. After uncoating, a viral RNA is translated into a large precursor polyprotein composed of 3,000 amino acids. This viral polyprotein is cleaved by signal peptidase (SP), signal peptide peptidase (SPP) and viral encoded proteases, resulting in 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 ribosomae entry site (IRES) that is essential for Cap-independent translation of viral RNA.
Although novel innovative agents in clinical development have been shown to have significant antiviral activity in patients with HCV, drug resistant viruses are easily emerged. Therefore, host proteins indispensable for HCV replication are ideal targets for the development of new therapeutics for chronic hepatitis C with a broad spectrum and a low possibility of emergence of breakthrough viruses against antiviral drugs. HCV belongs to the family of Flaviviridae which includes flavivirus such as Japanese encephalitis virus (JEV) that has a robust cell culture system and a small animal model. We are investigating the replication and pathogenesis of JEV as a surrogate model for HCV.
2. Development of baculoviral vectors
Viral vectors are essential tools for the studies on the replication deficient viral infectious diseases, such as HCV. Furthermore development of novel viral vectors is essential for future gene therapy. We are working on the baculovirus Autographa californica nucleopolyhedro virus (AcNPV) as a versatile viral vector for gene delivery not only in vitro but also in vivo. AcNPV is an insect virus possessing a 134-kb double-stranded circular DNA genome. Due to the strong promoters, baculovirus is commonly used as a tool for the large-scale production of recombinant protein in insect cells. Baculovirus is also capable of entering into a variety of mammalian cells to facilitate the expression of foreign genes under the control of the mammalian promoters without replication of the viral genome. Therefore, baculovirus is a useful viral vector, not only for the abundant expression of foreign genes in insect cells, but also for efficient gene delivery 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, the possibility of generating replication-competent revertants expressing baculoviral gene products, which can often lead to harmful immune responses against mammalian cells, is significantly lower than other viral vectors currently in use.
Furthermore, intranasal inoculation with AcNPV induces a strong innate immune response, protecting mice from lethal challenges of influenza viruses. We demonstrated that internalization of viral DNA via membrane fusion by the envelope glycoprotein in the endosome is required for the induction of innate immune response by AcNPV through a TLR9/MyD88-dependent pathway. This finding raises the possibility that AcNPV may be harnessed therapeutically to induce host immune response against various infectious diseases, especially caused by the pathogens invading from respiratory tract.
