Major Research Interests
I. Regulation and Control of RNA Virus Replication
Plus-stranded RNA viruses are the most popular human pathogenic viruses and include hepatitis C virus (HCV), Japanese encephalitis virus (JEV), and a newly emerged coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), which are the current research targets in my lab. Replication of plus-stranded RNA viruses is mediated by an RNA replicase complex formed with a virus-encoded RNA-dependent RNA polymerase (RdRp, a viral enzyme required for viral RNA replication) and viral and/or cellular proteins. Even though a great deal of information has been accumulated regarding biochemical properties of various viral RdRps, it is still not well characterized how RdRps initiate RNA synthesis, how their enzyme activities are regulated by cellular and/or viral proteins, and how the RNA replicase is formed and localize in a specific cellular compartment. My lab has been interested in characterizing the biochemical properties of RdRps from HCV, JEV, and SARS-CoV. In particular, we have been characterizing RNA synthesis initiation mechanisms with HCV, JEV, and SARS-CoV recombinant RdRps. In addition, we are interested in exploring: (1) How viral replication is regulated by cellular proteins and/or viral proteins?, (2) Where and how the RNA replicase is formed?, and (3) How HCV RdRp activity is controlled by host cellular responses? To answer these questions, we have been carrying out the projects as follows:
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(1) Hepatitis C virus RdRp
HCV is estimated to infect about 170 million people around the world, is a major cause of non-A and non-B hepatitis, and can lead liver cirrhosis and hepatocellular carcinoma. It is an enveloped virus with a positive-stranded RNA genome of approximately 9.6 kb belonging to the Hepacivirus genus in the Flaviviridae family. The HCV viral genome encodes a single polyprotein of ~3,010 amino acids, which is proteolytically processed by a combination of host and viral proteases into at least 10 distinct structural and nonstructural proteins. The structural proteins include C, E1, E2, and p7, and the nonstructural (NS) proteins include NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Among the nonstructural proteins, HCV NS5B is an RNA-dependent RNA polymerase (RdRp) that is important for replication of the HCV RNA genome.
Cellular kinase phosphorylating the viral RNA polymerase
Recently, we have shown that protein kinase C-related kinase 2 (PRK2) binds to and phosphorylates HCV RNA polymerase. PRK2-specific siRNA treatment or over-expression of PRK2 was shown to inhibit or enhance HCV replication, respectively. We are mapping the phosphorylation site on the polymerase by 2-dimnsional phosphoamino acid analysis and by MS analysis, to investigate further the function NS5B phosphorylation by a reverse genetics approach. Furthermore, we are currently investigating the anti-HCV effect of PRK2 inhibitors in an HCV subgenomic replicon cell line and in HCV-infected cells.
Characterization and cellular localization of HCV RNA replicase complex
We have been characterizing the cellular localization of HCV RNA replicase complex. Our results showed that the replicase complex appears to be associated with lipid raft-like domains of mitochondria. The complex purified from an HCV subgenomic replicon cells directed the synthesis RNA using the endogenous viral RNAs in the complex. By a proteomics approach, we recently identified several cellular proteins interacting with HCV RNA polymerase. One of proteins we have been characterizing seems to a component of the RNA replicase complex. Knock-down of expression of the identified protein by a specific siRNA or an antisense nucleic acid resulted in suppression of HCV RNA replication. We are investigation the roles of this cellular protein in assembly of the RNA replicase complex.
Roles of cellular factors in HCV entry
Currently, several HCV entry receptors, including CD81, scavenger receptor class B type I, claudin-1, glycosaminoglycans, and low-density lipoprotein receptor are postulated to be involved in HCV attachment to and entry into target cells. However, HCV specific, primary receptor(s) has not been yet identified. Furthermore, propagation of HCV for efficient production of infectious virus particles is still restricted to the Huh7 cell line or its derivatives. We are currently investigating the possibility that susceptibility of cells to HCV infection might be induced by regulation of cellular factor expression.
(2) Japanese encephalitis virus RdRp
We have been characterizing JEV RdRp as a model system for flavivirus RNA replication study. Our work is focused on analysis of cis-acting RNA elements required for viral RNA synthesis initiation. In addition, we have been investigating RNA synthesis initiation mechanism using a full-length, enzymatically active JEV RdRp expressed and purified from E. coli. We are also attempting to solve the three-dimensional structures of the purified RdRp and its N-terminal domain carrying a methyl transferase activity, by collaboration with other research groups. Another ongoing project is to identify the cellular proteins interacting with JEV RNA polymerase to study their roles in regulation of viral RNA synthesis and in the assembly or localization of JEV RNA replicase complex. Understanding of cis-acting region important for RNA replication and identification of cellular proteins involved in regulation of viral RNA synthesis will provide a novel target useful for development of anti-JEV agents.
(3) Severe acute respiratory syndrome coronavirus RdRp
Since its first appearance in Guangdong Province, China, SARS-CoV has been a threat to public health and economies worldwide. This newly emerged coronavirus has been isolated in various wild animals, and its popularity in various wild animals raises a possibility that we may have another outbreak in the near future. Recently, we for the first time successfully cloned and expressed a functionally active SARS-CoV RdRp, which could synthesis RNA in a primer dependent manner using a poly(A) RNA template or in a primer-independent manner using RNA templates derived from the viral genome. An exciting ongoing project is the analysis of cis-acting RNAs to understand how the viral RdRp initiate RNA synthesis from the 3’-ends of both plus-and minus viral RNA genome, and how viral subgenomic RNAs are synthesized. This functional RdRp will be used for development of anti-SARS drugs.
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II. Virus-Host Interactions
Virus-host interaction occurs in almost every step of virus infection and plays a crucial role in pathogenesis. In HCV infected cells, viral protein expression and viral RNA induce diverse cellular responses including innate immune responses, reactive oxygen species generation, and modulation of cellular signaling pathways. In addition, various viruses, including HCV, find a way to escape from host defense systems. For example, RNA virus infection is often associated with rapid induction of type I interferon response. Entry of virus and release of viral RNA genome induce the interferon expression by activation of RIG-I (a cellular helicase recognizing dsRNA) and its downstream signaling pathways including of phosphorylation of interferon regulatory factor 3 (IRF3), a transcriptional activator for interferon gene. Induction and release of interferon finally results in the production of interferon that suppresses general protein synthesis to inhibit virus propagation. This series of signal cascades can be interrupted by viral proteins. In HCV-infected cells, interferon production pathway seems to be impaired by cleavage of an adaptor molecule, IPS-1, by the protease activity of HCV NS3 protein.
We are interested in how persistent infection of RNA virus regulates interferon signaling pathway and how virus escapes from this host defense system. We are also interested in studying the effects of HCV-mediated ROS generation on viral propagation and on induction of host immune responses. |
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