Mami Matsuda

Proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin, in cells expressing hepatitis C virus core protein

The hepatitis C virus (HCV) core protein is involved in viral pathogenesis such as oxidative stress induction and lipid metabolism disturbance, and is primarily located in the cytoplasm and endoplasmic reticulum in association with lipid droplets as well as in the mitochondria. To clarify the impact of the core protein on mitochondria, we analyzed the expression pattern of mitochondrial proteins in core protein‐expressing cells by two‐dimensional polyacrylamide gel electrophoresis. Several proteins related to the mitochondrial respiratory chain or protein chaperons were identified by mass spectrometry. Among the identified proteins with consistently different expressions, prohibitin, a mitochondrial protein chaperon, was up‐regulated not only in core‐expressing cells but also in full‐genomic replicon cells and livers of core‐gene transgenic mice. The stability of prohibitin was increased through interaction with the core protein. Further analysis demonstrated that interaction of prohibitin with mitochondrial DNA‐encoded subunits of cytochrome c oxidase (COX) was disturbed by the core protein, resulting in a significant decrease in COX activity. Conclusion: The HCV core protein affects the steady‐state levels of a subset of mitochondrial proteins including prohibitin, which may lead to an impaired function of the mitochondrial respiratory chain with the overproduction of oxidative stress. (HEPATOLOGY 2009.)

Involvement of Hepatitis C Virus NS5A Hyperphosphorylation Mediated by Casein Kinase I-α in Infectious Virus Production

ABSTRACT Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) possesses multiple functions in the viral life cycle. NS5A is a phosphoprotein that exists in hyperphosphorylated and basally phosphorylated forms. Although the phosphorylation status of NS5A is considered to have a significant impact on its function, the mechanistic details regulating NS5A phosphorylation, as well as its exact roles in the HCV life cycle, are still poorly understood. In this study, we screened 404 human protein kinases via in vitro binding and phosphorylation assays, followed by RNA interference-mediated gene silencing in an HCV cell culture system. Casein kinase I-α (CKI-α) was identified as an NS5A-associated kinase involved in NS5A hyperphosphorylation and infectious virus production. Subcellular fractionation and immunofluorescence confocal microscopy analyses showed that CKI-α-mediated hyperphosphorylation of NS5A contributes to the recruitment of NS5A to low-density membrane structures around lipid droplets (LDs) and facilitates its interaction with core protein and the viral assembly. Phospho-proteomic analysis of NS5A with or without CKI-α depletion identified peptide fragments that corresponded to the region located within the low-complexity sequence I, which is important for CKI-α-mediated NS5A hyperphosphorylation. This region contains eight serine residues that are highly conserved among HCV isolates, and subsequent mutagenesis analysis demonstrated that serine residues at amino acids 225 and 232 in NS5A (genotype 2a) may be involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of virion production. These findings provide insight concerning the functional role of NS5A phosphorylation as a regulatory switch that modulates its multiple functions in the HCV life cycle. IMPORTANCE Mechanisms regulating NS5A phosphorylation and its exact function in the HCV life cycle have not been clearly defined. By using a high-throughput screening system targeting host protein kinases, we identified CKI-α as an NS5A-associated kinase involved in NS5A hyperphosphorylation and the production of infectious virus. Our results suggest that the impact of CKI-α in the HCV life cycle is more profound on virion assembly than viral replication via mediation of NS5A hyperphosphorylation. CKI-α-dependent hyperphosphorylation of NS5A plays a role in recruiting NS5A to low-density membrane structures around LDs and facilitating its interaction with the core for new virus particle formation. By using proteomic approach, we identified the region within the low-complexity sequence I of NS5A that is involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of infectious virus production. These findings will provide novel mechanistic insights into the roles of NS5A-associated kinases and NS5A phosphorylation in the HCV life cycle.

Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor.

Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research. Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier. Subsequent detection of increased HCV RNA suggested viral replication. Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant. Infectivity was shown by successful secondary passage to a new culture. Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible. The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection. It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy.

Production of Infectious Hepatitis C Virus by Using RNA Polymerase I-Mediated Transcription

ABSTRACT In this study, we used an RNA polymerase I (Pol I) transcription system for development of a reverse genetics protocol to produce hepatitis C virus (HCV), which is an uncapped positive-strand RNA virus. Transfection with a plasmid harboring HCV JFH-1 full-length cDNA flanked by a Pol I promoter and Pol I terminator yielded an unspliced RNA with no additional sequences at either end, resulting in efficient RNA replication within the cytoplasm and subsequent production of infectious virions. Using this technology, we developed a simple replicon trans-packaging system, in which transient transfection of two plasmids enables examination of viral genome replication and virion assembly as two separate steps. In addition, we established a stable cell line that constitutively produces HCV with a low mutation frequency of the viral genome. The effects of inhibitors of N-linked glycosylation on HCV production were evaluated using this cell line, and the results suggest that certain step(s), such as virion assembly, intracellular trafficking, and secretion, are potentially up- and downregulated according to modifications of HCV envelope protein glycans. This Pol I-based HCV expression system will be beneficial for a high-throughput antiviral screening and vaccine discovery programs.

Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

ABSTRACT Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.

Identification of annexin A1 as a novel substrate for E6AP-mediated ubiquitylation†

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of p53 in conjunction with the high‐risk human papillomavirus E6 proteins. However, the physiological functions of E6AP are poorly understood. To identify a novel biological function of E6AP, we screened for binding partners of E6AP using GST pull‐down and mass spectrometry. Here we identified annexin A1, a member of the annexin superfamily, as an E6AP‐binding protein. Ectopic expression of E6AP enhanced the degradation of annexin A1 in vivo. RNAi‐mediated downregulation of endogenous E6AP increased the levels of endogenous annexin A1 protein. E6AP interacted with annexin A1 and induced its ubiquitylation in a Ca2+‐dependent manner. GST pull‐down assay revealed that the annexin repeat domain III of annexin A1 is important for the E6AP binding. Taken together, our data suggest that annexin A1 is a novel substrate for E6AP‐mediated ubiquitylation. Our findings raise the possibility that E6AP may play a role in controlling the diverse functions of annexin A1 through the ubiquitin‐proteasome pathway. J. Cell. Biochem. 106: 1123–1135, 2009.

Chaperonin TRiC/CCT participates in replication of hepatitis C virus genome via interaction with the viral NS5B protein.

To identify the host factors implicated in the regulation of hepatitis C virus (HCV) genome replication, we performed comparative proteome analyses of HCV replication complex (RC)-rich membrane fractions prepared from cells harboring genome-length bicistronic HCV RNA at the exponential and stationary growth phases. We found that the eukaryotic chaperonin T-complex polypeptide 1 (TCP1)-ring complex/chaperonin-containing TCP1 (TRiC/CCT) plays a role in the replication possibly through an interaction between subunit CCT5 and the viral RNA polymerase NS5B. siRNA-mediated knockdown of CCT5 suppressed RNA replication and production of the infectious virus. Gain-of-function activity was shown following co-transfection with whole eight TRiC/CCT subunits. HCV RNA synthesis was inhibited by an anti-CCT5 antibody in a cell-free assay. These suggest that recruitment of the chaperonin by the viral nonstructural proteins to the RC, which potentially facilitate folding of the RC component(s) into the mature active form, may be important for efficient replication of the HCV genome.

Signal Peptidase Complex Subunit 1 Participates in the Assembly of Hepatitis C Virus through an Interaction with E2 and NS2

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a hydrophobic, transmembrane protein that is required not only for NS2-NS3 cleavage, but also for infectious virus production. To identify cellular factors that interact with NS2 and are important for HCV propagation, we screened a human liver cDNA library by split-ubiquitin membrane yeast two-hybrid assay using full-length NS2 as a bait, and identified signal peptidase complex subunit 1 (SPCS1), which is a component of the microsomal signal peptidase complex. Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired. Propagation of Japanese encephalitis virus was not affected by knockdown of SPCS1, suggesting that SPCS1 does not widely modulate the viral lifecycles of the Flaviviridae family. SPCS1 was found to interact with both NS2 and E2. A complex of NS2, E2, and SPCS1 was formed in cells as demonstrated by co-immunoprecipitation assays. Knockdown of SPCS1 impaired interaction of NS2 with E2. Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles.

Antiviral Activity of Glycyrrhizin against Hepatitis C Virus In Vitro

Glycyrrhizin (GL) has been used in Japan to treat patients with chronic viral hepatitis, as an anti-inflammatory drug to reduce serum alanine aminotransferase levels. GL is also known to exhibit various biological activities, including anti-viral effects, but the anti-hepatitis C virus (HCV) effect of GL remains to be clarified. In this study, we demonstrated that GL treatment of HCV-infected Huh7 cells caused a reduction of infectious HCV production using cell culture-produced HCV (HCVcc). To determine the target step in the HCV lifecycle of GL, we used HCV pseudoparticles (HCVpp), replicon, and HCVcc systems. Significant suppressions of viral entry and replication steps were not observed. Interestingly, extracellular infectivity was decreased, and intracellular infectivity was increased. By immunofluorescence and electron microscopic analysis of GL treated cells, HCV core antigens and electron-dense particles had accumulated on endoplasmic reticulum attached to lipid droplet (LD), respectively, which is thought to act as platforms for HCV assembly. Furthermore, the amount of HCV core antigen in LD fraction increased. Taken together, these results suggest that GL inhibits release of infectious HCV particles. GL is known to have an inhibitory effect on phospholipase A2 (PLA2). We found that group 1B PLA2 (PLA2G1B) inhibitor also decreased HCV release, suggesting that suppression of virus release by GL treatment may be due to its inhibitory effect on PLA2G1B. Finally, we demonstrated that combination treatment with GL augmented IFN-induced reduction of virus in the HCVcc system. GL is identified as a novel anti-HCV agent that targets infectious virus particle release.

Expression profiling of liver cell lines expressing entire or parts of hepatitis C virus open reading frame.

Although hepatitis C virus (HCV) is a causative agent of liver diseases, its mechanism of pathogenesis is still unclear, mainly because of the lack of adequate cell culture systems to support HCV infection and replication. In this report, we describe development and characterization of human hepatoma cell lines constitutively expressing entire (Hep394) or parts (Hep352, Hep3294) of the HCV open reading frame (ORF). The viral and cellular proteolytic machinery involved in the viral precursor processing was consistently functional, and processed HCV proteins were synthesized in these established cell lines. By using a cDNA microarray analysis coupled with semiquantitative reverse‐transcription polymerase chain reaction (RT‐PCR), we identified 12 genes up‐regulated and 4 genes down‐regulated in Hep394 cells. With regard to genes related to cell growth regulation, we found up‐regulation of forkhead transcription factor FREAC‐1, poly (A) binding protein PABP2, and Ras suppressor Rsu‐1. Another category of changes in gene expression includes MHC antigens, which play an important role in the T‐cell‐mediated immune reaction in the liver. In conclusion, functional genomic approaches comparing expression among the different cell lines expressing parts of the HCV genome may promote our understanding of the molecular basis of pathogenicity of HCV infection. (HEPATOLOGY2002;36:1431–1438).