Saneyuki Ujino

Heat-shock Protein 90 Is Essential for Stabilization of the Hepatitis C Virus Nonstructural Protein NS3

The hepatitis C virus (HCV) is a major cause of chronic liver disease. Here, we report a new and effective strategy for inhibiting HCV replication using 17-allylaminogeldanamycin (17-AAG), an inhibitor of heat-shock protein 90 (Hsp90). Hsp90 is a molecular chaperone with a key role in stabilizing the conformation of many oncogenic signaling proteins. We examined the inhibitory effects of 17-AAG on HCV replication in an HCV replicon cell culture system. In HCV replicon cells treated with 17-AAG, we found that HCV RNA replication was suppressed in a dose-dependent manner, and interestingly, the only HCV protein degraded in these cells was NS3 (nonstructural protein 3). Immunoprecipitation experiments showed that NS3 directly interacted with Hsp90, as did proteins expressed from ΔNS3 protease expression vectors. These results suggest that the suppression of HCV RNA replication is due to the destabilization of NS3 in disruption of the Hsp90 chaperone complex by 17-AAG.

Lipoprotein component associated with hepatitis C virus is essential for virus infectivity

Many chronic hepatitis patients with hepatitis C virus (HCV) are observed to have a degree of steatosis which is a factor in the progression of liver diseases. Transgenic mice expressing HCV core protein develop liver steatosis before the onset of hepatocellular carcinoma, suggesting active involvement of HCV in the de-regulation of lipid metabolism in host cells. However, the role of lipid metabolism in HCV life cycle has not been fully understood until the establishment of in vitro HCV infection and replication system. In this review we focus on HCV production with regard to modification of lipid metabolism observed in an in vitro HCV infection and replication system. The importance of lipid droplet to HCV production has been recognized, possibly at the stage of virus assembly, although the precise mechanism of lipid droplet for virus production remains elusive. Association of lipoprotein with HCV in circulating blood in chronic hepatitis C patients is observed. In fact, HCV released from culture medium is also associated with lipoprotein. The fact that treatment of HCV fraction with lipoprotein lipase (LPL) abolished infectivity indicates the essential role of lipoproteins association with virus particle in the virus life cycle. In particular, apolipoprotein E (ApoE), a component of lipoprotein associated with HCV plays a pivotal role in HCV infectivity by functioning as a virus ligand to lipoprotein receptor that also functions as HCV receptor. These results strongly suggest the direct involvement of lipid metabolism in the regulation of the HCV life cycle.

Hepatitis C virus utilizes VLDLR as a novel entry pathway.

Significance Hepatitis C virus (HCV) utilizes various host factors to enter host cells. During the process of HCV entry, cell surface-residing lipoprotein receptors such as scavenger receptor class B member 1 (SR-BI) play important roles through interactions with virus envelope protein E2 and virus-associated apolipoproteins such as apolipoprotein E (ApoE). CD81 plays a crucial role during this process in association with HCV. Here, we identified another pathway for HCV entry that does not use CD81. This pathway involves an association with the very-low-density lipoprotein receptor (VLDLR) and does not require previously reported host factors such as claudin, occludin, or CD81. This finding may shed new light on the process of HCV entry. Various host factors are involved in the cellular entry of hepatitis C virus (HCV). In addition to the factors previously reported, we discovered that the very-low-density lipoprotein receptor (VLDLR) mediates HCV entry independent of CD81. Culturing Huh7.5 cells under hypoxic conditions significantly increased HCV entry as a result of the expression of VLDLR, which was not expressed under normoxic conditions in this cell line. Ectopic VLDLR expression conferred susceptibility to HCV entry of CD81-deficient Huh7.5 cells. Additionally, VLDLR-mediated HCV entry was not affected by the knockdown of cellular factors known to act as HCV receptors or HCV entry factors. Because VLDLR is expressed in primary human hepatocytes, our results suggest that VLDLR functions in vivo as an HCV receptor independent of canonical CD81-mediated HCV entry.

Long noncoding RNA #32 contributes to antiviral responses by controlling interferon-stimulated gene expression.

Significance Here, we describe a key feature of the long noncoding RNA (lncRNA) involved in innate immunity. We identified 182 lncRNAs that were highly modulated in poly(I:C)-treated hepatocyte cells. Of these, lncRNA#32 regulated the level of IFN-stimulated genes under both unstimulated and type I IFN-stimulated conditions through interactions with hnRNPU and ATF2, and therefore plays an important role in antiviral immunity. Despite the breadth of knowledge that exists regarding the function of long noncoding RNAs (lncRNAs) in biological phenomena, the role of lncRNAs in host antiviral responses is poorly understood. Here, we report that lncRNA#32 is associated with type I IFN signaling. The silencing of lncRNA#32 dramatically reduced the level of IFN-stimulated gene (ISG) expression, resulting in sensitivity to encephalomyocarditis virus (EMCV) infection. In contrast, the ectopic expression of lncRNA#32 significantly suppressed EMCV replication, suggesting that lncRNA#32 positively regulates the host antiviral response. We further demonstrated the suppressive function of lncRNA#32 in hepatitis B virus and hepatitis C virus infection. lncRNA#32 bound to activating transcription factor 2 (ATF2) and regulated ISG expression. Our results reveal a role for lncRNA#32 in host antiviral responses.

Novel reporter system to monitor early stages of the hepatitis B virus life cycle

A recombinant hepatitis B virus (HBV) expressing NanoLuc (NL) (HBV/NL) was produced by cotransfecting a plasmid containing a 1.2‐fold HBV genome carrying the NL gene with a plasmid bearing a packaging‐defective 1.2‐fold HBV genome into a human hepatoma cell line, HepG2. We found that NL activity in HBV/NL‐infected primary hepatocytes or sodium taurocholate cotransporting polypeptide‐transduced human hepatocyte‐derived cell lines increased linearly for several days after infection and was concordant with HBV RNA levels in the cells. Treatment of the virus‐infected cells with HBV inhibitors reduced NL activity in a dose‐dependent manner. Detection of HBV/NL infection, monitored by NL activity, was highly sensitive and less expensive than detection using the conventional method to evaluate HBV infection. In addition, because we also studied host factors, this system is applicable not only for studying the HBV life cycle, but also for exploring agent(s) that regulate HBV proliferation.

The interaction between human initiation factor eIF3 subunit c and heat-shock protein 90: A necessary factor for translation mediated by the hepatitis C virus internal ribosome entry site

Heat-shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of various transcription factors and protein kinases in signal transduction. The hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA drives translation by directly recruiting the 40S ribosomal subunits that bind to eukaryotic initiation factor 3 (eIF3). Our data indicate that Hsp90 binds indirectly to eIF3 subunit c by interacting with it through the HCV IRES RNA, and the functional consequence of this Hsp90-eIF3c-HCV-IRES RNA interaction is the prevention of ubiquitination and the proteasome-dependent degradation of eIF3c. Hsp90 activity interference by Hsp90 inhibitors appears to be the result of the dissociation of eIF3c from Hsp90 in the presence of HCV IRES RNA and the resultant induction of the degradation of the free forms of eIF3c. Moreover, the interaction between Hsp90 and eIF3c is dependent on HCV IRES RNA binding. Furthermore, we demonstrate, by knockdown of eIF3c, that the silencing of eIF3c results in inhibitory effects on translation of HCV-derived RNA but does not affect cap-dependent translation. These results indicate that the interaction between Hsp90 and eIF3c may play an important role in HCV IRES-mediated translation.

The RNA-editing Enzyme APOBEC1 Requires Heterogeneous Nuclear Ribonucleoprotein Q Isoform 6 for Efficient Interaction with Interleukin-8 mRNA

Background: APOBEC1 stabilizes target mRNAs by suppressing nonsense- or AU-rich element-mediated decay; however, the mechanisms regulating target selection are unknown. Results: In the presence of hnRNPQ isoform 6, APOBEC1 stabilizes interleukin-8 mRNA independently of APOBEC1 complementation factor. Conclusion: APOBEC1 utilizes a complementing protein to select target mRNAs. Significance: These data shed light on the selective regulation of APOBEC1 target genes. Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC1) is an intestine-specific RNA-binding protein. However, inflammation or exposure to DNA-damaging agents can induce ectopic APOBEC1 expression, which can result in hepatocellular hyperplasia in animal models. To identify its RNA targets, FLAG-tagged APOBEC1 was immunoprecipitated from transfected HuH7.5 hepatocellular carcinoma cells and analyzed using DNA microarrays. The interleukin-8 (IL8) mRNA was the most abundant co-precipitated RNA. Exogenous APOBEC1 expression increased IL8 production by extending the half-life of the IL8 mRNA. A cluster of AU-rich elements in the 3′-UTR of IL8 was essential to the APOBEC1-mediated increase in IL8 production. Notably, IL8 mRNA did not co-immunoprecipitate with APOBEC1 from lysates of other cell types at appreciable levels; therefore, other factors may enhance the association between APOBEC1 and IL8 mRNA in a cell type-specific manner. A yeast two-hybrid analysis and siRNA screen were used to identify proteins that enhance the interaction between APOBEC1 and IL8 mRNA. Heterogeneous nuclear ribonucleoprotein Q (hnRNPQ) was essential to the APOBEC1/IL8 mRNA association in HuH7.5 cells. Of the seven hnRNPQ isoforms, only hnRNPQ6 enabled APOBEC1 to bind to IL8 mRNA when overexpressed in HEK293 cells, which expressed the lowest level of endogenous hnRNPQ6 among the cell types examined. The results of a reporter assay using a luciferase gene fused to the IL8 3′-UTR were consistent with the hypothesis that hnRNPQ6 is required for APOBEC1-enhanced IL8 production. Collectively, these data indicate that hnRNPQ6 promotes the interaction of APOBEC1 with IL8 mRNA and the subsequent increase in IL8 production.

Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus

Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies.

Combination Therapy for Hepatitis C Virus with Heat-Shock Protein 90 Inhibitor 17-AAG and Proteasome Inhibitor MG132

Background: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Here, we report a new and effective strategy for inhibiting HCV replication using an inhibitor of heat-shock protein 90, 17-AAG (17-allylamino-17demethoxygeldanamycin), and a proteasome inhibitor, MG132. Methods: To explore the virological basis of combination therapy, we analysed the effects of 17-AAG and MG132, singly and in combination on HCV replication in an HCV replicon cell system. Results: In HCV replicon cells, HCV RNA replication was suppressed by 17-AAG in a dose-dependent manner. As shown in the present study, the 50% inhibitory concentration values were 0.82 nM for 17-AAG and 0.21 nM for MG132. Low concentrations of MG132 had strong synergistic inhibitory effects with low toxicity on HCV replicon cells. Conclusions: The results of this study suggest that the different effects and synergistic actions of 17-AAG and MG132 could provide a new therapeutic approach to HCV infection.

Investigating the Hepatitis B Virus Life Cycle Using Engineered Reporter Hepatitis B Viruses

Chronic infection with hepatitis B virus (HBV) increases the risk of developing fibrosis, cirrhosis or hepatocellular carcinoma. Current therapies are limited to type‐I interferons and/or nucleos(t)ide analogues; however, these are only partially effective. The development of novel anti‐HBV agents for new treatment strategies has been hampered by the lack of a suitable system that allows the in vitro replication of HBV. Studies of virus infection/replication at the molecular level using wild‐type HBV are labor‐intensive and time‐consuming. To overcome these problems, we previously constructed a recombinant reporter HBV bearing the NanoLuc gene and showed its usefulness in identifying factors that affect HBV proliferation. Because this system mimics the early stage of the HBV life cycle faithfully, we conducted a quantitative analysis of HBV infectivity to several human hepatocyte cell lines as well as the effect of dimethyl sulfoxide and HBV protein X on the early stage of HBV proliferation using this system. Furthermore, we developed a system to produce a reporter HBV expressing a pol gene. These reporter HBV may provide an opportunity to enhance our understanding of the HBV life cycle and aid strategies for the development of new anti‐HBV agents.