SARS-CoV-2 papain-like protease inhibits ISGylation of the viral nucleocapsid protein to evade host anti-viral immunity.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.

Hepatitis C Virus Disrupts Annexin 5-Mediated Occludin Integrity through Downregulation of Protein Kinase Cα (PKCα) and PKCη Expression, Thereby Promoting Viral Propagation.

Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.

Oxidative stress sensor Keap1 recognizes HBx protein to activate the Nrf2/ARE signaling pathway, thereby inhibiting hepatitis B virus replication.

IMPORTANCE: The Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is one of the most important defense mechanisms against oxidative stress. We previously reported that a cellular hydrogen peroxide scavenger protein, peroxiredoxin 1, a target gene of transcription factor Nrf2, acts as a novel HBV X protein (HBx)-interacting protein and negatively regulates hepatitis B virus (HBV) propagation through degradation of HBV RNA. This study further demonstrates that the Nrf2/ARE signaling pathway is activated during HBV infection, eventually leading to the suppression of HBV replication. We provide evidence suggesting that Keap1 interacts with HBx, leading to Nrf2 activation and inhibition of HBV replication via suppression of HBV core promoter activity. This study raises the possibility that activation of the Nrf2/ARE signaling pathway is a potential therapeutic strategy against HBV. Our findings may contribute to an improved understanding of the negative regulation of HBV replication by the antioxidant response.

Hepatitis C Virus-Induced ROS/JNK Signaling Pathway Activates the E3 Ubiquitin Ligase Itch to Promote the Release of HCV Particles via Polyubiquitylation of VPS4A.

We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. However, the roles of ROS/JNK activation in the HCV life cycle remain unclear. We sought to identify a novel role of the ROS/JNK signaling pathway in the HCV life cycle. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of Itch, a HECT-type E3 ubiquitin ligase, leading to activation of Itch. The small interfering RNA (siRNA) knockdown of Itch significantly reduced the extracellular HCV infectivity titers, HCV RNA, and HCV core protein without affecting intracellular HCV infectivity titers, HCV RNA, and HCV proteins, suggesting that Itch is involved in the release of HCV particles. HCV-mediated JNK/Itch activation specifically promoted polyubiquitylation of an AAA-type ATPase, VPS4A, but not VPS4B, required to form multivesicular bodies. Site-directed mutagenesis revealed that two lysine residues (K23 and K121) on VPS4A were important for VPS4A polyubiquitylation. The siRNA knockdown of VPS4A, but not VPS4B, significantly reduced extracellular HCV infectivity titers. Coimmunoprecipitation analysis revealed that HCV infection specifically enhanced the interaction between CHMP1B, a subunit of endosomal sorting complexes required for transport (ESCRT)-III complex, and VPS4A, but not VPS4B, whereas VPS4A K23R/K121R greatly reduced the interaction with CHMP1B. HCV infection significantly increased ATPase activity of VPS4A, but not VPS4A K23R/K121R or VPS4B, suggesting that HCV-mediated polyubiquitylation of VPS4A contributes to activation of VPS4A. Taken together, we propose that the HCV-induced ROS/JNK/Itch signaling pathway promotes VPS4A polyubiquitylation, leading to enhanced VPS4A-CHMP1B interaction and promotion of VPS4A ATPase activity, thereby promoting the release of HCV particles. IMPORTANCE The ROS/JNK signaling pathway contributes to liver diseases, including steatosis, metabolic disorders, and hepatocellular carcinoma. We previously reported that HCV activates the ROS/JNK signaling pathway, leading to the enhancement of hepatic gluconeogenesis and apoptosis induction. This study further demonstrates that the HCV-induced ROS/JNK signaling pathway activates the E3 ubiquitin ligase Itch to promote release of HCV particles via polyubiquitylation of VPS4A. We provide evidence suggesting that HCV infection promotes the ROS/JNK/Itch signaling pathway and ESCRT/VPS4A machinery to release infectious HCV particles. Our results may lead to a better understanding of the mechanistic details of HCV particle release.

Complete genome analyses of G12P[8] rotavirus strains from hospitalized children in Surabaya, Indonesia, 2017-2018.

Rotavirus A (RVA) is a major viral cause of acute gastroenteritis (AGE) worldwide. G12 RVA strains have emerged globally since 2007. There has been no report of the whole genome sequences of G12 RVAs in Indonesia. We performed the complete genome analysis by the next-generation sequencing of five G12 strains from hospitalized children with AGE in Surabaya from 2017 to 2018. All five G12 strains were Wa-like strains (G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) and were clustered into lineage-III of VP7 gene phylogenetic tree. STM430 sample was observed as a mixed-infection between G12 and G1 strains: G12/G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. A phylogenetic tree analysis revealed that all five Indonesian G12 strains (SOEP379, STM371, STM413, STM430, and STM433) were genetically close to each other in all 11 genome segments with 98.0%-100% nucleotide identities, except VP3 and NSP4 of STM430, suggesting that these strains have originated from a similar ancestral G12 RVA. The VP3 and NSP4 genome segments of STM430-G12P[8] were separated phylogenetically from those of the other four G12 strains, probably due to intra-genotype reassortment between the G12 and G1 Wa-like strains. The change from G12P[6] lineage-II in 2007 to G12P[8] lineage-III 2017-2018 suggests the evolution and diversity of G12 RVAs in Indonesia over the past approximately 10 years.

A household survey of intrafamily norovirus transmission.

Norovirus (NoV) is a leading cause of epidemic and sporadic gastroenteritis in people of all ages. Humans are the primary source of NoV and household contact is one of the risk factors for NoV transmission. However, the mechanisms underlying person-to-person NoV transmission are poorly understood. Here we conducted a survey to profile the frequency and characteristics of intrafamily NoV transmission. Stool samples were collected every week from three households between 2016 and 2020; the total number of samples was 1105. The detection of NoV and the genotyping were performed by reverse transcription-polymerase chain reaction targeting the capsid region and direct sequencing methods. NoV was detected in 3.4% of all samples. Eight NoV genotypes were identified. The most common genotype was GII.17, followed in order by GII.6, GI.6, GII.4, GI.3, and GI.2/GI.8/GI.9. Most NoV-positive samples were obtained from asymptomatic individuals. The highest number of NoV transmissions was found in household 3 (6 infections), followed by household 2 (2 infections), while household 1 had no NoV transmission, suggesting that asymptomatic NoV carriers play a major role in infection as NoV reservoirs in the households. Further clarification of the mode of infection will contribute to improved understanding and an appropriate prevention.

HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein to promote viral replication.

Ubiquitin and ubiquitin-like protein modification play important roles in modulating the functions of viral proteins in many viruses. Here we demonstrate that hepatitis B virus (HBV) X protein (HBx) is modified by ISG15, which is a type I IFN-inducible, ubiquitin-like protein; this modification is called ISGylation. Immunoblot analyses revealed that HBx proteins derived from four different HBV genotypes accepted ISGylation in cultured cells. Site-directed mutagenesis revealed that three lysine residues (K91, K95 and K140) on the HBx protein, which are well conserved among all the HBV genotypes, are involved in acceptance of ISGylation. Using expression plasmids encoding three known E3 ligases involved in the ISGylation to different substrates, we found that HERC5 functions as an E3 ligase for HBx-ISGylation. Treatment with type I and type III IFNs resulted in the limited suppression of HBV replication in Hep38.7-Tet cells. When cells were treated with IFN-α, silencing of ISG15 resulted in a marked reduction of HBV replication in Hep38.7-Tet cells, suggesting a role of ISG15 in the resistance to IFN-α. In contrast, the silencing of USP18 (an ISG15 de-conjugating enzyme) increased the HBV replication in Hep38.7-Tet cells. Taken together, these results suggest that the HERC5-mediated ISGylation of HBx protein confers pro-viral functions on HBV replication and participates in the resistance to IFN-α-mediated antiviral activity.

ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A.

Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is covalently conjugated to many substrate proteins in order to modulate their functions; this conjugation is called ISGylation. Several groups reported that the ISGylation of hepatitis C virus (HCV) NS5A protein affects HCV replication. However, the ISG15 conjugation sites on NS5A are not well determined, and it is unclear whether the role of NS5A ISGylation in HCV replication is proviral or antiviral. Here, we investigated the role of NS5A ISGylation in HCV replication by using HCV RNA replicons that encode a mutation at each lysine (Lys) residue of the NS5A protein. Immunoblot analyses revealed that 5 Lys residues (K44, K68, K166, K215, and K308) of the 14 Lys residues within NS5A (genotype 1b, Con1) have the potential to accept ISGylation. We tested the NS5A ISGylation among different HCV genotypes and observed that the NS5A proteins of all of the HCV genotypes accept ISGylation at multiple Lys residues. Using an HCV luciferase reporter replicon assay revealed that residue K308 of NS5A is important for HCV (1b, Con1) RNA replication. We observed that K308, one of the Lys residues for NS5A ISGylation, is located within the binding region of cyclophilin A (CypA), which is the critical host factor for HCV replication. We obtained evidence derived from all of the HCV genotypes suggesting that NS5A ISGylation enhances the interaction between NS5A and CypA. Taken together, these results suggest that NS5A ISGylation functions as a proviral factor and promotes HCV replication via the recruitment of CypA.IMPORTANCE Host cells have evolved host defense machinery (such as innate immunity) to eliminate viral infections. Viruses have evolved several counteracting strategies for achieving an immune escape from host defense machinery, including type I interferons (IFNs) and inflammatory cytokines. ISG15 is an IFN-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation. Here, we demonstrate that HCV NS5A protein accepts ISG15 conjugation at specific Lys residues and that the HERC5 E3 ligase specifically promotes NS5A ISGylation. We obtained evidence suggesting that NS5A ISGylation facilitates the recruitment of CypA, which is the critical host factor for HCV replication, thereby promoting HCV replication. These findings indicate that E3 ligase HERC5 is a potential therapeutic target for HCV infection. We propose that HCV hijacks an intracellular ISG15 function to escape the host defense machinery in order to establish a persistent infection.

Peroxiredoxin 1, a Novel HBx-Interacting Protein, Interacts with Exosome Component 5 and Negatively Regulates Hepatitis B Virus (HBV) Propagation through Degradation of HBV RNA.

Hepatitis B virus (HBV) infection is a major risk factor for the development of chronic liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). A growing body of evidence suggests that HBV X protein (HBx) plays a crucial role in viral replication and HCC development. Here, we identified peroxiredoxin 1 (Prdx1), a cellular hydrogen peroxide scavenger, as a novel HBx-interacting protein. Coimmunoprecipitation analysis coupled with site-directed mutagenesis revealed that the region from amino acids 17 to 20 of the HBx, particularly HBx Cys17, is responsible for the interaction with Prdx1. Knockdown of Prdx1 by siRNA significantly increased the levels of intracellular HBV RNA, HBV antigens, and extracellular HBV DNA, whereas knockdown of Prdx1 did not increase the activities of HBV core, enhancer I (Enh1)/X, preS1, and preS2/S promoters. Kinetic analysis of HBV RNA showed that knockdown of Prdx1 inhibited HBV RNA decay, suggesting that Prdx1 reduces HBV RNA levels posttranscriptionally. The RNA coimmunoprecipitation assay revealed that Prdx1 interacted with HBV RNA. The exosome component 5 (Exosc5), a member of the RNA exosome complexes, was coimmunoprecipitated with Prdx1, suggesting its role in regulation of HBV RNA stability. Taken together, these results suggest that Prdx1 and Exosc5 play crucial roles in host defense mechanisms against HBV infection.IMPORTANCE Hepatitis B virus (HBV) infection is a major global health problem. HBx plays important roles in HBV replication and viral carcinogenesis through its interaction with host factors. In this study, we identified Prdx1 as a novel HBx-binding protein. We provide evidence suggesting that Prdx1 promotes HBV RNA decay through interaction with HBV RNA and Exosc5, leading to downregulation of HBV RNA. These results suggest that Prdx1 negatively regulates HBV propagation. Our findings may shed new light on the roles of Prdx1 and Exosc5 in host defense mechanisms in HBV infection.

Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1α via Chaperone-Mediated Autophagy.

Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1α caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1α in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1α matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1α was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1α disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1α is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1α with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1α protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1α protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1α. Immunofluorescence study revealed colocalization of NS5A and HNF-1α in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1α, thereby promoting the lysosomal degradation of HNF-1α via CMA.IMPORTANCE Many viruses use a protein degradation system, such as the ubiquitin-proteasome pathway or the autophagy pathway, for facilitating viral propagation and viral pathogenesis. We investigated the mechanistic details of the selective lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) induced by hepatitis C virus (HCV) NS5A protein. Using site-directed mutagenesis, we demonstrated that HNF-1α contains a pentapeptide chaperone-mediated autophagy (CMA)-targeting motif within the POU-specific domain of HNF-1α. The CMA-targeting motif is important for the association with HSC70. LAMP-2A is required for degradation of HNF-1α caused by NS5A. We propose that HCV NS5A interacts with HSC70, a key component of the CMA machinery, and recruits HSC70 to HNF-1α to target HNF-1α for CMA-mediated lysosomal degradation, thereby facilitating HCV pathogenesis. We discovered a role of HCV NS5A in CMA-dependent degradation of HNF-1α. Our results may lead to a better understanding of the role of CMA in the pathogenesis of HCV.

Unconjugated interferon-stimulated gene 15 specifically interacts with the hepatitis C virus NS5A protein via domain I.

Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is induced by type I INF. Although several groups have reported ISGylation of the HCV NS5A protein, it is still unclear whether ISGylation of NS5A has anti- or pro-viral effects in hepatitis C virus (HCV) infection. In the present study, the role of ISGylation-independent, unconjugated ISG15 in HCV infection was examined. Immunoprecipitation analyses revealed that ISG15 interacts specifically with NS5A domain I. ISG15 mutants lacking the C-terminal glycine residue that is essential for ISGylation still interacted with NS5A protein. Taken together, these results suggest that unconjugated ISG15 affects the functions of HCV NS5A through protein-protein interaction.

A single-amino-acid mutation in hepatitis C virus NS5A disrupts physical and functional interaction with the transcription factor HNF-1α.

Hepatitis C virus (HCV) infection often causes extrahepatic manifestations, such as type 2 diabetes. We previously reported that HCV infection induces the lysosomal degradation of the transcription factor HNF-1α via an interaction with viral NS5A, thereby suppressing GLUT2 gene expression. However, the molecular mechanism of NS5A-induced degradation of HNF-1α is largely unknown. We aimed to identify the determinants necessary for the degradation of HNF-1α induced by NS5A. Coimmunoprecipitation analysis revealed that the POU specific (POUs) domain spanning from aa 91 to 181 of HNF-1α is responsible for the interaction of NS5A. We also found that the region from aa 121 to 126 of NS5A, which is known as the binding motif of the HCV replication factor FKBP8, is important for the degradation of HNF-1α. A NS5A V121A mutation disrupted the NS5A-HNF-1α interaction as well as the degradation of HNF-1α. Our findings suggest that NS5A Val121 is crucial for viral pathogenesis.

Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B.

Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin-proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A-binding protein. Co-immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh-7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B-binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A-OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.

Hepatitis C virus-induced glucose metabolic disorder.

Hepatitis C virus (HCV) infection often causes intrahepatic diseases, such as chronic hepatitis, liver chirrohsis, and hepatocellular carcinoma (HCC). Moreover, HCV infection exhibits various extrahepatic manifestations, such as thyroiditis, glucose and lipid metabolic disorder, and iron metabolic disorder. HCV infection is often associated with type 2 diabetes, involving hepatic fibrosis and poor prognosis. Type 2 diabetes increases the risk of HCC. We have been investigating molecular mechanisms of HCV-induced glucose metabolic disorder and we reported that HCV infection promotes hepatic gluconeogenesis through forkhead box O1 (FoxO1)-dependent pathway and that HCV infection suppresses the cell surface expression of glucose transporter 2 (GLUT2), resulting in suppression of glucose uptake. We have found that HCV NS5A protein plays important roles in these two independent pathways. Here we discuss the roles of HCV NS5A in HCV-induced glucose metabolic disorder.

Elevated Serum Xanthine Oxidase and Its Correlation with Antioxidant Status in Patients with Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative movement disorder associated with a loss of dopamine neurons in the substantia nigra. The diagnosis of PD is sensitive since it shows clinical features that are common with other neurodegenerative diseases. In addition, most symptoms arise at the late stage of the disease, where most dopaminergic neurons are already damaged. Several studies reported that oxidative stress is a key modulator in the development of PD. This condition occurs due to excess reactive oxygen species (ROS) production in the cellular system and the incapability of antioxidants to neutralize it. In this study, we focused on the pathology of PD by measuring serum xanthine oxidase (XO) activity, which is an enzyme that generates ROS. Interestingly, the serum XO activity of patients with PD was markedly upregulated compared to patients with other neurological diseases (ONDs) as a control. Moreover, serum XO activity in patients with PD showed a significant correlation with the disease severity based on the Hoehn and Yahr (HY) stages. The investigation of antioxidant status also revealed that serum uric acid levels were significantly lower in the severe group (HY ≥ 3) than in the ONDs group. Together, these results suggest that XO activity may contribute to the development of PD and might potentially be a biomarker for determining disease severity in patients with PD.

Hepatitis C virus infection suppresses GLUT2 gene expression via downregulation of hepatocyte nuclear factor 1α.

Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including type 2 diabetes. We previously reported that HCV replication suppresses cellular glucose uptake by downregulation of cell surface expression of glucose transporter 2 (GLUT2) (D. Kasai et al., J. Hepatol. 50:883-894, 2009). GLUT2 mRNA levels were decreased in both HCV RNA replicon cells and HCV J6/JFH1-infected cells. To elucidate molecular mechanisms of HCV-induced suppression of GLUT2 gene expression, we analyzed transcriptional regulation of the GLUT2 promoter using a series of GLUT2 promoter-luciferase reporter plasmids. HCV-induced suppression of GLUT2 promoter activity was abrogated when the hepatocyte nuclear factor 1α (HNF-1α)-binding motif was deleted from the GLUT2 promoter. HNF-1α mRNA levels were significantly reduced in HCV J6/JFH1-infected cells. Furthermore, HCV infection remarkably decreased HNF-1α protein levels. We assessed the effects of proteasome inhibitor or lysosomal protease inhibitors on the HCV-induced reduction of HNF-1α protein levels. Treatment of HCV-infected cells with a lysosomal protease inhibitor, but not with a proteasome inhibitor, restored HNF-1α protein levels, suggesting that HCV infection promotes lysosomal degradation of HNF-1α protein. Overexpression of NS5A protein enhanced lysosomal degradation of HNF-1α protein and suppressed GLUT2 promoter activity. Immunoprecipitation analyses revealed that the region from amino acids 1 to 126 of the NS5A domain I physically interacts with HNF-1α protein. Taken together, our results suggest that HCV infection suppresses GLUT2 gene expression via downregulation of HNF-1α expression at transcriptional and posttranslational levels. HCV-induced downregulation of HNF-1α expression may play a crucial role in glucose metabolic disorders caused by HCV.

Transcription Factor JunB Suppresses Hepatitis C Virus Replication.

We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. Activation of JNK contributes to the development of liver diseases, including metabolic disorders, steatosis, liver cirrhosis and hepatocellular carcinoma. JNK is known to have numerous target genes, including JunB, a member of activator protein-1 transcription factor family. However, the roles of JunB in the HCV life cycle and HCV-associated pathogenesis remain unclear. To clarify a physiological role of JunB in HCV infection, we investigated the phosphorylation of JunB in HCV J6/JFH1-infected Huh-7.5 cells. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of JunB. The small interfering RNA (siRNA) knockdown of JunB significantly increased the amount of intracellular HCV RNA as well as the intracellular and extracellular HCV infectivity titers. Conversely, overexpression of JunB significantly reduced the amount of intracellular HCV RNA and the intracellular and extracellular HCV infectivity titers. These results suggest that JunB plays a role in inhibiting HCV propagation. Additionally, HCV-mediated JunB activation promoted hepcidin promoter activity and hepcidin mRNA levels, a key factor in modulating iron homeostasis, suggesting that JunB is involved in HCV-induced transcriptional upregulation of hepcidin. Taken together, we propose that the HCV-induced ROS/JNK/JunB signaling pathway plays roles in inhibiting HCV replication and contributing to HCV-mediated iron metabolism disorder.

Cellular Release of Infectious Hepatitis C Virus Particles via Endosomal Pathways.

Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The release of infectious HCV particles from infected hepatocytes is a crucial step in viral dissemination and disease progression. While the exact mechanisms of HCV particle release remain poorly understood, emerging evidence suggests that HCV utilizes intracellular membrane trafficking and secretory pathways. These pathways include the Golgi secretory pathway and the endosomal trafficking pathways, such as the recycling endosome pathway and the endosomal sorting complex required for transport (ESCRT)-dependent multivesicular bodies (MVBs) pathway. This review provides an overview of recent advances in understanding the release of infectious HCV particles, with a particular focus on the involvement of the host cell factors that participate in HCV particle release. By summarizing the current knowledge in this area, this review aims to contribute to a better understanding of endosomal pathways involved in the extracellular release of HCV particles and the development of novel antiviral strategies.

The Role of Chaperone-Mediated Autophagy in Hepatitis C Virus-Induced Pathogenesis.

Lysosome incorporate and degrade proteins in a process known as autophagy. There are three types of autophagy; macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Although autophagy is considered a nonselective degradation process, CMA is known as a selective degradation pathway. All proteins internalized in the lysosome via CMA contain a pentapeptide KFERQ-motif, also known as a CMA-targeting motif, which is necessary for selectivity. CMA directly delivers a substrate protein into the lysosome lumen using the cytosolic chaperone HSC70 and the lysosomal receptor LAMP-2A for degradation. Hepatitis C virus (HCV) NS5A protein interacts with hepatocyte-nuclear factor 1α (HNF-1α) together with HSC70 and promotes the lysosomal degradation of HNF-1α via CMA, resulting in HCV-induced pathogenesis. HCV NS5A promotes recruitment of HSC70 to the substrate protein HNF-1α. HCV NS5A plays a crucial role in HCV-induced CMA. Further investigations of HCV NS5A-interacting proteins containing CMA-targeting motifs may help to elucidate HCV-induced pathogenesis.