Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells.

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.

The kinesin KIF4 mediates HBV/HDV entry through the regulation of surface NTCP localization and can be targeted by RXR agonists in vitro.

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection. KIF4 knockdown suppressed both HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 mutant regained the surface localization of NTCP and significantly restored HBV permissiveness in these cells. IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study revealed that KIF4 interacts with NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent HBV infection in both HepG2-hNTCP and primary human hepatocyte (PXB) (Bexarotene, IC50 1.89 ± 0.98 µM) cultures. Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates targeting HBV and HDV entry.

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.

Predominance of norovirus GI.4 from children with acute gastroenteritis in Jambi, Indonesia, 2019.

Norovirus (NoV) is one of the most important viral causes of acute gastroenteritis (AGE) in children worldwide. Only a few studies have reported AGE with NoV-positive in some cities in Indonesia. This study aimed to investigate the incidence and clinical characteristic of NoV infection, and also genotype distribution of NoV in children with AGE in Jambi, as the capital and the largest city of Jambi province, Indonesia. Stool samples were collected from children (≤15 years of age) with AGE at three participating hospitals in Jambi from February to April 2019. The detection of NoV and its genotyping were carried out by reverse-transcriptase polymerase chain reaction and direct sequencing. Of the 91 stool samples collected, 14 (15.4%) were positive for NoV. Fever, vomiting, and severe diarrhea were commonly observed in AGE with NoV, while level of dehydration was statistically significant difference between children with NoV-positive and those with NoV-negative. The most prevalent genotype was GI.4 (42.9%), followed by GII.6 (28.6%) and some other genotypes. Interestingly, this study found the predominance of GI.4, differed from previous reports in Indonesia. Continuously investigation of the circulating genotype is needed to control the NoV-infected AGE.

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.

Cytosolic DNA-sensing immune response and viral infection.

How host cells recognize many kinds of RNA and DNA viruses and initiate innate antiviral responses against them has not yet been fully elucidated. Over the past decade, investigations into the mechanisms underlying these antiviral responses have focused extensively on immune surveillance sensors that recognize virus-derived components (such as lipids, sugars and nucleic acids). The findings of these studies have suggested that antiviral responses are mediated by cytosolic or intracellular compartment sensors and their adaptor molecules (e.g., TLR, myeloid differentiation primary response 88, retinoic acid inducible gene-I, IFN-ß promoter stimulator-1, cyclic GMP-AMP synthase and stimulator of IFN genes axis) for the primary sensing of virus-derived nucleic acids, leading to production of type I IFNs, pro-inflammatory cytokines and chemokines by the host cells. Thus, host cells have evolved an elaborate host defense machinery to recognize and eliminate virus infections. In turn, to achieve sustained viral infection and induce pathogenesis, viruses have also evolved several counteracting strategies for achieving immune escape by targeting immune sensors, adaptor molecules, intracellular kinases and transcription factors. In this review, we discuss recent discoveries concerning the role of the cytosolic nucleic acid-sensing immune response in viral recognition and control of viral infection. In addition, we consider the regulatory machinery of the cytosolic nucleic acid-sensing immune response because these immune surveillance systems must be tightly regulated to prevent aberrant immune responses to self and non-self-nucleic acids.

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.

Hepatitis C virus NS5A protein interacts with lysine methyltransferase SET and MYND domain-containing 3 and induces activator protein 1 activation.

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A-interacting protein, SET and MYND domain-containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon-harboring and HCV-infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N-SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A-SMYD3 interaction. NS5A co-localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP-1) activity, this being potentiated by co-expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP-1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP-1 activation in HCV-infected cells.

Interaction of the hepatitis B virus X protein with the lysine methyltransferase SET and MYND domain-containing 3 induces activator protein 1 activation.

Hepatitis B virus (HBV) is a widespread human pathogen that often causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The detailed mechanisms underlying HBV pathogenesis remain poorly understood. The HBV X protein (HBx) is a multifunctional regulator that modulates viral replication and host cell functions, such as cell cycle progression, apoptosis and protein degradation through interaction with a variety of host factors. Recently, the nonstructural protein 5A (NS5A) of hepatitis C virus has been reported to interact with methyltransferase SET and MYND domain-containing 3 (SMYD3), which is implicated in chromatin modification and development of cancer. Because HBx shares fundamental regulatory functions concerning viral replication and pathogenesis with NS5A, it was decided to examine whether HBx interacts with SMYD3. In the present study, it was demonstrated by co-immunoprecipitation analysis that HBx interacts with both ectopically and endogenously expressed SMYD3 in Huh-7.5 cells. Deletion mutation analysis revealed that the C-terminal region of HBx (amino acids [aa] 131-154) and an internal region of SMYD3 (aa 269-288) are responsible for their interaction. Immunofluorescence and proximity ligation assays showed that HBx and SMYD3 co-localize predominantly in the cytoplasm. Luciferase reporter assay demonstrated that the interaction between HBx and SMYD3 activates activator protein 1 (AP-1) signaling, but not that of nuclear factor-kappa B (NF-κB). On the other hand, neither overexpression nor knockdown of SMYD3 altered production of HBV transcripts and HBV surface antigen (HBsAg). In conclusion, a novel HBx-interacting protein, SMYD3, was identified, leading to proposal of a novel mechanism of AP-1 activation in HBV-infected cells.

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.

HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis.

We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the pro-apoptotic protein Bim and the protein expression of three major splice variants of Bim (BimEL, BimL and BimS). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.

14-3-3 proteins sequester a pool of soluble TRIM32 ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation.

Deregulated expression of tripartite motif-containing protein 32 (TRIM32, an E3 ubiquitin-protein ligase) contributes to various diseases. Here we report, using quantitative proteomics and biochemistry, that 14-3-3 proteins bind to phosphorylated TRIM32 and prevent TRIM32 autoubiquitylation and the formation of TRIM32-containing cytoplasmic bodies, which are potential autoregulatory mechanisms that can reduce the concentration of soluble free TRIM32. The 14-3-3-TRIM32 interaction is dependent on protein-kinase-A-catalyzed phosphorylation of TRIM32 at Ser651. We found that the inhibitory effect of 14-3-3 is, in part, a consequence of disrupting the propensity of TRIM32 to undergo higher-order self-association without affecting its dimerization. Consequently, dimerized TRIM32 bound to 14-3-3 was sequestered in a distinct cytoplasmic pool away from the microtubule network, whereas a TRIM32 mutant that cannot bind 14-3-3 underwent multimerization and was unavailable to facilitate cell growth. Our results reveal a novel connection between ubiquitylation and phosphorylation pathways, which could modulate a variety of cell events by stimulating the formation of the 14-3-3-TRIM32 signaling complex.

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.