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The norovirus genome consists of a single positive-stranded RNA. The mechanism by which this single-stranded RNA genome is replicated is not well understood. To reveal the mechanism underlying the initiation of the norovirus genomic RNA synthesis by its RNA-dependent RNA polymerase (RdRp), we used an in vitro assay to detect the complementary RNA synthesis activity. Results showed that the purified recombinant RdRp was able to synthesize the complementary positive-sense RNA from a 100-nt template corresponding to the 3'-end of the viral antisense genome sequence, but that the RdRp could not synthesize the antisense genomic RNA from the template corresponding to the 5'-end of the positive-sense genome sequence. We also predicted that the 31 nt region at the 3'-end of the RNA antisense template forms a stem-loop structure. Deletion of this sequence resulted in the loss of complementary RNA synthesis by the RdRp, and connection of the 31 nt to the 3'-end of the inactive positive-sense RNA template resulted in the gain of complementary RNA synthesis by the RdRp. Similarly, an electrophoretic mobility shift assay further revealed that the RdRp bound to the antisense RNA specifically, but was dependent on the 31 nt at the 3'-end. Therefore, based on this observation and further deletion and mutation analyses, we concluded that the predicted stem-loop structure in the 31 nt end and the region close to the antisense viral genomic stem sequences are both important for initiating the positive-sense human norovirus genomic RNA synthesis by its RdRp., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Down syndrome critical region (DSCR)-1 functions as a feedback modulator for calcineurin-nuclear factor for activated T cell (NFAT) signals, which are crucial for cell proliferation and inflammation. Stable expression of DSCR-1 inhibits pathological angiogenesis and septic inflammation. DSCR-1 also plays a critical role in vascular wall remodeling associated with aneurysm development that occurs primarily in smooth muscle cells. Besides, Dscr-1 deficiency promotes the M1-to M2-like phenotypic switch in macrophages, which correlates to the reduction of denatured cholesterol uptakes. However, the distinct roles of DSCR-1 in cholesterol and lipid metabolism are not well understood. Here, we show that loss of apolipoprotein (Apo) E in mice with chronic hypercholesterolemia induced Dscr-1 expression in the liver and aortic atheroma. In Dscr-1-null mice fed a high-fat diet, oxidative- and endoplasmic reticulum (ER) stress was induced, and sterol regulatory element-binding protein (SREBP) 2 production in hepatocytes was stimulated. This exaggerated ApoE -/- -mediated nonalcoholic fatty liver disease (NAFLD) and subsequent hypercholesterolemia. Genome-wide screening revealed that loss of both ApoE and Dscr-1 resulted in the induction of immune- and leukocyte activation-related genes in the liver compared with ApoE deficiency alone. However, expressions of inflammation-activated markers and levels of monocyte adhesion were suspended upon induction of the Dscr-1 null background in the aortic endothelium. Collectively, our study shows that the combined loss of Dscr-1 and ApoE causes metabolic dysfunction in the liver but reduces atherosclerotic plaques, thereby leading to a dramatic increase in serum cholesterol and the formation of sporadic vasculopathy., Competing Interests: Conflict of interest The authors have no competing financial interests to disclose., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatitis B, a viral infection that affects the liver, is thought to affect over 257 million people worldwide, and long-term infection can lead to life-threatening issues such as cirrhosis or liver cancer. Chronic hepatitis B develops by the interaction between hepatitis B virus (HBV) and host immune response. However, questions of how HBV-infected cells thwart immune system defenses remain unanswered. Extracellular vesicles (EVs) are used for cellular communication, carrying cargoes such as RNAs, proteins, and lipids and delivering them intracellularly after being endocytosed by target cells. HBV-infected liver cells secrete several types of EVs into body fluids such as complete and incomplete virions, and exosomes. We previously demonstrated that monocytes that incorporated EVs moved to immunoregulatory phenotypes via up-regulation of PD-L1, an immunocheckpoint molecule, and down-regulation of CD69, a leukocyte activation molecule. In this study, we transfected mice with HBV using hydrodynamic injection and studied the effects of EVs secreted by HBV-infected liver cells. EVs secreted from cells with HBV replication strongly suppressed the immune response, inhibiting the eradication of HBV-replicating cells in the mice transfected with HBV. EVs were systemically incorporated in multiple organs, including liver, bone marrow (BM), and intestine. Intriguingly, the BM cells that incorporated EVs acquired intestinal tropism and the dendritic cell populations in the intestine increased. These findings suggest that the EVs secreted by HBV-infected liver cells exert immunosuppressive functions, and that an association between the liver, bone marrow, and intestinal tract exists through EVs secreted from HBV-infected cells., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kakizaki et al.)

Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4β (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection., (© 2020 Iwamoto et al.)

Adenosine deaminases acting on RNA-1 (ADAR1) involves adenosine to inosine RNA editing and microRNA processing. ADAR1 is known to be involved in the replication of various viruses, including hepatitis C and D. However, the role of ADAR1 in hepatitis B virus (HBV) infection has not yet been elucidated. Here, for the first time, we demonstrated ADAR1 antiviral activity against HBV. ADAR1 has two splicing isoforms in human hepatocytes: constitutive p110 protein and interferon-α (IFN-α)-responsive p150 protein. We found that overexpression of ADAR1 decreased HBV RNA in an HBV culture model. A catalytic-site mutant ADAR1 also decreased HBV RNA levels, whereas another adenosine deaminases that act on the RNA (ADAR) family protein, ADAR2, did not. Moreover, the induction of ADAR1 by stimulation with IFN-α also reduced HBV RNA levels. Decreases in endogenous ADAR1 expression by knock-down or knock-out increased HBV RNA levels. A major hepatocyte-specific microRNA, miRNA-122, was found to be positively correlated with ADAR1 expression, and exogenous miRNA-122 decreased both HBV RNA and DNA, whereas, conversely, transfection with a miRNA-122 inhibitor increased them. The reduction of HBV RNA by ADAR1 expression was abrogated by p53 knock-down, suggesting the involvement of p53 in the ADAR1-mediated reduction of HBV RNA. This study demonstrated, for the first time, that ADAR1 plays an antiviral role against HBV infection by increasing the level of miRNA-122 in hepatocytes., (© 2019 Liu et al.)

Viruses hijack and modify host cell functions to maximize viral proliferation. Hepatitis C virus (HCV) reorganizes host cell metabolism to produce specialized membrane structures and to modify organelles such as double-membrane vesicles and enlarged lipid droplets (LDs), thereby enabling virus replication and assembly. However, the molecular bases of these host-HCV interactions are largely unknown. Here, using a chemical screen, we demonstrate that the benzamide derivative flutamide reduces the host capacity to produce infectious HCV. Flutamide disrupted the formation of enlarged LDs in HCV-infected cells, thereby abolishing HCV assembly. We also report that aryl hydrocarbon receptor (AhR), a known flutamide target, plays a key role in mediating LD accumulation and HCV production. This AhR function in lipid production was also observed in HCV-uninfected Huh-7 cells and primary human hepatocytes, suggesting that AhR signaling regulates lipid accumulation independently of HCV infection. We further observed that a downstream activity, that of cytochrome P450 1A1 (CYP1A1), was the primary regulator of AhR-mediated lipid production. Specifically, blockade of AhR-induced CYP1A1 up-regulation counteracted LD overproduction, and overproduction of CYP1A1, but not of CYP1B1, in AhR-inactivated cells restored lipid accumulation. Of note, HCV infection up-regulated the AhR-CYP1A1 pathway, resulting in the accumulation of enlarged LDs. In conclusion, we demonstrate that the AhR-CYP1A1 pathway has a significant role in lipid accumulation, a hallmark of HCV infection that maximizes progeny virus production. Our chemical-genetic analysis reveals a new strategy and lead compounds to control hepatic lipid accumulation as well as HCV infection., (© 2018 Ohashi et al.)

Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.

Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase Cγ-protein kinase C (PLCγ-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.

Activation-induced cytidine deaminase (AID) is essential to all three genetic alterations required for generation of antigen-specific immunoglobulin: class switch recombination, somatic hypermutation, and gene conversion. Here we demonstrate that AID molecules form a homodimer autonomously in the absence of RNA, DNA, other cofactors, or post-translational modifications. Studies on serial deletion mutants revealed the minimum region between Thr27 and His56 responsible for dimerization. Analyses of point mutations within this region revealed that the residues between Gly47 and Gly54 are most important for the dimer formation. Functional analyses of these mutations indicate that all mutations impairing the dimer formation are inefficient for class switching, suggesting that dimer formation is required for class switching activity. Dimer formation and its requirement for the function of AID are features that AID shares with APOBEC-1, an RNA editing enzyme of apolipoprotein B100 mRNA.

Sox2 is expressed at high levels in neuroepithelial stem cells and persists in neural stem/progenitor cells throughout adulthood. We showed previously that the Sox2 regulatory region 2 (SRR2) drives strong expression in these cells. Here we generated transgenic mouse strains with the beta-geo reporter gene under the control of the SRR2 in order to examine the spatiotemporal function of this regulatory region. We show that the SRR2 functions specifically in neural stem/progenitor cells. However, unlike Nestin 2nd intronic enhancer, the SRR2 shows strong regional specificity functioning only in restricted areas of the telencephalon but not in any other portions of the central nervous system such as the spinal cord. We also show by in vitro clonogenic assay that at least some of these SRR2-functioning cells possess the hallmark properties of neural stem cells. In adult brains, we could detect strong beta-geo expression in the subventricular zone of the lateral ventricle and along the rostral migrating stream where actively dividing cells reside. Chromatin immunoprecipitation assays reveal interactions of POU and Sox factors with SRR2 in neural stem/progenitor cells. Our data also suggest that the specific recruitment of these proteins to the SRR2 in the telencephalon defines the spatiotemporal activity of the enhancer in the developing nervous system.

The extraembryonic endoderm is derived from inner cell mass cells of the blastocyst during early mouse embryogenesis. Formation of the extraembryonic endoderm, which later contributes to the yolk sac, appears to be a prerequisite for subsequent differentiation of the inner cell mass. While embryonic stem cells can be induced to differentiate into extraembryonic endoderm cells in vitro, the molecular mechanisms underlying this process are poorly understood. We used a promoter trap approach to search for genes that are expressed in embryonic stem cells and are highly up-regulated during differentiation to the extraembryonic endoderm fate. We showed that fibronectin fits this expression profile. Moreover we identified an enhancer in the 12th intron of the fibronectin locus that recapitulated the endogenous pattern of fibronectin expression. This enhancer carries Sox protein-binding sequences, and our analysis demonstrated that Sox7 and Sox17, which are highly expressed in the extraembryonic endoderm, were involved in enhancer activity.

2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2) and has been shown to exhibit a variety of cannabimimetic activities in vitro and in vivo. Recently, we proposed that 2-arachidonoylglycerol is the true endogenous ligand for the cannabinoid receptors, and both receptors (CB1 and CB2) are primarily 2-arachidonoylglycerol receptors. The CB1 receptor is assumed to be involved in the attenuation of neurotransmission. On the other hand, the physiological roles of the CB2 receptor, which is abundantly expressed in several types of leukocytes such as macrophages, still remain unknown. In this study, we examined the effects of 2-arachidonoylglycerol on the motility of HL-60 cells differentiated into macrophage-like cells. We found that 2-arachidonoylglycerol induces the migration of differentiated HL-60 cells. The migration induced by 2-arachidonoylglycerol was blocked by treatment of the cells with either SR144528, a CB2 receptor antagonist, or pertussis toxin, suggesting that the CB2 receptor and Gi/Go are involved in the 2-arachidonoylglycerol-induced migration. Several intracellular signaling molecules such as Rho kinase and mitogen-activated protein kinases were also suggested to be involved. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, failed to induce the migration. The 2-arachidonoylglycerol-induced migration was also observed for two other types of macrophage-like cells, the U937 cells and THP-1 cells, as well as human peripheral blood monocytes. These results strongly suggest that 2-arachidonoylglycerol induces the migration of several types of leukocytes such as macrophages/monocytes through a CB2 receptor-dependent mechanism thereby stimulating inflammatory reactions and immune responses.

We examine in vitro nucleosome assembly by nucleosome assembly protein-1 (NAP-1) and ATP-utilizing chromatin assembly and remodeling factor (ACF). In contrast to previous studies that used relaxed, circular plasmids as templates, we have found that negatively supercoiled templates reveal the distinct roles of NAP-1 and ACF in histone deposition and the formation of an ordered nucleosomal array. NAP-1 can efficiently deposit histones onto supercoiled plasmids. Furthermore, NAP-1 exhibits a greater affinity for histones H2A-H2B than does naked DNA, but in the presence of H3-H4, H2A-H2B are transferred from NAP-1 to the plasmid templates. These observations underscore the importance of a high affinity between H2A-H2B and NAP-1 for ordered transfer of core histones onto DNA. In addition, recombinant ACF composed of imitation switch and Acf1 can extend closely packed nucleosomes, which suggests that recombinant ACF can mobilize nucleosomes. In the assembly reaction with a supercoiled template, ACF need not be added simultaneously with NAP-1. Regularly spaced nucleosomes are generated even when recombinant ACF is added after core histones are transferred completely onto the DNA. Atomic force microscopy, however, suggests that NAP-1 alone fails to accomplish the formation of fine nucleosomal core particles, which are only formed in the presence of ACF. These results suggest a model for the ordered deposition of histones and the arrangement of nucleosomes during chromatin assembly in vivo.

Although it is well established that estrogen deficiency causes osteoporosis among the postmenopausal women, the involvement of estrogen receptor (ER) in its pathogenesis still remains uncertain. In the present study, we have generated rats harboring a dominant negative ERalpha, which inhibits the actions of not only ERalpha but also recently identified ERbeta. Contrary to our expectation, the bone mineral density (BMD) of the resulting transgenic female rats was maintained at the same level with that of the wild-type littermates when sham-operated. In addition, ovariectomy-induced bone loss was observed almost equally in both groups. Strikingly, however, the BMD of the transgenic female rats, after ovariectomized, remained decreased even if 17beta-estradiol (E(2)) was administrated, whereas, in contrast, the decrease of littermate BMD was completely prevented by E(2). Moreover, bone histomorphometrical analysis of ovariectomized transgenic rats revealed that the higher rates of bone turnover still remained after treatment with E(2). These results demonstrate that the prevention from the ovariectomy-induced bone loss by estrogen is mediated by ER pathways and that the maintenance of BMD before ovariectomy might be compensated by other mechanisms distinct from ERalpha and ERbeta pathways.

Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM-binding peptide of alphaCaM-KK (residues 438-463), which suppressed the activity of constitutively active CaM-KK (84-434) in the absence of Ca(2+)/CaM but competitively with ATP. Truncation and site-directed mutagenesis of the CaM-binding region in CaM-KK reveal that Ile(441) is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu(440), exhibited enhanced Ca(2+)/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, M. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol. 6, 819-824), the chimeric CaM-KKs containing Ile(441) remained Ca(2+)/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile(441) for autoinhibition, which is located at the -3 position from the N-terminal anchoring residue (Trp(444)) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.

We have previously identified a silencer in the glutathione S-transferase P (GST-P) gene which is strongly and specifically expressed during chemical hepatocarcinogenesis. At least three trans-acting factors bind to multiple cis-elements in the silencer. One of them, Silencer Factor-B (SF-B), is identical with CCAAT/enhancer-binding protein beta (C/EBP beta) and binds to GST-P Silencer 1 (GPS1). Many C/EBP beta binding sites are recognized by each of the C/EBP isoforms. Western blot analyses of C/EBP isoforms during chemical hepatocarcinogenesis revealed a decrease of C/EBP alpha expression. However, there was no change in C/EBP beta level. In the nuclear extracts from normal liver, C/EBP alpha was the dominant form that bound to GPS1, whereas both C/EBP alpha and C/EBP beta bound to GPS1 in the nuclear extracts from carcinogenic liver. Furthermore, transfection assays showed that C/EBP alpha not only repressed the GST-P promoter activity but also attenuated the transcriptional stimulation by C/EBP beta. These observations strongly suggest that the ratio of C/EBP alpha to C/EBP beta is one of the important factors for the GST-P silencer activity, and the decrease of this ratio during hepatocarcinogenesis reduces the silencer activity and, consequently, increases the GST-P expression.