Identification and epidemiological study of an uncultured flavivirus from ticks using viral metagenomics and pseudoinfectious viral particles.

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.

Loxapine inhibits replication of hepatitis A virus in vitro and in vivo by targeting viral protein 2C.

No antiviral drugs currently are available for treatment of infection by hepatitis A virus (HAV), a causative agent of acute hepatitis, a potentially life-threatening disease. Chemical screening of a small-compound library using nanoluciferase-expressing HAV identified loxapine succinate, a selective dopamine receptor D2 antagonist, as a potent inhibitor of HAV propagation in vitro. Loxapine succinate did not inhibit viral entry nor internal ribosome entry site (IRES)-dependent translation, but exhibited strong inhibition of viral RNA replication. Blind passage of HAV in the presence of loxapine succinate resulted in the accumulation of viruses containing mutations in the 2C-encoding region, which contributed to resistance to loxapine succinate. Analysis of molecular dynamics simulations of the interaction between 2C and loxapine suggested that loxapine binds to the N-terminal region of 2C, and that resistant mutations impede these interactions. We further demonstrated that administration of loxapine succinate to HAV-infected Ifnar1-/- mice (which lack the type I interferon receptor) results in decreases in the levels of fecal HAV RNA and of intrahepatic HAV RNA at an early stage of infection. These findings suggest that HAV protein 2C is a potential target for antivirals, and provide novel insights into the development of drugs for the treatment of hepatitis A.

Dark accumulation of downstream glycolytic intermediates initiates robust photosynthesis in cyanobacteria.

Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies have shown that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid dynamic changes (on a time scale of seconds) in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enabled estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicated that downstream glycolytic intermediates, including phosphoglycerate and phosphoenolpyruvate, accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant decreases in the initial oxygen evolution rate were observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathways. Accordingly, the degree of decrease in the initial oxygen evolution rate was proportional to the accumulated pool size of glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.

Expression of dengue capsid-like particles in silkworm and display of envelope domain III of dengue virus serotype 2.

Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.

The Carbon Flow Shifts from Primary to Secondary Metabolism during Xylem Vessel Cell Differentiation in Arabidopsis thaliana.

Xylem vessel cell differentiation is characterized by the deposition of a secondary cell wall (SCW) containing cellulose, hemicellulose and lignin. VASCULAR-RELATED NAC-DOMAIN7 (VND7), a plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor, is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). Previous metabolome analysis using the VND7-inducible system in tobacco BY-2 cells successfully revealed significant quantitative changes in primary metabolites during xylem vessel cell differentiation. However, the flow of primary metabolites is not yet well understood. Here, we performed a metabolomic analysis of VND7-inducible Arabidopsis T87 suspension cells. Capillary electrophoresis-time-of-flight mass spectrometry quantified 57 metabolites, and subsequent data analysis highlighted active changes in the levels of UDP-glucose and phenylalanine, which are building blocks of cellulose and lignin, respectively. In a metabolic flow analysis using stable carbon 13 (13C) isotope, the 13C-labeling ratio specifically increased in 3-phosphoglycerate after 12 h of VND7 induction, followed by an increase in shikimate after 24 h of induction, while the inflow of 13C into lactate from pyruvate was significantly inhibited, indicating an active shift of carbon flow from glycolysis to the shikimate pathway during xylem vessel cell differentiation. In support of this notion, most glycolytic genes involved in the downstream of glyceraldehyde 3-phosphate were downregulated following the induction of xylem vessel cell differentiation, whereas genes for the shikimate pathway and phenylalanine biosynthesis were upregulated. These findings provide evidence for the active shift of carbon flow from primary metabolic pathways to the SCW polymer biosynthetic pathway at specific points during xylem vessel cell differentiation.

Macrophage Depletion Reactivates Fecal Virus Shedding following Resolution of Acute Hepatitis A in Ifnar1-/- Mice.

Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1-/- mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm3 in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4+ or CD8+ T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifnγ, Tnfα, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1-/- mice and that macrophages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1-/- mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1-/- mice and may have relevance to relapsing hepatitis A in humans.

IRF3-mediated pathogenicity in a murine model of human hepatitis A.

HAV-infected Ifnar1-/- mice recapitulate many of the cardinal features of hepatitis A in humans, including serum alanine aminotransferase (ALT) elevation, hepatocellular apoptosis, and liver inflammation. Previous studies implicate MAVS-IRF3 signaling in pathogenesis, but leave unresolved the role of IRF3-mediated transcription versus the non-transcriptional, pro-apoptotic activity of ubiquitylated IRF3. Here, we compare the intrahepatic transcriptomes of infected versus naïve Mavs-/- and Ifnar1-/- mice using high-throughput sequencing, and identify IRF3-mediated transcriptional responses associated with hepatocyte apoptosis and liver inflammation. Infection was transcriptionally silent in Mavs-/- mice, in which HAV replicates robustly within the liver without inducing inflammation or hepatocellular apoptosis. By contrast, infection resulted in the upregulation of hundreds of genes in Ifnar1-/- mice that develop acute hepatitis closely modeling human disease. Upregulated genes included pattern recognition receptors, interferons, chemokines, cytokines and other interferon-stimulated genes. Compared with Ifnar1-/- mice, HAV-induced inflammation was markedly attenuated and there were few apoptotic hepatocytes in livers of infected Irf3S1/S1Ifnar1-/- mice in which IRF3 is transcriptionally-inactive due to alanine substitutions at Ser-388 and Ser-390. Although transcriptome profiling revealed remarkably similar sets of genes induced in Irf3S1/S1Ifnar1-/- and Ifnar1-/- mice, a subset of genes was differentially expressed in relation to the severity of the liver injury. Prominent among these were both type 1 and type III interferons and interferon-responsive genes associated previously with apoptosis, including multiple members of the ISG12 and 2'-5' oligoadenylate synthetase families. Ifnl3 and Ifnl2 transcript abundance correlated strongly with disease severity, but mice with dual type 1 and type III interferon receptor deficiency remained fully susceptible to liver injury. Collectively, our data show that IRF3-mediated transcription is required for HAV-induced liver injury in mice and identify key IRF3-responsive genes associated with pathogenicity, providing a clear distinction from the transcription-independent role of IRF3 in liver injury following binge exposure to alcohol.

Enhanced production of 3,4-dihydroxybutyrate from xylose by engineered yeast via xylonate re-assimilation under alkaline condition.

To realize lignocellulose-based bioeconomy, efficient conversion of xylose into valuable chemicals by microbes is necessary. Xylose oxidative pathways that oxidize xylose into xylonate can be more advantageous than conventional xylose assimilation pathways because of fewer reaction steps without loss of carbon and ATP. Moreover, commodity chemicals like 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone can be produced from the intermediates of xylose oxidative pathway. However, successful implementations of xylose oxidative pathway in yeast have been hindered because of the secretion and accumulation of xylonate which is a key intermediate of the pathway, leading to low yield of target product. Here, high-yield production of 3,4-dihydroxybutyrate from xylose by engineered yeast was achieved through genetic and environmental perturbations. Specifically, 3,4-dihydroxybutyrate biosynthetic pathway was established in yeast through deletion of ADH6 and overexpression of yneI. Also, inspired by the mismatch of pH between host strain and key enzyme of XylD, alkaline fermentations (pH ≥ 7.0) were performed to minimize xylonate accumulation. Under the alkaline conditions, xylonate was re-assimilated by engineered yeast and combined product yields of 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone resulted in 0.791 mol/mol-xylose, which is highest compared with previous study. These results shed light on the utility of the xylose oxidative pathway in yeast.

Hepatocellular organellar abnormalities following elimination of hepatitis C virus.

BACKGROUND AND AIMS: The future development of hepatocellular carcinoma (HCC) in patients after sustained virologic response (SVR) is an important issue. The purposes of this study were to investigate pathological alterations in organelle of the liver of SVR patients and to characterize organelle abnormalities that may be related to carcinogenesis after SVR. METHODS: The ultrastructure of liver biopsy specimens from patients with chronic hepatitis C (CHC) and SVR were compared to cell and mouse models and assessed semi-quantitatively using transmission electron microscopy. RESULTS: Hepatocytes in patients with CHC showed abnormalities in the nucleus, mitochondria, endoplasmic reticulum, lipid droplet, and pericellular fibrosis, comparable to those seen in hepatitis C virus (HCV)-infected mice and cells. DAA treatment significantly reduced organelle abnormalities such as the nucleus, mitochondria, and lipid droplet in the hepatocytes of patients and mice after SVR, and cured cells, but it did not change dilated/degranulated endoplasmic reticulum and pericellular fibrosis in patients and mice after SVR. Further, samples from patients with a post-SVR period of >1 year had significantly larger numbers of abnormalities in the mitochondria and endoplasmic reticulum than those of <1 year. A possible cause of organelle abnormalities in patients after SVR could be oxidative stress of the endoplasmic reticulum and mitochondria associated with abnormalities of the vascular system due to fibrosis. Interestingly, abnormal endoplasmic reticulum was associated with patients with HCC for >1 year after SVR. CONCLUSIONS: These results indicate that patients with SVR exhibit a persistent disease state and require long-term follow-up to detect early signs of carcinogenesis.

Enhanced supply of acetyl-CoA by exogenous pantothenate kinase promotes synthesis of poly(3-hydroxybutyrate).

BACKGROUND: Coenzyme A (CoA) is a carrier of acyl groups. This cofactor is synthesized from pantothenic acid in five steps. The phosphorylation of pantothenate is catalyzed by pantothenate kinase (CoaA), which is a key step in the CoA biosynthetic pathway. To determine whether the enhancement of the CoA biosynthetic pathway is effective for producing useful substances, the effect of elevated acetyl-CoA levels resulting from the introduction of the exogenous coaA gene on poly(3-hydroxybutyrate) [P(3HB)] synthesis was determined in Escherichia coli, which express the genes necessary for cyanobacterial polyhydroxyalkanoate synthesis (phaABEC). RESULTS: E. coli containing the coaA gene in addition to the pha genes accumulated more P(3HB) compared with the transformant containing the pha genes alone. P(3HB) production was enhanced by precursor addition, with P(3HB) content increasing from 18.4% (w/w) to 29.0% in the presence of 0.5 mM pantothenate and 16.3%-28.2% by adding 0.5 mM ß-alanine. Strains expressing the exogenous coaA in the presence of precursors contained acetyl-CoA in excess of 1 nmol/mg of dry cell wt, which promoted the reaction toward P(3HB) formation. The amount of acetate exported into the medium was three times lower in the cells carrying exogenous coaA and pha genes than in the cells carrying pha genes alone. This was attributed to significantly enlarging the intracellular pool size of CoA, which is the recipient of acetic acid and is advantageous for microbial production of value-added materials. CONCLUSIONS: Enhancing the CoA biosynthetic pathway with exogenous CoaA was effective at increasing P(3HB) production. Supplementing the medium with pantothenate facilitated the accumulation of P(3HB). ß-Alanine was able to replace the efficacy of adding pantothenate.

Identification of Two Critical Neutralizing Epitopes in the Receptor Binding Domain of Hepatitis B Virus preS1.

Hepatitis B virus (HBV) infection is a major public health problem. Human hepatocytes are infected with HBV via binding between the preS1 region in the large envelope protein of HBV and sodium taurocholate cotransporting polypeptide. Although several monoclonal antibodies (MAbs) that recognize the receptor binding domain in preS1 and neutralize HBV infection have been isolated, details of neutralizing epitopes are not understood. In this study, we generated 13 MAbs targeting the preS1 receptor binding domain from preS1-specific memory B cells derived from DNA immunized mice. The MAbs were classified into three groups according to the epitope regions, designated epitopes I-III. A virus neutralization assay revealed that MAbs recognizing epitopes I and III neutralized HBV infection, suggesting that these domains are critical epitopes for viral neutralization. In addition, a neutralization assay against multiple genotypes of HBV revealed that epitope I is a semi-pangenotypic neutralizing epitope, whereas epitope III is a genotype-specific epitope. We also showed that neutralizing MAbs against preS1 could neutralize HBV bearing vaccine-induced escape mutation. These findings provide insight into novel immunoprophylaxis for the prevention and treatment of HBV infection.IMPORTANCE The HBV preS1 2-47 aa region (preS1/2-47) is essential for virus binding with sodium taurocholate cotransporting polypeptide. Several MAbs targeting preS1/2-47 have been reported to neutralize HBV infection; however, which region in preS1/2-47 contains the critical neutralizing epitope for HBV infection is unclear. Here, we generated several MAbs targeting preS1/2-47 and found that MAbs recognizing the N- or C-terminus of preS1/2-47 remarkably neutralized HBV infection. We further confirmed the neutralizing activity of anti-preS1 MAbs against HBV with vaccine escape mutation. These data clarified the relationship between the antibody epitope and the virus neutralizing activity and also suggested the potential ability of a vaccine antigen containing the preS1 region to overcome the weakness of current HB vaccines comprising the small S protein.

N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus.

BACKGROUND AND AIMS: An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. APPROACH AND RESULTS: We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. CONCLUSIONS: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.

Surfeit 4 Contributes to the Replication of Hepatitis C Virus Using Double-Membrane Vesicles.

A number of positive-strand RNA viruses, such as hepatitis C virus (HCV) and poliovirus, use double-membrane vesicles (DMVs) as replication sites. However, the role of cellular proteins in DMV formation during virus replication is poorly understood. HCV NS4B protein induces the formation of a "membranous web" structure that provides a platform for the assembly of viral replication complexes. Our previous screen of NS4B-associated host membrane proteins by dual-affinity purification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and small interfering RNA (siRNA) methods revealed that the Surfeit 4 (Surf4) gene, which encodes an integral membrane protein, is involved in the replication of the JFH1 subgenomic replicon. Here, we investigated in detail the effect of Surf4 on HCV replication. Surf4 affects HCV replication in a genotype-independent manner, whereas HCV replication does not alter Surf4 expression. The influence of Surf4 on HCV replication indicates that while Surf4 regulates replication, it has no effect on entry, translation, assembly, or release. Analysis of the underlying mechanism showed that Surf4 is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs and the structural integrity of RNA replication complexes. Surf4 also participates in the replication of poliovirus, which uses DMVs as replication sites, but it has no effect on the replication of dengue virus, which uses invaginated/sphere-type vesicles as replication sites. These findings clearly show that Surf4 is a novel cofactor that is involved in the replication of positive-strand RNA viruses using DMVs as RNA replication sites, which provides valuable clues for DMV formation during positive-strand RNA virus replication.IMPORTANCE Hepatitis C virus (HCV) NS4B protein induces the formation of a membranous web (MW) structure that provides a platform for the assembly of viral replication complexes. The main constituents of the MW are double-membrane vesicles (DMVs). Here, we found that the cellular protein Surf4, which maintains endoplasmic reticulum (ER)-Golgi intermediate compartments and the Golgi compartment, is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs. Moreover, Surf4 participates in the replication of poliovirus, which uses DMVs as replication sites, but has no effect on the replication of dengue virus, which uses invaginated vesicles as replication sites. These results indicate that the cellular protein Surf4 is involved in the replication of positive-strand RNA viruses that use DMVs as RNA replication sites, providing new insights into DMV formation during virus replication and potential targets for the diagnosis and treatment of positive-strand RNA viruses.

Optimization of 1,2,4-butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance.

1,2,4-Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2-ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2-keto-3-deoxy-xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2-ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.

Adenosine deaminase acting on RNA-1 (ADAR1) inhibits hepatitis B virus (HBV) replication by enhancing microRNA-122 processing.

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.

Involvement of the 3' Untranslated Region in Encapsidation of the Hepatitis C Virus.

Although information regarding morphogenesis of the hepatitis C virus (HCV) is accumulating, the mechanism(s) by which the HCV genome encapsidated remains unknown. In the present study, in cell cultures producing HCV, the molecular ratios of 3' end- to 5' end-regions of the viral RNA population in the culture medium were markedly higher than those in the cells, and the ratio was highest in the virion-rich fraction. The interaction of the 3' untranslated region (UTR) with Core in vitro was stronger than that of the interaction of other stable RNA structure elements across the HCV genome. A foreign gene flanked by the 3' UTR was encapsidated by supplying both viral NS3-NS5B proteins and Core-NS2 in trans. Mutations within the conserved stem-loops of the 3' UTR were observed to dramatically diminish packaging efficiency, suggesting that the conserved apical motifs of the 3´ X region are important for HCV genome packaging. This study provides evidence of selective packaging of the HCV genome into viral particles and identified that the 3' UTR acts as a cis-acting element for encapsidation.

Temperature enhanced succinate production concurrent with increased central metabolism turnover in the cyanobacterium Synechocystis sp. PCC 6803.

Succinate is a versatile petrochemical compound that can be produced by microorganisms, often from carbohydrate based carbon sources. Phototrophic cyanobacteria including Synechocystis sp. PCC 6803 can more efficiently produce organic acids such as succinate without sugar supplementation, via photosynthetic production of glycogen followed by glycogen utilization, typically under dark conditions. In this study, Synechocystis 6803 bioproduction of organic acids under dark anoxic conditions was found to increase with elevation of temperature from 30 °C to 37 °C. The further enhancement of succinate bioproduction by overexpression of the rate limiting enzyme phosphoenolpyruvate carboxylase resulted in improved glycogen utilization. To gain more insight into the mechanisms underlying the increased organic acid output, a novel temperature dependent metabolomics analysis was performed. Adenylate energy charge was found to decrease along with elevating temperature, while central metabolites glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, glycerol 3-phosphate, malate, fumarate and succinate increased. Temperature dependent 13C-labeling metabolomics analysis further revealed a glycolysis to TCA bottleneck, which could be overcome by addition of CO2, leading to even higher organic acid production. Optimization of initial cell concentration to 25 g-dry cell weight/L, in combination with 100 mM NaHCO3 supplementation, afforded a succinate titer of over 1.8 g/L, the highest reported autotrophic succinate titer. Succinate titers remained high after additional knockout of ackA, resulting in the highest reported autotrophic D-lactate titer as well. The optimization of Synechocystis 6803 organic acid production therefore holds significant promise for CO2 capture and utilization.

Widespread effect of N-acetyl-D-glucosamine assimilation on the metabolisms of amino acids, purines, and pyrimidines in Scheffersomyces stipitis.

BACKGROUND: Following cellulose, chitin is the most abundant renewable resource and is composed of the monomeric amino sugar N-acetyl-D-glucosamine (GlcNAc). Although many yeasts, including Saccharomyces cerevisiae, have lost their ability to utilize GlcNAc, some yeasts are able to use GlcNAc as a carbon source. However, our understanding of the effects of GlcNAc on the intracellular metabolism of nitrogen-containing compounds in these yeast species is limited. RESULTS: In the present study, we quantitatively investigated the metabolic responses to GlcNAc in the GlcNAc-assimilating yeast Scheffersomyces stipitis (formerly known as Pichia stipitis) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The comprehensive analysis of the metabolites extracted from S. stipitis cells grown in glucose, xylose, or GlcNAc revealed increased intracellular accumulation of a wide range of nitrogen-containing compounds during GlcNAc assimilation in this yeast. The levels of aromatic, branched-chain, and sulfur-containing amino acids and adenine, guanine, and cytosine nucleotides were the highest in GlcNAc-grown cells. CONCLUSIONS: The CE-TOFMS analysis revealed a positive effect for GlcNAc on the intracellular concentration of a wide range of nitrogen-containing compounds. The metabolomic data gathered in this study will be useful for designing effective genetic engineering strategies to develop novel S. stipitis strains for the production of valuable nitrogen-containing compounds from GlcNAc.

Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B.

UNLABELLED: It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCE: The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors.

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements.

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell's metabolic activity for the biosynthesis of secondary wall polymers.