Hepatitis C virus utilizes VLDLR as a novel entry pathway.

Various host factors are involved in the cellular entry of hepatitis C virus (HCV). In addition to the factors previously reported, we discovered that the very-low-density lipoprotein receptor (VLDLR) mediates HCV entry independent of CD81. Culturing Huh7.5 cells under hypoxic conditions significantly increased HCV entry as a result of the expression of VLDLR, which was not expressed under normoxic conditions in this cell line. Ectopic VLDLR expression conferred susceptibility to HCV entry of CD81-deficient Huh7.5 cells. Additionally, VLDLR-mediated HCV entry was not affected by the knockdown of cellular factors known to act as HCV receptors or HCV entry factors. Because VLDLR is expressed in primary human hepatocytes, our results suggest that VLDLR functions in vivo as an HCV receptor independent of canonical CD81-mediated HCV entry.

[Optimization of Scan Parameters for Patient without Breath Hold in Chest by Computed Tomography].

This study aims to establish optimal scan parameters by high temporal resolution computed tomography (CT) scan for emergency patients who cannot hold their breath. First, we investigated scan parameters that can reduce the effect of motion by evaluating motion artifacts from the moving phantom scan and the temporal sensitivity profile (TPS) measurement. Second, we confirmed the standard deviation (SD) of the CT values as well as the operating time and exposure dose. As the results, plan C [rotation time: 0.275 s, detector rows: 80, pitch factor (PF): 1.100] and plan E (rotation time: 0.275 s, detector rows: 100, PF: 0.880) demonstrated high temporal resolution. The difference between the two is PF. The noise of plan C increased because PF is higher than plan E. This is also evident from the results of SD measurement. Our study demonstrates that the optimal parameters for patients who cannot hold their breath in the emergency care are plan C and plan E. In conclusion, we clarified necessary optimal scan parameters to provide clinical image that has more diagnostic information by reducing the effect of breath motion for emergency patients.

ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2.

Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation.

The RNA-editing enzyme APOBEC1 requires heterogeneous nuclear ribonucleoprotein Q isoform 6 for efficient interaction with interleukin-8 mRNA.

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

Hepatitis C virus infection induces inflammatory cytokines and chemokines mediated by the cross talk between hepatocytes and stellate cells.

Inflammatory cytokines and chemokines play important roles in inflammation during viral infection. Hepatitis C virus (HCV) is a hepatotropic RNA virus that is closely associated with chronic liver inflammation, fibrosis, and hepatocellular carcinoma. During the progression of HCV-related diseases, hepatic stellate cells (HSCs) contribute to the inflammatory response triggered by HCV infection. However, the underlying molecular mechanisms that mediate HSC-induced chronic inflammation during HCV infection are not fully understood. By coculturing HSCs with HCV-infected hepatocytes in vitro, we found that HSCs stimulated HCV-infected hepatocytes, leading to the expression of proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), IL-8, macrophage inflammatory protein 1α (MIP-1α), and MIP-1ß. Moreover, we found that this effect was mediated by IL-1α, which was secreted by HSCs. HCV infection enhanced production of CCAAT/enhancer binding protein (C/EBP) ß mRNA, and HSC-dependent IL-1α production contributed to the stimulation of C/EBPß target cytokines and chemokines in HCV-infected hepatocytes. Consistent with this result, knockdown of mRNA for C/EBPß in HCV-infected hepatocytes resulted in decreased production of cytokines and chemokines after the addition of HSC conditioned medium. Induction of cytokines and chemokines in hepatocytes by the HSC conditioned medium required a yet to be identified postentry event during productive HCV infection. The cross talk between HSCs and HCV-infected hepatocytes is a key feature of inflammation-mediated, HCV-related diseases.

Structure, interaction and real-time monitoring of the enzymatic reaction of wild-type APOBEC3G.

Human APOBEC3G exhibits anti-human immunodeficiency virus-1 (HIV-1) activity by deaminating cytidines of the minus strand of HIV-1. Here, we report a solution structure of the C-terminal deaminase domain of wild-type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real-time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3' --> 5' order. Virus infectivity factor (Vif) of HIV-1 counteracts the anti-HIV-1 activity of APOBEC3G. The structure of the N-terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species-specific sensitivity of APOBEC3G to Vif action.

HIV-1 RT-dependent DNAzyme expression inhibits HIV-1 replication without the emergence of escape viruses.

DNAzymes are easier to prepare and less sensitive to chemical and enzymatic degradation than ribozymes; however, a DNA enzyme expression system has not yet been developed. In this study, we exploited the mechanism of HIV-1 reverse transcription (RT) in a DNA enzyme expression system. We constructed HIV-1 RT-dependent lentiviral DNAzyme expression vectors including the HIV-1 primer binding site, the DNA enzyme, and either a native tRNA (Lys-3), tR(M)DtR(L), or one of two truncated tRNAs (Lys-3), tR(M)DΔARMtR(L) or tR(M)D3'-endtR(L). Lentiviral vector-mediated DNAzyme expression showed high levels of inhibition of HIV-1 replication in SupT1 cells. We also demonstrated the usefulness of this approach in a long-term assay, in which we found that the DNAzymes prevented escape from inhibition of HIV. These results suggest that HIV-1 RT-dependent lentiviral vector-derived DNAzymes prevent the emergence of escape mutations.

Heat shock protein 70 inhibits HIV-1 Vif-mediated ubiquitination and degradation of APOBEC3G.

The cytidine deaminase APOBEC3G, which is incorporated into nascent virus particles, possesses potent antiviral activity and restricts Vif-deficient HIV-1 replication at the reverse transcription step through deamination-dependent and -independent effects. HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inducing APOBEC3G polyubiquitination and its subsequent proteasomal degradation. In this study, we show that overexpression of heat shock protein 70 (HSP70) blocked the degradation of APOBEC3G in the ubiquitin-proteasome pathway by HIV-1 Vif, rendering the viral particles non-infectious. In addition, siRNA targeted knock-down of HSP70 expression enhanced the Vif-mediated degradation of APOBEC3G. A co-immunoprecipitation study revealed that overexpression of HSP70 inhibited APOBEC3G binding to HIV-1 Vif. Thus, we provide evidence for a host protein-mediated suppression of HIV-1 replication in an APOBEC3G-dependent manner.

Evaluating target silencing by short hairpin RNA mediated by the group I intron in cultured mammalian cells.

BACKGROUND: The group I intron, a ribozyme that catalyzes its own splicing reactions in the absence of proteins in vitro, is a potential target for rational engineering and attracted our interest due to its potential utility in gene repair using trans-splicing. However, the ribozyme activity of a group I intron appears to be facilitated by RNA chaperones in vivo; therefore, the efficiency of self-splicing could be dependent on the structure around the insert site or the length of the sequence to be inserted. To better understand how ribozyme activity could be modulated in cultured mammalian cells, a group I intron was inserted into a short hairpin RNA (shRNA), and silencing of a reporter gene by the shRNA was estimated to reflect self-splicing activity in vivo. In addition, we appended a theophylline-binding aptamer to the ribozyme to investigate any potential effects caused by a trans-effector. RESULTS: shRNA-expression vectors in which the loop region of the shRNA was interrupted by an intron were constructed to target firefly luciferase mRNA. There was no remarkable toxicity of the shRNA-expression vectors in Cos cells, and the decrease in luciferase activity was measured as an index of the ribozyme splicing activity. In contrast, the expression of the shRNA through intron splicing was completely abolished in 293T cells, although the silencing induced by the shRNA-expressing vector alone was no different from that in the Cos cells. The splicing efficiency of the aptamer-appended intron also had implications for the potential of trans-factors to differentially promote self-splicing among cultured mammalian cells. CONCLUSIONS: Silencing by shRNAs interrupted by a group I intron could be used to monitor self-splicing activity in cultured mammalian cells, and the efficiency of self-splicing appears to be affected by cell-type specific factors, demonstrating the potential effectiveness of a trans-effector.

Infectivity of hepatitis C virus is influenced by association with apolipoprotein E isoforms.

Hepatitis C virus (HCV) is a causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV in circulating blood associates with lipoproteins such as very low density lipoprotein (VLDL) and low-density lipoprotein (LDL). Although these associations suggest that lipoproteins are important for HCV infectivity, the roles of lipoproteins in HCV production and infectivity are not fully understood. To clarify the roles of lipoprotein in the HCV life cycle, we analyzed the effect of apolipoprotein E (ApoE), a component of lipoprotein, on virus production and infectivity. The production of infectious HCV was significantly reduced by the knockdown of ApoE. When an ApoE mutant that fails to be secreted into the culture medium was used, the amount of infectious HCV in the culture medium was dramatically reduced; the infectious HCV accumulated inside these cells, suggesting that infectious HCV must associate with ApoE prior to virus release. We performed rescue experiments in which ApoE isoforms were ectopically expressed in cells depleted of endogenous ApoE. The ectopic expression of the ApoE2 isoform, which has low affinity for the LDL receptor (LDLR), resulted in poor recovery of infectious HCV, whereas the expression of other isoforms, ApoE3 and ApoE4, rescued the production of infectious virus, raising it to an almost normal level. Furthermore, we found that the infectivity of HCV required both the LDLR and scavenger receptor class B, member I (SR-BI), ligands for ApoE. These findings indicate that ApoE is an essential apolipoprotein for HCV infectivity.

Genetic analysis of hepatitis C virus with defective genome and its infectivity in vitro.

Replication and infectivity of hepatitis C virus (HCV) with a defective genome is ambiguous. We molecularly cloned 38 HCV isolates with defective genomes from 18 patient sera. The structural regions were widely deleted, with the 5' untranslated, core, and NS3-NS5B regions preserved. All of the deletions were in frame, indicating that they are translatable to the authentic terminus. Phylogenetic analyses showed self-replication of the defective genomes independent of full genomes. We generated a defective genome of chimeric HCV to mimic the defective isolate in the serum. By using this, we demonstrated for the first time that the defective genome, as it is circulating in the blood, can be encapsidated as an infectious particle by trans complementation of the structural proteins.

Baculovirus activates murine dendritic cells and induces non-specific NK cell and T cell immune responses.

We previously reported that the baculovirus induced a strong host immune response against infections and malignancies. Among the immune cells, the dendritic cells were most strongly infected and activated by the baculovirus, although the exact mechanism remained unclear. Here, we evaluated the non-specific immune responses of bone marrow-derived dendritic cells (BMDCs) after infection by a wild-type baculovirus. MHC class I and II molecules and co-stimulation molecules (CD40, CD80, and CD86) on BMDCs were up-regulated by baculovirus infection. At the same time, the BMDCs produced pre-inflammatory cytokines (IL-6, IL12p70, and TNF-alpha) and IFN-alpha. NK cells showed IFN-gamma production, CD69 up-regulation, and enhanced cytotoxicity when they were co-cultured with baculovirus-infected BMDCs. T cells showed IFN-gamma production, CD69 up-regulation, and cell proliferation. Ex vivo analysis performed in vitro produced similar results. These findings suggested that baculovirus-infected dendritic cells induce non-specific immune responses and can be used as an immunotherapeutic agent against viral infections and malignancies, together with present therapeutic drug regimens.

Short-hairpin RNAs synthesized by T7 phage polymerase do not induce interferon.

RNA interference (RNAi) mediated by small-interfering RNAs (siRNAs) is a highly effective gene-silencing mechanism with great potential for gene-therapeutic applications. siRNA agents also exert non-target-related biological effects and toxicities, including immune-system stimulation. Specifically, siRNA synthesized from the T7 RNA polymerase system triggers a potent induction of type-I interferon (IFN) in a variety of cells. Single-stranded RNA also stimulates innate cytokine responses in mammals. We found that pppGn (n = 2,3) associated with the 5'-end of the short-hairpin RNA (shRNA) from the T7 RNA polymerase system did not induce detectable amounts of IFN. The residual amount of guanine associated with the 5'-end and hairpin structures of the transcript was proportional to the reduction of the IFN response. Here we describe a T7 pppGn (n = 2,3) shRNA synthesis that does not induce the IFN response, and maintains the full efficacy of siRNA.

Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy.

BACKGROUND: Hepatitis C virus (HCV) is one of the main causes of liver-related morbidity and mortality. Although combined interferon-alpha-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA) expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1) and the origin of latent viral DNA replication (OriP) sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV) replicon cells. RESULTS: We constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector. CONCLUSION: These findings indicate that we successfully constructed a long-term transgene (shRNA) expression vector (Ac-EP-shRNA452) using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA) expression vector was evaluated with the view of establishing highly effective therapeutic agents that can be further developed for HCV gene therapy applications.

Impact of age at marriage and migration on HIV and AIDS epidemics in Japan.

The causes of wide variation in the rates of HIV and AIDS epidemics among Japanese and non-Japanese nationals are not well understood. So, this paper examines the associations and assesses the potential roles of mean age at marriage, and migration in the HIV and AIDS epidemics in Japan. For the purpose, bivariate and multivariate regression analysis have been performed using epidemiological panel data to build up the relationships among overall HIV and AIDS prevalence, mean age at marriage, and migration. The same analyses have done for non-Japanese nationals living with HIV and AIDS separately. These indicators were significantly correlated with mean age at marriage, and migration. Multivariate linear regression analysis identified non-Japanese nationals' HIV and AIDS prevalence and mean age at marriage as the two most prominent factors linked with the national HIV and AIDS epidemics. The findings of this study supported the hypotheses that a high average age at marriage in the population leads to long period of premarital sex and the non-Japanese nationals' high prevalence facilitating the spread of the HIV and AIDS epidemics in Japan.

Induction of natural killer cell-dependent antitumor immunity by the Autographa californica multiple nuclear polyhedrosis virus.

Wild-type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a variety of mammalian cell types in vitro, but does not replicate in these cells. We investigated the effects of AcMNPV in the induction of the immune response and tumor metastasis in mice. After intravenous injection, AcMNPV was taken up by the liver and spleen, and preferentially infected dendritic cells (DCs) and B cells in the spleen; costimulatory molecules CD40, CD80, and CD86 were upregulated in the DCs. The hepatic mononuclear cells (MNCs) in these animals were highly cytotoxic to natural killer (NK)-sensitive YAC-1 and B16 melanoma cells, but not to NK-resistant EL4 cells. Intravenous injection of AcMNPV-induced NK cell proliferation in the liver and spleen, and enhanced antitumor immunity in mice with B16 liver metastases. Furthermore, such treatment increased the survival of C57BL/6, J alpha 281 (-/-), and interferon (IFN)-gamma (-/-) mice that were previously injected with B16 tumor cells. AcMNPV injection did not enhance the survival of NK cell-depleted mice. Moreover, one AcMNPV treatment effectively prolonged survival in a B16 liver metastasis model, and was equivalent to five treatments with recombinant interleukin-12 (IL-12) protein. These findings suggest that AcMNPV efficiently stimulates NK cell-mediated antitumor immunity.

HIV/AIDS acquisition and transmission in Bangladesh: turning to the concentrated epidemic.

A seventh round behavioral and serological surveillance found that the HIV epidemic had remarkably increased to 7% among intravenous drug users (IDU) in Central Bangladesh, indicating the urgent need to increase prevention. The main purposes of this study were to find out, by collecting data and the necessary information from sero-surveillances, published reports, and articles, what the prevalence of HIV/AIDS is, and what the acquisition and transmission routes are. In addition, trends in HIV-related risk behaviors among recognized high risk groups were observed, and estimations and projections of HIV transmission up to the year 2020 presented. The Estimation and Projection Package was used to estimate and project HIV transmission. The study results reveal that Bangladesh is a low prevalence country which is turning into one with a concentrated epidemic due to the high HIV prevalence rate of IDU (7%) among the most-at-risk groups. Within this at-risk population, IDU have the highest prevalence rate of HIV transmission, followed by female sex workers, clients of sex workers, and men who have sex with men. If the transmission rate continues to increase, the situation will be uncontrolled. Therefore, there is an urgent need for a comprehensive prevention program to control the spread of HIV.

Recombinant baculovirus expressing the FrC-OVA protein induces protective antitumor immunity in an EG7-OVA mouse model.

BACKGROUND: The baculovirus (BV) Autographa californica multiple nuclear polyhedrosis virus has been used in numerous protein expression systems because of its ability to infect insect cells and serves as a useful vaccination vector with several benefits, such as its low clinical risks and posttranslational modification ability. We recently reported that dendritic cells (DCs) infected with BV stimulated antitumor immunity. The recombinant BV (rBV) also strongly stimulated peptide-specific T-cells and antitumor immunity. In this study, the stimulation of an immune response against EG7-OVA tumors in mice by a recombinant baculovirus-based combination vaccine expressing fragment C-ovalbumin (FrC-OVA-BV; rBV) was evaluated. RESULTS: We constructed an rBV expressing fragment C (FrC) of tetanus toxin containing a promiscuous MHC II-binding sequence and a p30-ovalbumin (OVA) peptide that functions in the MHC I pathway. The results showed that rBV activated the CD8+ T-cell-mediated response much more efficiently than the wild-type BV (wtBV). Experiments with EG7-OVA tumor mouse models showed that rBV significantly decreased tumor volume and increased survival compared with those in the wild-type BV or FrC-OVA DNA vaccine groups. In addition, a significant antitumor effect of classic prophylactic or therapeutic vaccinations was observed for rBV against EG7-OVA-induced tumors compared with that in the controls. CONCLUSION: Our findings showed that FrC-OVA-BV (rBV) induced antitumor immunity, paving the way for its use in BV immunotherapy against malignancies.

A 30-year record reveals re-equilibration rates of 137Cs in marine biota after the Fukushima Dai-ichi nuclear power plant accident: Concentration ratios in pre- and post-event conditions.

Concentration ratios (CRs), expressed by dividing 137Cs activity in seawater by that in marine biota (mainly fish), were obtained from the monitoring of 137Cs in coastal areas around Japan between 1984 and 2016. Before the TEPCO Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident (1984-2010), mean CRs of 137Cs, mainly from global fallout (i.e. CRGF), were almost constant for each species throughout the monitoring period, but were different among species, while the values for several species were dependent on their length (i.e. CRGF-SIZE). Thus, CRGF and CRGF-SIZE values for 29 of marketable species are given here as references for conditions where marine biota are in approximate equilibrium (or steady state) with their host water with respect to 137Cs activities in the marine environment. After the FDNPP accident (2011-2016), the impact of the accident has been sustained in eastern Japan waters as indicated by apparent CRs (CRas) which are being used here as indicators of disequilibrium between organisms and their host water. The recession rates of this disequilibrium (the effective CRa half-lives) ranged from 100 to 1100 days. The identified distinct variation was due to the sample locations, even for the same species, because of the change in 137Cs activity concentrations in their host water and diet preference differences. Variation among species, even those captured from the same area, was mainly due to diet differences as well as metabolic-physiological differences in 137Cs retention. Thus, our results from >30 years of systematically monitoring have helped quantify the recession rates of post-FDNPP disequilibrium of 137Cs in biota for assessment of how long term is required from contaminated condition by underlying spatial, inter- and intra-species factors.

Suppression of hepatitis C virus replication by baculovirus vector-mediated short-hairpin RNA expression.

Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. Here, we report on the inhibition, by baculovirus-based vector-derived shRNAs, of core-protein expression in full-length hepatitis C virus (HCV) replicon cells. shRNAs were designed to target the highly conserved core region of the HCV genome. In particular, the core-shRNA452 containing nucleotides 452-472, as the target in the HCV core gene, dramatically inhibited the expression of the HCV core protein in replicon cells. Furthermore, HCV core-protein expression was inhibited more strongly by the vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped baculovirus vector than by the wild-type baculovirus vector.