Suppression of Hepatitis C Virus Core Protein by Short Hairpin RNA Expression Vectors in the Core Protein Expression HUH-7 Cells

Short hairpin RNAs (shRNAs) efficiently inhibit gene expression by RNA interference. Here, we report the efficient inhibition by DNA-based vector-derived shRNAs of core protein expression in Huh-7 cells. The shRNAs were designed to target the core region of the hepatitis C virus (HCV) genome. The core region is the most conserved region in the HCV genome, making it an ideal target for shRNAs. We identified an effective site on the core region for suppression of the HCV core protein. The HCV core protein in core protein-expressing Huh-7 cells was downregulated by core protein-shRNA expression vectors (core-shRNA-452, 479, and 503). Our results support the feasibility of using shRNA-based gene therapy to inhibit HCV core protein production

Mathematical modeling and experimental validation of the spatial distribution of boron in the root of arabidopsis thaliana identify high boron accumulation in the tip and predict a distinct root tip uptake function

Boron, an essential micronutrient, is transported in roots of Arabidopsis thaliana mainly by two different types of transporters, BORs and NIPs (nodulin26-like intrinsic proteins). Both are plasma membrane localized, but have distinct transport properties and patterns of cell type-specific accumulation with different polar localizations, which are likely to affect boron distribution. Here, we used mathematical modeling and an experimental determination to address boron distributions in the root. A computational model of the root is created at the cellular level, describing the boron transporters as observed experimentally. Boron is allowed to diffuse into roots, in cells and cell walls, and to be transported over plasma membranes, reflecting the properties of the different transporters. The model predicts that a region around the quiescent center has a higher concentration of soluble boron than other portions. To evaluate this prediction experimentally, we determined the boron distribution in roots using laser ablation-inductivity coupled plasma-mass spectrometry. The analysis indicated that the boron concentration is highest near the tip and is lower in the more proximal region of the meristem zone, similar to the pattern of soluble boron distribution predicted by the model. Our model also predicts that upward boron flux does not continuously increase from the root tip toward the mature region, indicating that boron taken up in the root tip is not efficiently transported to shoots. This suggests that root tip-absorbed boron is probably used for local root growth, and that instead it is the more mature root regions which have a greater role in transporting boron toward the shoots

Neutralization of hepatitis B virus with vaccine-escape mutations by hepatitis B vaccine with large-HBs antigen

優れたB型肝炎予防ワクチン開発に成功 --既存ワクチンの弱点克服へ--. 京都大学プレスリリース. 2022-09-07.Although the current hepatitis B (HB) vaccine comprising small-HBs antigen (Ag) is potent and safe, attenuated prophylaxis against hepatitis B virus (HBV) with vaccine-escape mutations (VEMs) has been reported. We investigate an HB vaccine consisting of large-HBsAg that overcomes the shortcomings of the current HB vaccine. Yeast-derived large-HBsAg is immunized into rhesus macaques, and the neutralizing activities of the induced antibodies are compared with those of the current HB vaccine. Although the antibodies induced by the current HB vaccine cannot prevent HBV infection with VEMs, the large-HBsAg vaccine-induced antibodies neutralize those infections. The HBV genotypes that exhibited attenuated neutralization via these vaccines are different. Here, we show that the HB vaccine consisting of large-HBsAg is useful to compensate for the shortcomings of the current HB vaccine. The combined use of these HB vaccines may induce antibodies that can neutralize HBV strains with VEMs or multiple HBV genotypes

The Progression of Liver Fibrosis Is Related with Overexpression of the miR-199 and 200 Families

Chronic hepatitis C (CH) can develop into liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Liver fibrosis and HCC development are strongly correlated, but there is no effective treatment against fibrosis because the critical mechanism of progression of liver fibrosis is not fully understood. microRNAs (miRNAs) are now essential to the molecular mechanisms of several biological processes. In order to clarify how the aberrant expression of miRNAs participates in development of the liver fibrosis, we analyzed the liver fibrosis in mouse liver fibrosis model and human clinical samples

Hepatic microRNA expression is associated with the response to interferon treatment of chronic hepatitis C

<p>Abstract</p> <p>Background</p> <p>HCV infection frequently induces chronic liver diseases. The current standard treatment for chronic hepatitis (CH) C combines pegylated interferon (IFN) and ribavirin, and is less than ideal due to undesirable effects. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that control gene expression by degrading or suppressing the translation of target mRNAs. In this study we administered the standard combination treatment to CHC patients. We then examined their miRNA expression profiles in order to identify the miRNAs that were associated with each patient's drug response.</p> <p>Methods</p> <p>99 CHC patients with no anti-viral therapy history were enrolled. The expression level of 470 mature miRNAs found their biopsy specimen, obtained prior to the combination therapy, were quantified using microarray analysis. The miRNA expression pattern was classified based on the final virological response to the combination therapy. Monte Carlo Cross Validation (MCCV) was used to validate the outcome of the prediction based on the miRNA expression profile.</p> <p>Results</p> <p>We found that the expression level of 9 miRNAs were significantly different in the sustained virological response (SVR) and non-responder (NR) groups. MCCV revealed an accuracy, sensitivity, and specificity of 70.5%, 76.5% and 63.3% in SVR and non-SVR and 70.0%, 67.5%, and 73.7% in relapse (R) and NR, respectively.</p> <p>Conclusions</p> <p>The hepatic miRNA expression pattern that exists in CHC patients before combination therapy is associated with their therapeutic outcome. This information can be utilized as a novel biomarker to predict drug response and can also be applied to developing novel anti-viral therapy for CHC patients.</p

MicroRNA-3145 as a potential therapeutic target for hepatitis B virus: inhibition of viral replication via downregulation of HBS and HBX

Current treatments for hepatitis B virus (HBV), such as interferons and nucleic acid analogs, have limitations due to side effects like depression and the development of drug-resistant mutants, highlighting the need for new therapeutic approaches. In this study, we identified microRNA-3145 (miR-3145) as a host-derived miRNA with antiviral activity that is upregulated in primary hepatocytes during HBV infection. The expression of its precursor, pri-miR-3145, increased in response to the the virus infection, and miR-3145 downregulated the hepatitis B virus S (HBS) antigen and hepatitis B virus X (HBX), thereby inhibiting viral replication. The binding site for miR-3145 was located in the HBV polymerase (pol) region, as experimentally confirmed. Moreover, overexpression of HBS and HBX induced pri-miR-3145 expression through endoplasmic reticulum stress. The expression of pri-miR-3145 showed a strong correlation with the Nance–Horan syndrome-like 1 (NHSL1) gene, as it is encoded within an intron of NHSL1, and higher NHSL1 expression in hepatocellular carcinoma patients with HBV infection was associated with better prognosis. These findings suggest that miR-3145-3p, along with small molecules targeting its binding sites, holds promise as a potential therapeutic candidate for HBV treatment

Development of Mouse Hepatocyte Lines Permissive for Hepatitis C Virus (HCV)

The lack of a suitable small animal model for the analysis of hepatitis C virus (HCV) infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. Human and mouse harbor a comparable system for antiviral type I interferon (IFN) induction and amplification, which regulates viral infection and replication. Using hepatocytes from knockout (ko) mice, we determined the critical step of the IFN-inducing/amplification pathways regulating HCV replication in mouse. The results infer that interferon-beta promoter stimulator (IPS-1) or interferon A receptor (IFNAR) were a crucial barrier to HCV replication in mouse hepatocytes. Although both IFNARko and IPS-1ko hepatocytes showed a reduced induction of type I interferons in response to viral infection, only IPS-1-/- cells circumvented cell death from HCV cytopathic effect and significantly improved J6JFH1 replication, suggesting IPS-1 to be a key player regulating HCV replication in mouse hepatocytes. We then established mouse hepatocyte lines lacking IPS-1 or IFNAR through immortalization with SV40T antigen. Expression of human (h)CD81 on these hepatocyte lines rendered both lines HCVcc-permissive. We also found that the chimeric J6JFH1 construct, having the structure region from J6 isolate enhanced HCV replication in mouse hepatocytes rather than the full length original JFH1 construct, a new finding that suggests the possible role of the HCV structural region in HCV replication. This is the first report on the entry and replication of HCV infectious particles in mouse hepatocytes. These mouse hepatocyte lines will facilitate establishing a mouse HCV infection model with multifarious applications