High serum gamma-glutamyltransferase level after hepatitis C virus elimination is a risk factor for the development of hepatocellular carcinoma.

AIM: Gamma-glutamyltransferase (GGT) is known as an oxidative stress marker, induced by alcohol consumption and metabolic disorders, and is reported as a predictor of hepatocellular carcinoma (HCC) development after hepatitis C virus (HCV) elimination. However, it is not clear whether GGT serves simply as a surrogate marker for overlapping metabolic diseases or reflects HCV-specific carcinogenicity. We investigated the association between GGT and hepatocarcinogenesis after achieving a sustained viral response (SVR), accounting for drinking habits or diabetes, and examined predisposing factors associated with GGT levels after SVR. METHODS: This is a prospective, multicenter, and observational study using the database of 1001 patients after HCV eradication with direct-acting antiviral agents. The association of GGT at SVR with cumulative HCC development was examined in a multivariate analysis using Cox proportional hazard models after adjustment for covariates including alcohol and diabetes. The association between oxidative stress markers or genetic factors and GGT levels was analyzed. RESULTS: High GGT levels at SVR were associated with HCC development (HR] 2.38, 95% CI 1.10-5.17). This association was also significant when restricted to patients without alcohol consumption or diabetes (HR 8.38, 95% CI 2.87-24.47). GGT levels were correlated with serum growth differentiation factor 15 levels, a marker of mitochondrial dysfunction. Single-nucleotide polymorphisms of ZNF827 and GDF15 were associated with high GGT levels. CONCLUSIONS: High GGT levels at SVR were associated with HCC development after accounting for alcohol consumption and diabetes. GGT levels are influenced by genetic predisposition and may reflect mitochondrial dysfunction after HCV eradication.

Disseminated Bacillus Calmette-Guérin Infection with Rhabdomyolysis, Acute Kidney Injury, and Interstitial Pneumonia after Bacillus Calmette-Guérin Intravesical Instillation Therapy.

A 79-year-old man experienced a fever and immobility after receiving 6 doses of Bacillus Calmette-Guérin (BCG) intravesical instillation therapy for bladder tumor. Rhabdomyolysis and acute kidney injury occurred; therefore, hemodialysis was performed. His kidney function was restored. However, he exhibited an inflammatory reaction that was resistant to broad-spectrum antibiotics and eventually developed interstitial pneumonia. Corticosteroid treatment partially relieved the symptoms of interstitial pneumonia, although disuse syndrome persisted. He was diagnosed with disseminated BCG infection through sputum culture. BCG infection shows various symptoms and is difficult to diagnose microbiologically. It should be suspected when systemic symptoms occur after BCG intravesical instillation therapy.

[Endoscopic variceal ligation and percutaneous transhepatic obliteration difficulty in esophageal variceal bleeding: a case report].

A 48-year-old male patient with a history of alcoholic cirrhosis was admitted to our hospital due to hematemesis with a 7-day history of melena. Emergency esophagogastroduodenoscopy revealed esophageal variceal bleeding. We attempted hemostasis with endoscopic variceal ligation (EVL). The esophageal mucosa was not aspirated into the EVL device although the patient had no history of endoscopic injection sclerotherapy or EVL. Percutaneous transhepatic obliteration (PTO) was performed and esophageal variceal bleeding was successfully hemostasis. PTO is a viable option for refractory esophageal bleeding.

Central Diabetes Insipidus after Syndrome of Inappropriate Antidiuretic Hormone Secretion with Severe Hyponatremia in a Patient with Rathke's Cleft Cyst.

A 49-year-old man developed severe hyponatremia associated with transient headache and was diagnosed with syndrome of inappropriate antidiuretic hormone secretion (SIADH). Fluid restriction and sodium supplementation corrected the hyponatremia. However, several days later, the patient exhibited hypernatremia with thirst and polyuria. A detailed examination indicated central diabetes insipidus (CDI) with an intrasellar cystic lesion indicative of Rathke's cleft cyst (RCC). A case of RCC exhibiting headache, hyponatremia, and subsequent hypernatremia has been reported. Our case shows that CDI may appear after SIADH in patients with RCC, especially in those with serum sodium levels that unexpectedly increase rapidly beyond the reference range.

Prolonged Postoperative Pyrexia and Transient Nonnephrogenic Vasopressin-Analogue-Resistant Polyuria following Endoscopic Transsphenoidal Resection of an Infundibular Epidermoid Cyst.

Prolonged postoperative pyrexia (PPP) due to Mollaret's meningitis following endoscopic transsphenoidal surgery (eTSS) for an intracranial epidermoid cyst can be confused with postoperative meningeal infection after transsphenoidal resection, especially in the middle of the COVID-19 pandemic. Anosmia, as well as dysgeusia, cannot be evaluated in patients of eTSS for a while after surgery. We report a case of an infundibular epidermoid cyst with post-eTSS Mollaret's meningitis (MM). The post-eTSS MM caused vasopressin-analogue-resistant polyuria (VARP) in synchronization with PPP. A 59-year-old man experiencing recurrent headaches and irregular bitemporal hemianopsia over three months was diagnosed with a suprasellar tumor. The suprasellar tumor was an infundibular cyst from the infundibular recess to the posterior lobe of the pituitary, which was gross-totally resected including the neurohypophysis via an extended eTSS. Since awakening from general anesthesia after the gross total resection (GTR) of the tumor, the patient continuously had suffered from headache until the 13th postoperative day (POD13). The patient took analgesics once a day before the surgery and three times a day after the surgery until POD11. Pyrexia (37.5-39.5 degree Celsius) in synchronization with nonnephrogenic VARP remitted on POD18. Intravenous antibiotics had little effect on changes of pyrexia. Serum procalcitonin values (reference range <0.5 ng/mL) are 0.07 ng/mL on POD12 and 0.06 ng/mL on POD18. His polyuria came to react with sublingual desmopressin after alleviation of pyrexia. He left the hospital under hormone replacement therapy without newly added neurological sequelae other than hypopituitarism. After GTR of an infundibular epidermoid cyst, based on values of serum procalcitonin, post-eTSS MM can be distinguished from infection and can be treated with symptomatic treatments. The postoperative transient nonnephrogenic VARP that differs from usual central diabetes insipidus can react with sublingual desmopressin after alleviation of PPP in the clinical course of post-eTSS MM. An infundibular epidermoid cyst should be sufficiently resected in one sitting to minimize comorbidities, its recurrence, or postoperative MM to the utmost.

Improvements in the water dispersibility of paclitaxel by complexing with synthetic peptides derived from ß-casein.

Recently, digestive peptides prepared as a casein hydrolysate have been found to be an effective dispersant for the poorly water-soluble drug paclitaxel (Ptx). A major hydrophobic peptide in the digested peptides was identified as YQEPVLGPVRGPFPIIV (PepY) by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry with the "LIFT" technique. In the present study, three peptides PepY, VVVPPFLQPEVMGVSKV (PepV), and KFQSEEQQQTEDELQDK (PepK) were chemically synthesized by Fmoc solid-phase synthesis to compare their function as dispersants for Ptx. PepV and PepK are the most hydrophobic and hydrophilic peptides, respectively, in the sequence of ß-casein, which are the same length as PepY (PepY, PepV, and PepK are abbreviated as Peps). The complex between Ptx and Peps (Ptx-Peps) was prepared by mixing an ethanol solution of Ptx and an aqueous solution of Peps, followed by lyophilization. The complex with PepV, which is estimated to be the most hydrophobic of the peptides, had the greatest ability to improve the water dispersibility of Ptx. The water dispersibility of the complexes between Ptx and PepY and PepV increased as the amount of the peptides increased, whereas PepK was not effective in enhancing the dispersibility of Ptx. Furthermore, a peptide mixture obtained from a casein hydrolysate [Pep (fraction A)] was more effective for the enhancement of Ptx dispersibility than the single peptide PepY. These results suggests that a variety of peptides in the casein hydrolysate contribute toward complexation with Ptx.

Serial measurement of Wisteria floribunda agglutinin positive Mac-2-binding protein is useful for predicting liver fibrosis and the development of hepatocellular carcinoma in chronic hepatitis C patients treated with IFN-based and IFN-free therapy.

AIM: Wisteria floribunda agglutinin positive (WFA+) Mac-2-binding protein (M2BPGi) is a noninvasive glyco-marker for liver fibrosis. This study evaluated the utility of serial measurement of serum M2BPGi and total M2BP as a predictor of fibrosis and the development of hepatocellular carcinoma (HCC). METHODS: This study included 119 patients with chronic hepatitis C (CHC). Of these patients, 97 were treated with IFN-based therapy and 22 were treated with daclatasvir and asunaprevir. Serum M2BPGi values were measured prior to, at the end of, and at 24 weeks after the completion of treatment. As subanalysis, serum total M2BP levels were measured in patients treated with pegylated-interferon and ribavirin. RESULTS: In patients treated with IFN-based therapy, M2BPGi levels were elevated at the end of treatment but decreased afterwards. In contrast, M2BPGi levels in patients treated with IFN-free therapy decreased immediately after starting the treatment without transient elevation. Though pre-treatment M2BPGi levels significantly correlated with fibrosis in both patients with a sustained virological response (SVR) and non-SVR, post-treatment M2BPGi levels decreased regardless of the degree of fibrosis in patients with SVR. In multivariate analysis, non-SVR and HCC development were independent factors associated with M2BPGi level ≥2.2. In patients treated with pegylated-interferon and ribavirin, total M2BP levels were positively correlated with fibrosis and HCC development. CONCLUSION: Real-time monitoring of the serum M2BPGi level after antiviral therapy for CHC patients could be a helpful screening tool for assessing the risk of HCC. M2BP and its glycan structure could be associated together with hepatocarcinogenesis.

Evaluation of Interferon Resistance in Newly Established Genotype 1b Hepatitis C Virus Cell Culture System.

BACKGROUND AND AIMS: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. METHODS: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. RESULTS were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. RESULTS: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). CONCLUSIONS: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment.

Efficacy of additional radiofrequency ablation after transcatheter arterial chemoembolization for intermediate hepatocellular carcinoma.

AIM: For intermediate hepatocellular carcinoma (HCC), transcatheter arterial chemoembolization (TACE) therapy is recommended in the guidelines as a monotherapy, although TACE is a non-curative therapy. The aims of the present study were to evaluate the efficacy of adding radiofrequency ablation (RFA) to TACE in patients with intermediate HCC, and to identify the factors that were associated with favorable survival in these patients. METHODS: Fifty-nine patients with intermediate HCC were enrolled in this retrospective study. Thirty-nine patients were treated with TACE alone and 20 patients were treated with additional RFA after TACE. RESULTS: The recurrence-free survival rates at 0.5, 1 and 2 years for the additional RFA group were 32%, 19% and 13%, respectively, and these were significantly higher than those of the TACE group (8%, 3% and 0%, respectively; log-rank test, P = 0.001). The cumulative survival rates of the additional RFA group were significantly higher than those of the TACE group (log-rank test, P = 0.002), although this significant difference was not found in the subgroup of treatment naive patients because of small sample size. Multivariate analysis indicated male sex, lower total bilirubin, lower α-fetoprotein, lower des-γ-carboxyprothrombin, newly recurrent HCC nodules within the last 12 months and additional RFA as independent factors that were significantly associated with favorable overall survival. CONCLUSION: Additional RFA of nodules insufficiently treated by TACE is effective therapy for obtaining favorable disease-free survival in patients with intermediate HCC, and leads to better overall survival particularly in recurrent patients.

Comprehensive analyses of mutations and hepatitis B virus integration in hepatocellular carcinoma with clinicopathological features.

BACKGROUND AND AIMS: Genetic alterations in specific genes are critical events in carcinogenesis and hepatocellular carcinoma (HCC) progression. However, the genetic alterations responsible for HCC development, progression, and survival are unclear. METHODS: We investigated the essential difference in genetic alterations between HCC and adjacent non-HCC tissues using next-generation sequencing technology. RESULTS: We found recurrent mutations in several genes such as telomerase reverse transcriptase (TERT; 65% of the total 104 HCCs), TP53 (38%), CTNNB1 (30%), AXIN1 (2%), PTEN (2%), and CDKN2A (2%). TERT promoter mutations were associated with older age (p = 0.005), presence of hepatitis C virus (HCV) infection (p = 0.003), and absence of hepatitis B virus (HBV) infection (p < 0.0001). In hepatitis B surface antigen (HBs Ag)-positive HCC without TERT promoter mutations, HBV integration into TERT locus was found in 47% patients and was mutually exclusive to TERT promoter mutations. Most (89%) HBV integrants were in the HBx region. TP53 mutations were associated with HBV infection (p = 0.0001) and absence of HCV infection (p = 0.002). CTNNB1 mutations were associated with absence of HBV infection (p = 0.010). Moreover, TERT promoter mutation was significantly associated with shorter disease-free survival (p = 0.005) and poor overall survival (p = 0.024). CONCLUSIONS: Gene alterations in TERT promoter, TP53, CTNNB1, and HBV integration were closely associated with HCC development, and mutations in TERT promoter are related to poor prognosis. These results are useful for understanding the underlying mechanism of hepatocarcinogenesis, diagnosis, and predicting outcomes of patients with HCC.

MRI of Perfusion: Principles and Clinical Applications.

The principle and clinical application of measurement of cerebral blood perfusion (CBP) using MRI are described. Purposes of measuring CBP using MRI are wide-ranging. Generally, it is used to diagnose cerebro-vascular disorders or brain tumors. There are two types of measuring methods. One is dynamic susceptibility contrast (DSC) method using a contrast agent as a tracer. Another is an arterial spin labeling (ASL) method using protons in arterial blood as an endogenous tracer, instead of bio-exogenous tracer. Basic theory of ASL method was published in the 1990s, recently, its clinical application has been spreading rapidly by the technological innovations. ASL method is attractive as a way to measure CBP because of its non-invasiveness (no radiation-exposure, not need intravenous injection or blood sampling), and the imaging time is about 5 minutes, thereby the measurement can be repeated. The analysis of DSC method has not been standardized, though various valuable parameters are provided. And the prerequisite of DSC method is uncertain in vivo. On the other hand, the result of ASL is affected by the post labeling delay, and limited to the arterial information.

Primary hepatic neuroendocrine carcinoma with a cholangiocellular carcinoma component in one nodule.

Primary neuroendocrine carcinomas (NECs) in the liver are very rare; however, several reports have described cases of a primary hepatic NEC combined with a hepatocellular carcinoma (HCC). We present the first report of a primary hepatic NEC with a cholangiocellular carcinoma (CCC) component in one nodule in a patient with a metachronous liver HCC. A 73-year old man who had received partial hepatectomy surgery because of a primary HCC and a primary CCC two years prior was diagnosed with a primary hepatic NEC after surgical treatment. Histological analysis of the resected tumor revealed that the tumor consisted of a predominant NEC area with a partial CCC component in one nodule and that the NEC cells were negative for markers of pancreatic NEC. Neoplastic cells in both the NEC and CCC component focally expressed CD44, a representative marker for cancer-initiating cells, and the CD44-positive cells in the NEC component were seen in the vicinity of those in the CCC component of one nodule. This case report provides suggestive information for the origin of primary hepatic NECs.

Hepatitis C virus NS4B protein targets STING and abrogates RIG-I-mediated type I interferon-dependent innate immunity.

UNLABELLED: Hepatitis C virus (HCV) infection blocks cellular interferon (IFN)-mediated antiviral signaling through cleavage of Cardif by HCV-NS3/4A serine protease. Like NS3/4A, NS4B protein strongly blocks IFN-ß production signaling mediated by retinoic acid-inducible gene I (RIG-I); however, the underlying molecular mechanisms are not well understood. Recently, the stimulator of interferon genes (STING) was identified as an activator of RIG-I signaling. STING possesses a structural homology domain with flaviviral NS4B, which suggests a direct protein-protein interaction. In the present study, we investigated the molecular mechanisms by which NS4B targets RIG-I-induced and STING-mediated IFN-ß production signaling. IFN-ß promoter reporter assay showed that IFN-ß promoter activation induced by RIG-I or Cardif was significantly suppressed by both NS4B and NS3/4A, whereas STING-induced IFN-ß activation was suppressed by NS4B but not by NS3/4A, suggesting that NS4B had a distinct point of interaction. Immunostaining showed that STING colocalized with NS4B in the endoplasmic reticulum. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays demonstrated that NS4B specifically bound STING. Intriguingly, NS4B expression blocked the protein interaction between STING and Cardif, which is required for robust IFN-ß activation. NS4B truncation assays showed that its N terminus, containing the STING homology domain, was necessary for the suppression of IFN-ß promoter activation. NS4B suppressed residual IFN-ß activation by an NS3/4A-cleaved Cardif (Cardif1-508), suggesting that NS3/4A and NS4B may cooperate in the blockade of IFN-ß production. CONCLUSION: NS4B suppresses RIG-I-mediated IFN-ß production signaling through a direct protein interaction with STING. Disruption of that interaction may restore cellular antiviral responses and may constitute a novel therapeutic strategy for the eradication of HCV.

Identification of novel N-(morpholine-4-carbonyloxy) amidine compounds as potent inhibitors against hepatitis C virus replication.

To identify novel compounds that possess antiviral activity against hepatitis C virus (HCV), we screened a library of small molecules with various amounts of structural diversity using an HCV replicon-expressing cell line and performed additional validations using the HCV-JFH1 infectious-virus cell culture. Of 4,004 chemical compounds, we identified 4 novel compounds that suppressed HCV replication with 50% effective concentrations of ranging from 0.36 to 4.81 µM. N'-(Morpholine-4-carbonyloxy)-2-(naphthalen-1-yl) acetimidamide (MCNA) was the most potent and also produced a small synergistic effect when used in combination with alpha interferon. Structure-activity relationship (SAR) analyses revealed 4 derivative compounds with antiviral activity. Further SAR analyses revealed that the N-(morpholine-4-carbonyloxy) amidine moiety was a key structural element for antiviral activity. Treatment of cells with MCNA activated nuclear factor κB and downstream gene expression. In conclusion, N-(morpholine-4-carbonyloxy) amidine and other related morpholine compounds specifically suppressed HCV replication and may have potential as novel chemotherapeutic agents.

Serum interleukin-6 levels correlate with resistance to treatment of chronic hepatitis C infection with pegylated-interferon-α2b plus ribavirin.

BACKGROUND: Interleukin (IL)-6, a pleiotropic cytokine, is increased in various types of chronic liver disease, including chronic hepatitis C (CHC). It was reported recently that IL-6 is associated with insulin resistance, iron metabolism and interferon resistance, which may affect the outcome of antiviral treatment. In this study, we investigated the association of serum IL-6 levels with outcomes of pegylated interferon (PEG-IFN) plus ribavirin (RBV) combination therapy. METHODS: We included 149 CHC patients and measured serum IL-6 levels at baseline and at 4, 8 and 12 weeks, and the end of treatment in 49 patients. We performed univariate and multivariate regression analyses for the association of IL-6 levels and clinical and laboratory parameters and treatment responses. RESULTS: Serum IL-6 levels were significantly higher in CHC patients than healthy subjects. Pretreatment IL-6 levels of male patients were inversely correlated with sustained virological response (SVR) in univariate analysis (P=0.012). In male patients with SVR, serum IL-6 levels decreased significantly at 4 weeks of treatment (P=0.029) and remained significantly lower than those of non-SVR patients after 4, 8 and 12 weeks of PEG-IFN plus RBV therapy. CONCLUSIONS: Our results suggest that baseline levels of IL-6, as well as their decrease during treatment, are correlated to outcomes of PEG-IFN plus RBV therapy in male patients. Further analyses of IL-6 may provide new strategies for difficult-to-treat CHC patients and prevention of hepatocarcinogenesis.

Inhibitory effect of a triterpenoid compound, with or without alpha interferon, on hepatitis C virus infection.

A lack of patient response to alpha interferon (α-IFN) plus ribavirin (RBV) treatment is a major problem in eliminating hepatitis C virus (HCV). We screened chemical libraries for compounds that enhanced cellular responses to α-IFN and identified a triterpenoid, toosendanin (TSN). Here, we studied the effects and mechanisms of action of TSN on HCV replication and its effect on α-IFN signaling. We treated HCV genotype 1b replicon-expressing cells and HCV-J6/JFH-infected cells with TSN, with or without α-IFN, and the level of HCV replication was quantified. To study the effects of TSN on α-IFN signaling, we detected components of the interferon-stimulated gene factor 3 (ISGF3), phosphorylated signal transducer and activator of transcription 1 (STAT1), and STAT2 by Western blotting analysis; expression levels of mRNA of interferon regulatory factor 9 using real-time reverse transcription-PCR (RT-PCR); and interferon-stimulated response element reporter activity and measured the expression levels of interferon-inducible genes for 2',5'-oligoadenylate synthetase, MxA, protein kinase R, and p56 using real-time RT-PCR. TSN alone specifically inhibited expression of the HCV replicon (50% effective concentration = 20.6 nM, 50% cytotoxic concentration > 3 µM, selectivity index > 146). Pretreatment with TSN prior to α-IFN treatment was more effective in suppressing HCV replication than treatment with either drug alone. Although TSN alone did not activate the α-IFN pathway, it significantly enhanced the α-IFN-induced increase of phosphorylated STATs, interferon-stimulated response element activation, and interferon-stimulated gene expression. TSN significantly increased baseline expression of interferon regulatory factor 9, a component of interferon-stimulated gene factor 3. Antiviral effects of treatment with α-IFN can be enhanced by pretreatment with TSN. Its mechanisms of action could potentially be important to identify novel molecular targets to treat HCV infection.

Inhibition of hepatitis C virus replication by a specific inhibitor of serine-arginine-rich protein kinase.

Splicing of messenger RNAs is regulated by site-specific binding of members of the serine-arginine-rich (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate overall activity of the SR proteins by phosphorylation of their RS domains. We have reported that specifically designed SRPK inhibitors suppressed effectively several DNA and RNA viruses in vitro and in vivo. Here, we show that an SRPK inhibitor, SRPIN340, suppressed in a dose-dependent fashion expression of a hepatitis C virus (HCV) subgenomic replicon and replication of the HCV-JFH1 clone in vitro. The inhibitory effects were not associated with antiproliferative or nonspecific cytotoxic effects on the host cells. Overexpression of SRPK1 or SRPK2 resulted in augmentation of HCV replication, while small interfering RNA (siRNA) knockdown of the SRPKs suppressed HCV replication significantly. Immunocytochemistry showed that SRPKs and the HCV core and NS5A proteins colocalized to some extent in the perinuclear area. Our results demonstrate that SRPKs are host factors essential for HCV replication and that functional inhibitors of these kinases may constitute a new class of antiviral agents against HCV infection.

Antiviral effects of the interferon-induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein.

UNLABELLED: Interferons (IFNs) and the interferon-stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We have reported previously that ISGs, including guanylate binding protein 1 (GBP-1), interferon alpha inducible protein (IFI)-6-16, and IFI-27, inhibit HCV subgenomic replication. In this study we investigated the effects of these ISGs against HCV in cell culture and their direct molecular interaction with viral proteins. HCV replication and virus production were suppressed significantly by overexpression of GBP-1, IFI-6-16, or IFI-27. Knockdown of the individual ISGs enhanced HCV RNA replication markedly. A two-hybrid panel of molecular interaction of the ISGs with HCV proteins showed that GBP-1 bound HCV-NS5B directly. A protein truncation assay showed that the guanine binding domain of GBP-1 and the finger domain of NS5B were involved in the interaction. Binding of NS5B with GBP-1 inhibited its guanosine triphosphatase GTPase activity, which is essential for its antiviral effect. Taken together, interferon-induced GBP-1 showed antiviral activity against HCV replication. CONCLUSION: Binding of the HCV-NS5B protein to GBP-1 countered the antiviral effect by inhibition of its GTPase activity. These mechanisms may contribute to resistance to innate, IFN-mediated antiviral defense and to the clinical persistence of HCV infection.

Inhibition of hepatitis C virus infection and expression in vitro and in vivo by recombinant adenovirus expressing short hairpin RNA.

BACKGROUND AND AIM: We have reported previously that synthetic small interfering RNA (siRNA) and DNA-based siRNA expression vectors efficiently and specifically suppress hepatitis C virus (HCV) replication in vitro. In this study, we investigated the effects of the siRNA targeting HCV-RNA in vivo. METHODS: We constructed recombinant retrovirus and adenovirus expressing short hairpin RNA (shRNA), and transfected into replicon-expressing cells in vitro and transgenic mice in vivo. RESULTS: Retroviral transduction of Huh7 cells to express shRNA and subsequent transfection of an HCV replicon into the cells showed that the cells had acquired resistance to HCV replication. Infection of cells expressing the HCV replicon with an adenovirus expressing shRNA resulted in efficient vector delivery and expression of shRNA, leading to suppression of the replicon in the cells by approximately 10(-3). Intravenous delivery of the adenovirus expressing shRNA into transgenic mice that can be induced to express HCV structural proteins by the Cre/loxP switching system resulted in specific suppression of virus protein synthesis in the liver. CONCLUSION: Taken together, our results support the feasibility of utilizing gene targeting therapy based on siRNA and/or shRNA expression to counteract HCV replication, which might prove valuable in the treatment of hepatitis C.

Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity.

HCV culture in vitro results in massive cell death, which suggests the presence of HCV-induced cytopathic effects. Therefore, we investigated its mechanisms and viral nucleotide sequences involved in this effect using HCV-JFH1 cell culture and a newly developed HCV plaque assay technique. The plaque assay developed cytopathic plaques, depending on the titer of the inoculum. In the virus-infected cells, the ER stress markers, GRP78 and phosphorylated eIF2-alpha, were overexpressed. Cells in the plaques were strongly positive for an apoptosis marker, annexin V. Isolated virus subclones from individual plaque showed greater replication efficiency and cytopathogenicity than the parental virus. The plaque-purified virus had 9 amino acid substitutions, of which 5 were clustered in the C terminal of the NS5B region. Taken together, the cytopathic effect of HCV infection involves ER-stress-induced apoptotic cell death. Certain HCV genomic structures may determine the viral replication capacity and cytopathogenicity.