Genetic Variation of the IL-28B Promoter Affecting Gene Expression

The current standard of care for the treatment of chronic hepatitis C is pegylated interferon-α (PEG-IFNα) and ribavirin (RBV). The treatment achieves a sustained viral clearance in only approximately 50% of patients. Recent whole genome association studies revealed that single nucleotide polymorphisms (SNPs) around IL-28B have been associated with response to the standard therapy and could predict treatment responses at approximately 80%. However, it is not clear which SNP is most informative because the genomic region containing significant SNPs shows strong linkage disequilibrium. We focused on SNPs in close proximity to the IL-28B gene to evaluate the function of each and identify the SNP affecting the IL-28B expression level most. The structures of IL-28A/B from 5′ to 3′-UTR were determined by complete cDNA cloning. Both IL-28A and 28B genes consisted of 6 exons, differing from the CCDS data of NCBI. Two intron SNPs and a nonsynonymous SNP did not affect IL-28B gene function and expression levels but a SNP located in the proximal promoter region influenced gene expression. A (TA) dinucleotide repeat, rs72258881, located in the promoter region was discovered by our functional studies of the proximal SNPs upstream of IL-28B; the transcriptional activity of the promoter increased gradually in a (TA)n length-dependent manner following IFN-α and lipopolysaccharide stimulation. Healthy Japanese donors exhibited a broad range of (TA) dinucleotide repeat numbers from 10 to 18 and the most prevalent genotype was 12/12 (75%), differing from the database (13/13). However, genetic variation of IL-28A corresponding to that of IL-28B was not detected in these Japanese donors. These findings suggest that the dinucleotide repeat could be associated with the transcriptional activity of IL-28B as well as being a marker to improve the prediction of the response to interferon-based hepatitis C virus treatment

Replication of a hepatitis C virus replicon clone in mouse cells

BACKGROUND: Hepatitis C Virus (HCV) is a significant public health burden and small animal models are needed to study the pathology and immunobiology of the virus. In effort to develop experimental HCV mouse models, we screened a panel of HCV replicons to identify clones capable of replicating in mouse hepatocytes. RESULTS: We report the establishment of stable HCV replication in mouse hepatocyte and fibroblast cell lines using replicons derived from the JFH-1 genotype 2a consensus sequence. Viral RNA replication efficiency in mouse cells was comparable to that observed in human Huh-7 replicon cells, with negative-strand HCV RNA and the viral NS5A protein being readily detected by Northern and Western Blot analysis, respectively. Although HCV replication was established in the absence of adaptive mutations that might otherwise compromise the in vitro infectivity of the JFH-1 clone, no infectious virus was detected when the culture medium from full length HCV RNA replicating mouse cells was titrated on Huh-7 cells, suggesting that the mouse cells were unable to support production of infectious progeny viral particles. Consistent with an additional block in viral entry, infectious JFH-1 particles produced in Huh-7 cells were not able to establish detectable HCV RNA replication in naïve mouse cells. CONCLUSION: Thus, this report expands the repertoire of HCV replication systems and possibly represents a step toward developing mouse models of HCV replication, but it also highlights that other species restrictions might continue to make the development of a purely murine HCV infectious model challenging

Rab13 Is Involved in the Entry Step of Hepatitis C Virus Infection

Membrane transport probably participates in the lifecycle of hepatitis C virus (HCV). Rab proteins are essential host factors for HCV RNA replication, but these proteins’ roles in other steps of the HCV lifecycle are not clear. The tight junction (TJ) plays a key role in HCV infection. Rab13 regulates the endocytic recycling of the TJ-associated proteins. Here we investigated whether Rab13 is involved in the HCV entry step. We used HuH-7-derived RSc cells and Li23-derived D7 cells. To evaluate the effect of Rab13 in HCV infection, we transfected the cells with siRNA targeting Rab13 before HCV infection. The down-regulation of Rab13 inhibited HCV infection. The D7 cells had showed a greater inhibitory effect against HCV infection compared to that in the RSc cells by Rab13 knockdown. Next, to evaluate the effect of Rab13 after infection, we inoculated the cells with HCV before transfection of the siRNA. The down-regulation of Rab13 did not show any effects after HCV infection. We further examined whether Rab13 would influence HCV RNA replication by using HCV replicon-harboring cells. The results revealed that Rab13 did not affect the step of HCV RNA replication. These results suggest that Rab13 plays an important role in the step of HCV entry

Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

Adenosine 5′-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome

Annexin A1 Negatively Regulates Viral RNA Replication of Hepatitis C Virus

Persistent infection with hepatitis C virus (HCV) often causes chronic hepatitis, and then shows a high rate of progression to liver cirrhosis and hepatocellular carcinoma. To clarify the mechanism of the persistent HCV infection is considered to be important for the discovery of new target(s) for the development of anti-HCV strategies. In the present study, we found that the expression level of annexin A1 (ANXA1) in human hepatoma cell line Li23-derived D7 cells was remarkably lower than that in parental Li23 cells, whereas the susceptibility of D7 cells to HCV infection was much higher than that of Li23 cells. Therefore, we hypothesized that ANXA1 negatively regulates persistent HCV infection through the inhibition of viral RNA replication. The results revealed that HCV production was significantly inhibited without a concomitant reduction in the amount of lipid droplets in the D7 cells stably expressing exogenous ANXA1. Further, we demonstrated that ANXA1 negatively regulated the step of viral RNA replication rather than that of viral entry in human hepatocytes. These results suggest that ANXA1 would be a novel target for the development of anti-HCV strategies

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a highly sensitive detection of enterovirus in the stool samples of acute flaccid paralysis cases

<p>Abstract</p> <p>Background</p> <p>In the global eradication program for poliomyelitis, the laboratory diagnosis plays a critical role by isolating poliovirus (PV) from the stool samples of acute flaccid paralysis (AFP) cases. In this study, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a rapid and highly sensitive detection of enterovirus including PV to identify stool samples positive for enterovirus including PV.</p> <p>Methods</p> <p>A primer set was designed for RT-LAMP to detect enterovirus preferably those with PV-like 5'NTRs of the viral genome. The sensitivity of RT-LAMP system was evaluated with prototype strains of enterovirus. Detection of enterovirus from stool extracts was examined by using RT-LAMP system.</p> <p>Results</p> <p>We detected at least 400 copies of the viral genomes of PV(Sabin) strains within 90 min by RT-LAMP with the primer set. This RT-LAMP system showed a preference for <it>Human enterovirus species C </it>(HEV-C) strains including PV, but exhibited less sensitivity to the prototype strains of HEV-A and HEV-B (detection limits of 7,400 to 28,000 copies). Stool extracts, from which PV, HEV-C, or HEV-A was isolated in the cell culture system, were mostly positive by RT-LAMP method (positive rates of 15/16 (= 94%), 13/14 (= 93%), and 4/4 (= 100%), respectively). The positive rate of this RT-LAMP system for stool extracts from which HEV-B was isolated was lower than that of HEV-C (positive rate of 11/21 (= 52%)). In the stool samples, which were negative for enterovirus isolation by the cell culture system, we found that two samples were positive for RT-LAMP (positive rates of 2/38 (= 5.3%)). In these samples, enterovirus 96 was identified by sequence analysis utilizing a seminested PCR system.</p> <p>Conclusions</p> <p>RT-LAMP system developed in this study showed a high sensitivity comparable to that of the cell culture system for the detection of PV, HEV-A, and HEV-C, but less sensitivity to HEV-B. This RT-LAMP system would be useful for the direct detection of enterovirus from the stool extracts.</p

Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines

　A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann-Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication

Inhibitory effects on HAV IRES-mediated translation and replication by a combination of amantadine and interferon-alpha

Hepatitis A virus (HAV) causes acute hepatitis and sometimes leads to fulminant hepatitis. Amantadine is a tricyclic symmetric amine that inhibits the replication of many DNA and RNA viruses. Amantadine was reported to suppress HAV replication, and the efficacy of amantadine was exhibited in its inhibition of the internal ribosomal entry site (IRES) activities of HAV. Interferon (IFN) also has an antiviral effect through the induction of IFN stimulated genes (ISG) and the degradation of viral RNA. To explore the mechanism of the suppression of HAV replication, we examined the effects of the combination of amantadine and IFN-alpha on HAV IRES-mediated translation, HAV replicon replication in human hepatoma cell lines, and HAV KRM003 genotype IIIB strain replication in African green monkey kidney cell GL37. IFN-alpha seems to have no additive effect on HAV IRES-mediated translation inhibition by amantadine. However, suppressions of HAV replicon and HAV replication were stronger with the combination than with amantadine alone. In conclusion, amantadine, in combination of IFN-alpha, might have a beneficial effect in some patients with acute hepatitis A

New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

BACKGROUND: Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed. 　 METHODOLOGY/PRINCIPAL FINDINGS: 　 　 Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.　 　 CONCLUSIONS/SIGNIFICANCE:　 　 We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin