Simplified RNA secondary structure mapping by automation of SHAPE data analysis

SHAPE (Selective 2′-hydroxyl acylation analysed by primer extension) technology has emerged as one of the leading methods of determining RNA secondary structure at the nucleotide level. A significant bottleneck in using SHAPE is the complex and time-consuming data processing that is required. We present here a modified data collection method and a series of algorithms, embodied in a program entitled Fast Analysis of SHAPE traces (FAST), which significantly reduces processing time. We have used this method to resolve the secondary structure of the first ∼900 nt of the hepatitis C virus (HCV) genome, including the entire core gene. We have also demonstrated the ability of SHAPE/FAST to detect the binding of a small molecule inhibitor to the HCV internal ribosomal entry site (IRES). In conclusion, FAST allows for high-throughput data processing to match the current high-throughput generation of data possible with SHAPE, reducing the barrier to determining the structure of RNAs of interest

A Nucleotide Binding Motif in Hepatitis C Virus (HCV) NS4B Mediates HCV RNA Replication

Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. We have identified a nucleotide binding motif (NBM) in one of the virus's nonstructural proteins, NS4B. This structural motif binds and hydrolyzes GTP and is conserved across HCV isolates. Genetically disrupting the NBM impairs GTP binding and hydrolysis and dramatically inhibits HCV RNA replication. These results have exciting implications for the HCV life cycle and novel antiviral strategies

An N-Terminal Amphipathic Helix in Hepatitis C Virus (HCV) NS4B Mediates Membrane Association, Correct Localization of Replication Complex Proteins, and HCV RNA Replication

Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the virus's nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AH's ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4B's N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies

Potential for Hepatitis C Virus Resistance to Nitazoxanide or Tizoxanide▿

Nitazoxanide and its primary metabolite, tizoxanide, inhibit hepatitis C virus (HCV) replication in HCV replicon systems. To study the potential for resistance, we subjected Huh7 cells harboring HCV replicons to serial passage in 250 μM G418 and increasing concentrations of nitazoxanide or tizoxanide. Passage of the replicon-containing cell lines in either compound resulted in increases in the 50% effective concentrations (EC50s) (7- to 13-fold), EC90s (14- to 36-fold), and 50% cytotoxic concentrations (2- to 4-fold) of both compounds. Serial passage in either compound did not alter the susceptibility of HCV replicons to ribavirin or 2′-C-methylcytidine. Interestingly, serial passage in nitazoxanide or tizoxanide resulted in increased sensitivity to alpha interferon 2b: EC50s and EC90s were reduced three- and eightfold, respectively. Replicons isolated from these cell lines had no greater ability to confer tizoxanide resistance, or increased susceptibility to alpha interferon, than replicons isolated from the parental cell line that had not previously been exposed to nitazoxanide or tizoxanide. These findings are indicative of a cell-mediated activity differing from that of other anti-HCV drugs but complementary with interferon and are consistent with the enhanced response rates observed clinically when nitazoxanide is combined with pegylated interferon therapy. Finally, unlike data for other compounds in advanced clinical development for HCV, these data are consistent with resistance in HCV replicon-containing cell lines conferred by changes in the host and not by mutations in the virus

Amphipathic Helix-Dependent Localization of NS5A Mediates Hepatitis C Virus RNA Replication

We identified an N-terminal amphipathic helix (AH) in one of hepatitis C virus (HCV)’s nonstructural proteins, NS5A. This AH is necessary and sufficient for membrane localization and is conserved across isolates. Genetically disrupting the AH impairs HCV replication. Moreover, an AH peptide-mimic inhibits the membrane association of NS5A in a dose-dependent manner. These results have exciting implications for the HCV life cycle and novel antiviral strategies

Long-term culture of human liver tissue with advanced hepatic functions

A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver’s natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450–mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.NMRC (Natl Medical Research Council, S’pore)NRF (Natl Research Foundation, S’pore)Published versio