6 research outputs found

    The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

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    Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 μM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 μM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development. © 2013 Tavis et al

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    Self-association mediated by the Ras association 1 domain of AF6 activates the oncogenic potential of MLL-AF6

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    MLL is a common target for chromosomal translocations associated with acute leukemia resulting in its fusion with a large variety of nuclear or cytoplasmic proteins that may activate its oncogenic properties by distinct but poorly understood mechanisms. The MLL-AF6 fusion gene represents the most common leukemogenic fusion of mixed lineage leukemia (MLL) to a cytoplasmic partner protein. Here, we identified a highly conserved Ras association (RA1) domain at the amino-terminus of AF6 as the minimal region sufficient for MLL-AF6 mediated myeloid progenitor immortalization in vitro and short latency leukemogenesis in vivo. Moreover, the ability of RA1 to activate MLL oncogenesis is conserved with its Drosophila ortholog, Canoe. Although the AF6 RA1 domain has previously been defined as an interaction surface for guanosine triphosphate–bound Ras, single amino acid substitutions known to abolish the AF6-Ras interaction did not abrogate MLL-AF6–mediated oncogenesis. Furthermore, fusion of MLL to heterologous RA domains of c-Raf1 or RalGDS, or direct fusion of MLL to constitutively active K-RAS, H-RAS, or RAP1 was not sufficient for oncogenic activation of MLL. Rather, the AF6 RA1 domain efficiently mediated self-association, suggesting that constitutive MLL self-association is a more common pathogenic mechanism for MLL oncogenesis than indicated by previous studies of rare MLL fusion partners

    Modularized CRISPR/dCas9 Effector Toolkit for Target-Specific Gene Regulation

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    The ability to control mammalian genes in a synergistic mode using synthetic transcription factors is highly desirable in fields of tissue engineering, stem cell reprogramming and fundamental research. In this study, we developed a standardized toolkit utilizing an engineered CRISPR/Cas9 system that enables customizable gene regulation in mammalian cells. The RNA-guided dCas9 protein was implemented as a programmable transcriptional activator or repressor device, including targeting of endogenous loci. For facile assembly of single or multiple CRISPR RNAs, our toolkit comprises a modular RNAimer plasmid, which encodes the required noncoding RNA components

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