Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the current study, we further characterized silymarin's antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA-dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti-HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV-infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell-to-cell spread of virus. Conclusion: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarin's antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell.

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.

Optical molecular imaging resulting from Cerenkov radiation has become a motivating topic recently and will potentially open new avenues for the study of small animal imaging. Cerenkov-based optical imaging taken from living animals in vivo has been studied with two-dimensional (2D) planar geometry and three-dimensional (3D) homogeneous mouse model. In this study, we performed 3D Cerenkov-based luminescence tomography (CLT) using a heterogeneous mouse model with an implanted Na(131)I radioactive source, which provided the accurate location for the reconstructed source. Furthermore, single photon emission computed tomography (SPECT) was utilized to verify the results of 3D CLT. We reconstructed the localization and intensity of an embedded radioactive source with various concentrations, and established a quantitative relationship between the radiotracer activity and the reconstructed intensity. The results showed the ability of in vivo CLT to recover the radioactive probe distribution in the heterogeneous mouse model and the potential of a SPECT imaging validation strategy to verify the results of optical molecular tomography.

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The hepadnaviral polymerase (P) functions in a complex with viral nucleic acids and cellular chaperones. To begin to identify contacts between P and its partners, we assessed the exposure of the epitopes of six monoclonal antibodies (MAbs) to the terminal protein domain of the duck hepatitis B virus P protein in a partially denaturing buffer (RIPA) and a physiological buffer (IPP150). All MAbs immunoprecipitated in vitro translated P well in RIPA, but three immunoprecipitated P poorly in IPP150. Therefore, the epitopes for these MAbs were obscured in the native conformation of P but were exposed when P was in RIPA. Epitopes for MAbs that immunoprecipitated P poorly in IPP150 were between amino acids (aa) 138 and 202. Mutation of a highly conserved motif within this region (T3; aa 176 to 183) improved the immunoprecipitation of P by these MAbs and simultaneously inhibited DNA priming by P. Peptides containing the T3 motif inhibited DNA priming in a dose-dependent manner, whereas eight irrelevant peptides did not. T3 function appears to be conserved among the hepadnaviruses because mutating T3 ablated DNA synthesis in both duck hepatitis B virus and hepatitis B virus. These results indicate that (i) the conserved T3 motif is a molecular contact point whose ligand can be competed by soluble T3 peptides, (ii) the occupancy of T3 obscures the epitopes for three MAbs, and (iii) proper occupancy of T3 by its ligand is essential for DNA priming. Therefore, small-molecule ligands that compete for binding to T3 with its natural ligand could form a novel class of antiviral drugs.Hepatitis B virus (HBV) is a small DNA virus that replicates by reverse transcription (reviewed in reference 9). It has a lipid envelope studded with viral glycoproteins that surrounds an icosahedral core particle composed of the core protein. Within the core particle are the viral nucleic acids and reverse transcriptase (P). Other hepadnaviruses infect woolly monkeys, woodchucks, ground squirrels, ducks, geese, and herons (6,17,29,31,32). Significant differences exist among the hepadnaviruses, but they share a high degree of hepatotropism, follow the same replication cycle, and have a nearly identical genetic organization.Hepadnaviral reverse transcription (34) occurs within cytoplasmic capsid particles. Reverse transcription begins with binding of P to an RNA stem-loop (ε) on the pregenomic RNA, and then this complex is encapsidated. Reverse transcription is primed by P itself, so minus-strand DNA is covalently linked to P (36, 40). Complexes containing P and the viral nucleic acids must be dynamic because three strand transfers are required to produce the mature circular viral DNA (21,22,38,41).The binding of P to ε requires the active participation of a molecular chaperone complex (13). In vitro reconstitution studies with recombinant duck hepatitis B virus (DHBV) P revealed that P-ε binding requires HSP90, HSP70, HSP40, HSP23, and HOP (2,11,14), although under certain circumstances only HSC70 and HSP40 are necessary (3). Because chaperones mod...