Acetophenone 4-nitrophenylhydrazone inhibits Hepatitis B virus replication by modulating capsid assembly.

Hepatitis B virus (HBV) is the causative agent of chronic liver disease and is correlated with the development of subsequent hepatic cirrhosis and hepatocellular carcinoma. Current antiviral therapy using nucleos(t)ide analogs is effective in suppressing viral replication and interrupting disease progression, but HBV is rarely cured completely. Thus, there remains an unmet need for the development of novel anti-HBV drugs. Here, we report the identification of N-(4-Nitrophenyl)-1-phenylethanone hydrazone (ANPH) as a novel structural class of selective inhibitors targeting the replication of the HBV genome using adenovirus vector-mediated HBV genome transduction. ANPH inhibited viral genome replication in HepG2.2.15 cells by inducing the formation of empty capsids devoid of pregenomic RNA without affecting its transcription and translation. Biochemical assays using a truncated core protein consisting of the assembly domain showed that ANPH accelerates the formation of morphologically intact capsids. Taken together, we propose that ANPH might provide a new structural scaffold to design a new anti-HBV drug in medicinal chemistry as well as chemical probes for HBV core protein functions in the future.

Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit.

The complicated replication mechanisms of hepatitis B virus (HBV) have impeded HBV studies and anti-HBV therapy development as well. Herein we report efficient genome replication of HBV applying adenovirus vectors (AdVs) showing high transduction efficiency. Even in primary hepatocytes derived from humanized mice the transduction efficiencies using AdVs were 450-fold higher compared than those using plasmids. By using an expression unit consisting of the CMV promoter, 1.03-copy HBV genome and foreign poly(A) signal, we successfully generated an improved AdV (HBV103-AdV) that efficiently provided 58 times more pregenomic RNA than previously reported AdVs. The HBV103-AdV-mediated HBV replication was easily and precisely detected using quantitative real-time PCR in primary hepatocytes as well as in HepG2 cells. Notably, when the AdV containing replication-defective HBV genome of 1.14 copy was transduced, we observed that HBV DNA-containing circular molecules (pseudo-ccc DNA) were produced, which were probably generated through homologous recombination. However, the replication-defective HBV103-AdV hardly yielded the pseudo-ccc, probably because the repeated sequences are vey short. Additionally, the efficacies of entecavir and lamivudine were quantitatively evaluated using this system at only 4 days postinfection with HBV103-AdVs. Therefore, this system offers high production of HBV genome replication and thus could become used widely.

Receptor (CD155)-dependent endocytosis of poliovirus and retrograde axonal transport of the endosome.

Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic mice carrying the human PV receptor (hPVR/CD155) gene. Here, we demonstrated by using an immunoelectron microscope that PV particles exist on vesicle structures in nerve terminals of neuromuscular junctions. We also demonstrated in glutathione S-transferase pull-down experiments that the dynein light chain, Tctex-1, interacts directly with the cytoplasmic domain of hPVR. In the axons of differentiated rat PC12 cells transfected with expression vectors for hPVRs, vesicles composed of PV and hPVR alpha, as well as a mutant hPVR alpha (hPVRM alpha) that had a reduced ability to bind Tctex-1, colocalized with Tctex-1. However, vesicles containing PV, dextran, and hPVR alpha had only retrograde motion, while those containing PV, dextran, and hPVRM alpha had anterograde or retrograde motion. Topical application of the antimicrotubule agent vinblastine to the sciatic nerve reduced the amount of virus transported from the calf to the spinal cord. These results suggest that direct efficient interaction between the cytoplasmic domain and Tctex-1 is essential for the efficient retrograde transport of PV-containing vesicles along microtubules in vivo.