Despite its implication in the progression of hepatitis B virus (HBV)‐associated liver disease, the pro‐apoptotic function of HBx protein remains poorly understood. We show that the expression of HBx leads to hyperactivation of caspase‐8 and caspase‐3 upon treatment with tumor necrosis factor‐α (TNF‐α) or anti‐Fas antibody, and this activation is correlated with the sensitivity to apoptosis. We demonstrate cytoplasmic co‐localization and direct interaction between HBx and the cellular FLICE inhibitory protein (c‐FLIP), a key regulator of the death‐inducing signaling complex (DISC). Deletion analysis shows that the death effector domain 1 (DED1) of c‐FLIP is important for the observed interaction. Overexpression of c‐FLIP rescued the cells from HBx‐mediated apoptosis, with both the full‐length HBV genome and HBx expression vectors. Moreover, c‐FLIP and caspase‐8 inhibitor considerably protected cells from HBx‐mediated apoptosis. These data suggest that HBx abrogates the apoptosis‐inhibitory function of c‐FLIP and renders the cell hypersensitive towards the TNF‐α apoptotic signal even below threshold concentration. This provides a novel mechanism for deregulation of hepatic cell growth in HBV patients and a new target for intervention in HBV‐associated liver cancer and disease.

In research relating to the development of new drugs, hit identification and validations are critical for successful optimization of candidates. To achieve rapid identification of new lead compounds, high-throughput screening assays have been employed in many pharmaceutical companies and laboratories. However, their success depends on the assay system relevant to in vivo conditions and they are physically limited by the repertoire of compounds. As an alternative or complementary approach to high-throughput screening assays, virtual screening is an efficient method to identify drug candidates in silico from large chemical compound databases. Its usefulness has been verified by current applications that successfully retrieved hit and lead identifications against various disease targets. However, for better application, the scoring functions for distinguishing possible active and inactive compounds must beimproved. In this review, we provide an overview of pharmacophore-based virtual screening methods with a special focus on their successful application towards finding hits against various diseasetargets. Readers will rapidly gain insight into the recent successful applications of pharmacophore-based virtual screening. They will acknowledge that this technique is a powerful and cost-effective alternative to high-throughput assays. Although there are many hurdles yet to be resolved, virtual screening techniques will emerge as essential infrastructure and as a prerequisite for developing new lead compounds with therapeuticapplications.

Hepatitis B e antigen (HBeAg) is a secreted version of hepatitis B virus (HBV) core protein that promotes immune tolerance and persistent infection. It is derived from a translation product of the precore/core gene by two proteolytic cleavage events: removal of the amino-terminal signal peptide and removal of the carboxyl-terminal arginine-rich sequence. Four RXXR motifs are present at the carboxyl terminus of the HBeAg precursor, with the first two fused as¹⁵¹RRGRSPR¹⁵⁷. Genotype A possesses two extra amino acids at the first motif (¹⁵¹RRDRGRSPR¹⁵⁹), which weakens the first motif and separates it from the second one. Western blot analysis of patient sera revealed a single HBeAg form for genotypes B to D but two additional forms of larger sizes for genotype A. Site-directed mutagenesis and transfection experiments with human hepatoma cell lines indicated that HBeAg of genotype B is derived from cleavage at the first (¹⁵¹RRGR¹⁵⁴) motif. The major HBeAg form of genotype A corresponds to cleavage at the second (¹⁵⁶RSPR¹⁵⁹) motif, and the other two forms are cleavage products of the first (¹⁵¹RRDR¹⁵⁴) and third (¹⁶⁶RRRR¹⁶⁹) motifs, respectively. Only the cleavage product of the third motif of genotype A was observed in furin-deficient LoVo cells, and an inhibitor of furin-like proprotein convertases blocked cleavage of the first and second motifs in human hepatoma cells. In conclusion, our study reveals genotypic differences in HBeAg processing and implicates furin as the major enzyme involved in the cleavage of the first and second RXXR motifs.

While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo.

More than 300 million people worldwide are chronically infected with hepatitis B virus (HBV). Considering the very short generation time for a virus, and the high error rate associated with the reverse transcription step of HBV replication, decades of HBV infection are probably equivalent to million years of human evolution. The most important selective force during the natural course of HBV infection appears to be the immune response. The development of anti-HBe antibody in hepatitis B patients usually correlates with reduction of HBV viremia. As a consequence, escape mutants of anti-HBe are selected. The core promoter mutants express less HBe antigen (HBeAg) through transcriptional down regulation, while precore mutants express truncated products. We recently identified additional mutations that modulate HBeAg translation initiation, proteolytic cleavage, and secondary structure maintenance through a disulfide bond. The core promoter mutants have been associated with the development of fulminant hepatitis during acute infection and liver cancer during chronic infection. Consistent with their enhanced pathogenicity, core promoter mutants were found to replicate at up to 10-fold higher levels in transfected human hepatoma cells than the wild-type virus. Moreover, some core promoter mutants are impaired in virion secretion due to missense mutations in the envelope gene. These virological properties may help explain enhanced pathogenicity of core promoter mutants in vivo.