The role and functional domain of hepatitis B virus (HBV) X protein (HBx) in regulating HBV transcription and replication were investigated with a transient transfection system in the human hepatoma cell line HepG2 using wild-type or HBx-minus HBV genome constructs and a series of deletion or mutation HBx expression plasmids. We show here that HBx has augmentation effects on HBV transcription and replication as a HBV mutant genome with defective X gene led to decreased levels of 3.5-kb HBV RNA and HBV replication intermediates and that these decreases can be restored Hepatitis B virus (HBV) is the prototypic member of the Hepadnavirdae family, which contains a group of closely related hepatotropic small DNA viruses that infect their respective animal hosts (13,29). The HBV genome is a 3.2-kb, circular, partially double-stranded DNA molecule with four overlapping open reading frames (ORFs) named PC-C, PS-S, P, and X (35, 38). Upon HBV infection of the hepatocyte, the HBV genome is converted to covalently closed circular DNA in the nucleus. The covalently closed circular DNA serves as the template for transcription by the host RNA polymerase II, generating the 3.5-, 2.4-, 2.1-, and 0.7-kb viral transcripts that encode the HBV core and polymerase polypeptides, the large surface antigen polypeptide, the middle and major surface antigen polypeptides, and the HBx polypeptide. The expression of those transcripts are directed by four HBV promoters (Cp, PS1p, Sp, and Xp), respectively, and influenced by two HBV enhancers (Enh I and Enh II) (14,29,35,38,43). HBV replicates by reverse transcription of the viral pregenomic 3.5-kb RNA (pgRNA) using the HBV polymerase that catalyzes RNA-dependent DNA synthesis and DNA-dependent DNA synthesis (36,40). Besides encoding for the HBV core polypeptide and HBV DNA polymerase that compose the viral capsid, the greater-than-genome-length 3.5-kb pgRNA is also encapsidated and serves as the template for reverse transcription. The encapsidated pgRNA is converted into the 3.2-kb partially double-stranded genomic DNA inside the viral capsid in the cytoplasm of the hepatocytes (35,36,38,40). There are several putative regulatory steps for HBV replication, e.g., pgRNA synthesis, encapsidation of pgRNA, and reverse transcription of pgRNA. As the pgRNA encodes both the HBV polymerase and core polypeptides and serves an additional function as the replication template, regulation of the synthesis of this RNA is therefore a critical step in the viral life cycle (35,38,39).HBV infection is a worldwide health problem and is one of the major causes of hepatocellular carcinoma (HCC). The crucial role of HBV in hepatocarcinogenesis is established, while the mechanism by which HBV causes transformation of hepatocytes remains unclear (1, 2, 6). HBV X protein (HBx) has long been suspected of playing a positive role in hepatocarcinogenesis, as avian hepadnaviruses missing the X ORF seem not to be associated with HCC, and some HBx transgenic mice appear to develop HCC (15, 44) or be more sensitive to a car...

Ultrafast endocytosis generates vesicles from the plasma membrane as quickly as 50 ms in hippocampal neurons following synaptic vesicle fusion. The molecular mechanism underlying the rapid maturation of these endocytic pits is not known. Here we demonstrate that synaptojanin-1, and its partner endophilin-A, function in ultrafast endocytosis. In the absence of synaptojanin or endophilin, the membrane is rapidly invaginated, but pits do not become constricted at the base. The 5-phosphatase activity of synaptojanin is involved in formation of the neck, but 4-phosphatase is not required. Nevertheless, these pits are eventually cleaved into vesicles; within a 30-s interval, synaptic endosomes form and are resolved by clathrin-mediated budding. Then synaptojanin and endophilin function at a second step to aid with the removal of clathrin coats from the regenerated vesicles. These data together suggest that synaptojanin and endophilin can mediate membrane remodeling on a millisecond timescale during ultrafast endocytosis.

We demonstrate a methodology that utilizes the specificity of enzyme-substrate biomolecular interactions to trigger miniaturized tools under biocompatible conditions. Miniaturized grippers were constructed using multilayer hinges that employed intrinsic strain energy and biopolymer triggers, as well as ferromagnetic elements. This composition obviated the need for external energy sources, and allowed for remote manipulation of the tools. Selective enzymatic degradation of biopolymer hinge components triggered closing of the grippers; subsequent reopening was achieved with an orthogonal enzyme. We highlight the utility of these enzymatically triggered tools by demonstrating the biopsy of liver tissue from a model organ system and gripping and releasing an alginate bead. This strategy suggests an approach for the development of smart materials and devices that autonomously reconfigure in response to extremely specific biological environments.

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