Virtually all DNA viruses including hepatitis B viruses (HBV) replicate their genome inside the nucleus. In non-dividing cells, the genome has to pass through the nuclear pore complexes (NPCs) by the aid of nuclear transport receptors as e.g. importin β (karyopherin). Most viruses release their genome in the cytoplasm or at the cytosolic face of the NPC, as the diameter of their capsids exceeds the size of the NPC. The DNA genome of HBV is derived from reverse transcription of an RNA pregenome. Genome maturation occurs in cytosolic capsids and progeny capsids can deliver the genome into the nucleus causing nuclear genome amplification. The karyophilic capsids are small enough to pass the NPC, but nuclear entry of capsids with an immature genome is halted in the nuclear basket on the nuclear side of the NPC, and the genome remains encapsidated. In contrast, capsids with a mature genome enter the basket and consequently liberate the genome. Investigating the difference between immature and mature capsids, we found that mature capsids had to disintegrate in order to leave the nuclear basket. The arrest of a karyophilic cargo at the nuclear pore is a rare phenomenon, which has been described for only very few cellular proteins participating in nuclear entry. We analyzed the interactions causing HBV capsid retention. By pull-down assays and partial siRNA depletion, we showed that HBV capsids directly interact with nucleoporin 153 (Nup153), an essential protein of the nuclear basket which participates in nuclear transport via importin β. The binding sites of importin β and capsids were shown to overlap but capsid binding was 150-fold stronger. In cellulo experiments using digitonin-permeabilized cells confirmed the interference between capsid binding and nuclear import by importin β. Collectively, our findings describe a unique nuclear import strategy not only for viruses but for all karyophilic cargos.

Assembly and disassembly of viral capsids are essential steps in the viral life cycle. Studies on their kinetics are mostly performed in vitro, allowing application of biochemical, biophysical and visualizing techniques. In vivo kinetics are poorly understood and the transferability of the in vitro models to the cellular environment remains speculative. We analyzed capsid disassembly of the hepatitis B virus in digitonin-permeabilized cells which support nuclear capsid entry and subsequent genome release. Using gradient centrifugation, size exclusion chromatography and immune fluorescence microscopy of digitonin-permeabilized cells, we showed that capsids open and close reversibly. In the absence of RNA, capsid re-assembly slows down; the capsids remain disintegrated and enter the nucleus as protein dimers or irregular polymers. Upon the presence of cellular RNA, capsids re-assemble in the nucleus. We conclude that reversible genome release from hepatitis B virus capsids is a unique strategy different from that of other viruses, which employs irreversible capsid destruction for genome release. The results allowed us to propose a model of HBV genome release in which the unique environment of the nuclear pore favors HBV capsid disassembly reaction, while both cytoplasm and nucleus favor capsid assembly.

To take advantage of the large number of well-characterized mouse immunoglobulins (IgGs) for the study of antibody-dependent cell-mediated cytotoxicity (ADCC) in human cells, we armed human cytotoxic lymphocytes with a mouse receptor for the Fc portion of IgG antibodies. The human ΝΚ-92 natural killer cell line was transduced with a mouse receptor gene (mCD16), which was stably expressed on the cell surface (referred to as NK-92 (mCD16) ). When tested against a B-lymphoblastoid cell line (BLCL) coated with mouse anti-CD20 IgG1, IgG2a or IgG2b monoclonal antibodies (mAbs), the newly expressed mouse Fc receptor enabled the NK-92 (mCD16) cells to kill the BLCL by ADCC. Next, using the NK-92 (mCD16) we compared mouse mAbs directed at B lineage specific CD antigens for their ability to induce ADCC against human Epstein-Barr virus- infected B lymphoblastoid (for anti-CD19, -CD20 and -CD21) or against myeloma (for anti-CD38 and -CD138) target cells. Our results demonstrated that the "NK-92 (mCD16) assay" allows convenient and sensitive discrimination of mouse mAbs for their ability to mediate ADCC in a human cellular system. In addition, our results provide examples of dissociation between opsonization and target cell killing through ADCC. These "murinized" human effector cells thus represent a convenient cellular tool for the study of ADCC.