The chaperone dynein LL1 mediates cytoplasmic transport of empty and mature hepatitis B virus capsids.

BACKGROUND & AIMS: Hepatitis B virus (HBV) has a DNA genome but replicates within the nucleus by reverse transcription of an RNA pregenome, which is converted to DNA in cytoplasmic capsids. Capsids in this compartment are correlated with inflammation and epitopes of the capsid protein core (Cp) are a major target for T cell-mediated immune responses. We investigated the mechanism of cytoplasmic capsid transport, which is important for infection but also for cytosolic capsid removal. METHODS: We used virion-derived capsids containing mature rcDNA (matC) and empty capsids (empC). RNA-containing capsids (rnaC) were used as a control. The investigations comprised pull-down assays for identification of cellular interaction partners, immune fluorescence microscopy for their colocalization and electron microscopy after microinjection to determine their biological significance. RESULTS: matC and empC underwent active transport through the cytoplasm towards the nucleus, while rnaC was poorly transported. We identified the dynein light chain LL1 as a functional interaction partner linking capsids to the dynein motor complex and showed that there is no compensatory transport pathway. Using capsid and dynein LL1 mutants we characterized the required domains on the capsid and LL1. CONCLUSIONS: This is the first investigation on the detailed molecular mechanism of how matC pass the cytoplasm upon infection and how empC can be actively removed from the cytoplasm into the nucleus. Considering that hepatocytes with cytoplasmic capsids are better recognized by the T cells, we hypothesize that targeting capsid DynLL1-interaction will not only block HBV infection but also stimulate elimination of infected cells. LAY SUMMARY: In this study, we identified the molecular details of HBV translocation through the cytoplasm. Our evidence offers a new drug target which could not only inhibit infection but also stimulate immune clearance of HBV infected cells.

Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket.

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 beta (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 beta. The binding sites of importin beta 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 beta. Collectively, our findings describe a unique nuclear import strategy not only for viruses but for all karyophilic cargos.

Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation.

Capsids of hepatitis B virus and other hepadnaviruses contain a cellular protein kinase, which phosphorylates the capsid protein. Some phosphorylation sites are shown to be essential for distinct steps of viral replication as pregenome packaging or plus strand DNA synthesis. Although different protein kinases have been reported to phosphorylate the capsid protein, varying experimental approaches do not allow direct comparison. Furthermore, the activity of a specific protein kinase has not yet been correlated to steps in the hepadnaviral life cycle. In this study we show that capsids from various sources encapsidate active protein kinase Calpha, irrespective of hepatitis B virus genotype and host cell. Treatment of a virion expressing cell line with a pseudosubstrate inhibitor showed that inhibition of protein kinase C phosphorylation did not affect genome maturation but resulted in capsid accumulation and inhibited virion release to the medium. Our results imply that different protein kinases have distinct functions within the hepadnaviral life cycle.

Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids.

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.

Intracellular transport of hepatitis B virus.

For genome multiplication hepadnaviruses use the transcriptional machinery of the cell that is found within the nucleus. Thus the viral genome has to be transported through the cytoplasm and nuclear pore. The intracytosolic translocation is facilitated by the viral capsid that surrounds the genome and that interacts with cellular microtubules. The subsequent passage through the nuclear pore complexes (NPC) is mediated by the nuclear transport receptors importin alpha and beta. Importin alpha binds to the C-terminus of the capsid protein that comprises a nuclear localization signal (NLS). The exposure of the NLS is regulated and depends upon genome maturation and/or phosphorylation of the capsid protein. As for other karyophilic cargos using this pathway importin alpha interacts with importin beta that facilitates docking of the import complex to the NPC and the passage through the pore. Being a unique strategy, the import of the viral capsid is incomplete in that it becomes arrested inside the nuclear basket, which is a cage-like structure on the karyoplasmic face of the NPC. Presumably only this compartment provides the factors that are required for capsid disassembly and genome release that is restricted to those capsids comprising a mature viral DNA genome.

Lipid-mediated introduction of hepatitis B virus capsids into nonsusceptible cells allows highly efficient replication and facilitates the study of early infection events.

The hepatitis B virus (HBV) is an enveloped DNA virus which is highly infectious in vivo. In vitro, only primary hepatocytes of humans and Tupaia belangeri or the novel HepaRG cell line are susceptible to HBV, but infection is inefficient and study of early infection events in single cells is unsatisfactory. Since hepatoma cells replicate the virus efficiently after transfection, this limited infection efficiency must be related to the initial entry phase. Here, we describe the lipid-based delivery of HBV capsids into nonsusceptible cells, circumventing the natural entry pathway. Successful infection was monitored by observing the emergence of the nuclear viral covalently closed circular DNA and the production of progeny virus and subviral particles. Lipid-mediated transfer initiated productive infection that was at least 100-fold more effective than infection of permissive cell cultures. High-dose capsid transfer showed that the uptake was not receptor limited and allowed the intracellular transport of capsids and genomes to be examined microscopically. The addition of inhibitors confirmed an entry pathway by fusion of the lipid with the plasma membrane. By indirect immune fluorescence and native fluorescence in situ hybridization, we followed the pathway of capsids and viral genomes in individual cells. We observed an active microtubule-dependent capsid transfer to the nucleus and a subsequent release of the viral genomes exclusively into the karyoplasm. Lipid-mediated transfer of viral capsids thus appears to allow efficient introduction of genetic information into target cells, facilitating studies of early infection events which are otherwise impeded by the small number of viruses entering the cell.

Nuclear import of hepatitis B virus capsids and release of the viral genome.

While studying the import of the hepatitis B virus genome into the nucleus of permeabilized tissue culture cells, we found that viral capsids were imported in intact form through the nuclear pore into the nuclear basket. Import depended on phosphorylation of the capsid protein and was mediated by the cellular transport receptors importin alpha and beta. Virus-derived capsids that contained the mature viral genome were able to release the viral DNA and capsid protein into the nucleoplasm. The uncoating reaction was independent of Ran, a GTP-binding enzyme responsible for dissociating other imported cargo from the inner face of the nuclear pore. Immature capsids that did not contain the mature viral genome reached the basket but did not release capsid proteins nor immature genomes into the nucleoplasm. The different fate of mature and immature capsids after passing the nuclear pore indicates that the outcome of a nuclear import event may be regulated within the nuclear basket.