Amongst the depicted viruses human hepatitis B virus (HBV) is particular (figure 2). HBV is an enveloped pararetrovirus that multiplies via RNA synthesis. It enters the cell by a receptor(s) that have not been unequivocally identified yet. Independent upon acidification it releases the viral capsid into the cytoplasm. Subsequently the capsid surrounding the partially double stranded viral DNA is transported to the nucleus in which the genome becomes released. Cellular enzymes convert the viral genome to the covalently closed circular DNA (cccDNA) that is the template for mRNA synthesis. One mRNA of supergenomic length, the pregenomic RNA (PG) is bicistronic and encodes for the capsid protein and the viral polymerase (pol).  Pol binds speci-fically to PG thereby facilitatin - ing encapsidation into the assembling capsid. Being only active in the capsid environment Pol converts the encapsidated RNA into the partially double stranded viral DNA. In contrast to all other viruses (exception: caulimoviruses of plants) the HBV capsids derived from cellular entry are identical to the mature progeny capsids.
Hence not only incoming
capsids but also the progeny ones are targeted to the nucleus (fig.1, red arrow) leading to amplification of the nuclear cccDNA. Only after sufficient amounts of surface proteins are synthesized the mature capsids become enveloped by the surface proteins leading to secretion of progeny HBV. |
Nuclear transport. As depicted, the capsids release the genome exclusively into the nucleus implying a tight regulation of capsid disintegration. We analyzed the interactions at the nucleus in more detail using Digitonin-permeabilized cells. Digitonin permeabilizes the plasma membrane leaving the nuclear membrane integer.
We showed that the capsids interact with the NPC via the cellular nuclear import receptors importin a and ß. However RNA-containing capsids did not interact with the NPC (figure 4A), capsid with an immature DNA genome interact with the NPC without becoming imported (figure 4B) into the karyoplasm, while mature capsids containing the partially double stranded DNA caused nuclear capsid stain (figure 4C)  and released genomes. Consistently only those capsids capable to interact with the nucleus exposed a nuclear localization signal (NLS) on their surface, while RNA-containing capsids hide the NLS in their interior. NLS are stretches of basic amino acids that interact with importin a. Nuclear transport mechanisms are phylogenetically well conserved. Consequently electron microscopy after microinjection of capsids into the cytoplasm of Xenopus laevis oocytes confirmed the interaction with the NPCs (Figure 5; collaboration with Nelly Panté, UBC, Canada). Further immature and mature capsids were depicted to enter the nuclear basket integer leading to a redefinition of the maximal nuclear pore size to be 39 nm (Panté & Kann, MBC, 2002).
The basket is a cage-like structure on the karyoplasmic side of the NPC where cargo and import receptors dissociate, allowing that the cargo (in this case the capsid) diffuse deeper into the karyoplasm. So why do immature capsids arrest in the basket? And why do mature capsids enter the karyoplasm? Cross-linked mature capsids, unable to dissociate, behave like immature ones. They become arrested, implying that both capsid types interact with a protein of the nuclear basket. In collaboration with my lab at Giessen University, Germany, this protein could be identified to be nucleoporin 153 (Nup153), which is essential for cell viability.
Having these assays allows us (i) to evaluate Nup153 on import and export processes, (ii) to identify its function in capsid disassembly and (iii) evaluate the further fate of the genome. |
The studies are complemented by studying other viruses or subviral structures. In order to learn more about cargo-motor protein interactions we are currently evaluating the intracytoplasmic transport of the HIV integrase (HIV IN) in collaboration with Michel Fournier and Sébastien Desfarges in our department. HIV IN is not only of interest as it is an essential protein of the HIV preintegration complex but also as its small size allows a rapid identification of interaction domains.
Further nuclear interactions are investigated using parvoviruses. Like HBV capsids parvoviruses replicate in the nucleus, are small enough to pass the nuclear pore and contain a potential NLS hidden on one capsid protein. Nonetheless the nuclear entry of the parvoviral genome is controversial. Our analyses showed that despite of the potential NLS different parvoviruses cause local nuclear envelope break-down (NEBD). Physiologically NEBD occurs in mitosis, meiosis and apoptosis and our cell-free assays may help to unravel the signalling and nuclear degradation in these essential processes. |
For replication viruses have to transport their genome to the place where multiplication of the genomic information occurs. Viruses that make use of the nuclear replication processes and infect non-dividing cells have to deliver their genome through cytoplasm towards the nuclear envelope followed by entry into the karyoplasm by passing the nuclear pore complex (NPC) (for overview see figure 1). As nucleic acids are not karyophilic per se attached viral proteins have to mediate distinct and well coordinated interactions with the different cellular transport machineries. Evidently the location of the virus (or its subviral structure) has to be related with genome release in order to prevent premature abortion of the viral life cycle.
How ever after transfection of viral DNA hepatoma cell lines produce in vivo-likeamounts of virus indicating restrictions in viral entry. We there-fore replaced the viral envelope by lipids allowing the analysis of HBV travel towards the nucleus. Figure 3 depicts the time course of capsid transport and genome release. This system allowed determining the intracytoplasmic transport system and participating motor complexes. Detailed analysis shall identify the domains on capsid and motor proteins.