Capsids and the enclosed DNA of adenoviruses, including the species C viruses adenovirus type 2 (Ad2) and Ad5, and herpesviruses, such as herpes simplex virus type 1 (HSV-1), are targeted to the nuclei of epithelial, endothelial, fibroblastic, and neuronal cells. Cytoplasmic transport of fluorophore-tagged Ad2 and immunologically detected HSV-1 capsids required intact microtubules and the microtubule-dependent minus-enddirected motor complex dynein-dynactin. A recent study with epithelial cells suggested that Ad5 was transported to the nucleus and expressed its genes independently of a microtubule network. To clarify the mechanisms by which Ad2 and, as an independent control, HSV-1 were targeted to the nucleus, we treated epithelial cells with nocodazole ( Many viruses, including adenoviruses (Ads) and herpesviruses, spread by intracellular transport within infected host cells, thus increasing the viral load in target organs and possibly causing severe disease (44). The 51 human Ad serotypesclassified into six species (A to F)-have distinct tropisms (19). For example, Ad type 2 (Ad2) and Ad5 (species C) and Ad3 (species B) are associated with upper-airway infections. Other serotypes are linked to epidemic keratoconjunctivitis (species D), pneumonia (species E), enteric infections (species A and F), or infections of hematopoietic cells (41). The Herpesviridae family consists of the alpha-, beta-and gammaherpesviruses. Like Ads, alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), infect different cell types both in cultures and in their hosts. After infection of mucosal or damaged cutaneous epithelium, these neurotropic viruses establish latent infections, primarily in sensory ganglia, that, upon reactivation, lead to recurrent epidermal lesions (40, 55). The ability to infect a broad range of postmitotic cells has made both Ads and herpesviruses useful gene delivery vehicles (21) that are currently being evaluated in clinical trials (29,39). For their application as therapeutic vectors and to identify new potential targets for antiviral therapy, it is crucial to understand how the genomes are targeted to the nucleus.The entry mechanisms for Ads and herpesviruses have been well studied. Ads are internalized by receptor-mediated endocytosis that is dependent on F actin and leave the endosomal pathway at various sites (recently reviewed in reference 11). The species C Ads, including Ad2 and Ad5, exit from a slightly acidic compartment of pH 6 at about 10 min postentry (16, 42), whereas Ad7 (species B) has been reported to escape from acidic late endosomes and lysosomes (32). In contrast, HSV-1 delivers its capsids into the cytosol upon fusion of the viral envelope with the plasma membrane (45,46). Both viruses then target their capsids to the cell nucleus, uncoat, and inject the enclosed linear double-stranded DNA genomes through the nuclear pores into the nucleoplasm for replication (14,35,53). A number of electron microscopy studies have shown that cytoplasmic capsids of both species C Ads and alpha...
In order to characterize the hepatitis B virus (HBV) hepatocellular receptor, several proteins have previously been identified in HepG2 hepatoma cells and in primary cultured normal human hepatocytes (PCHs) that reacted with an anti-idiotypic antibody against a preS1(21-47)-specific MAb (F35.25). Here, we report the identification of one of these preS1-binding proteins, a 35 kDa protein (preS1-BP35), as glyceraldehyde-3-phosphate dehydrogenase (GAPD). GAPD is well-known as a key enzyme involved in glycolysis and gluconeogenesis. Nevertheless, GAPD has also been shown to have many other functions such as protein kinase activity (GAPD-PK). HBV core particles derived from infected hepatocytes possess an associated kinase activity that phosphorylates HBcAg, and the nucleocapsid
Hepadnaviruses are DNA viruses but, as pararetroviruses, their morphogenesis initiates with the encapsidation of an RNA pregenome, and these viruses have therefore evolved mechanisms to exclude nucleocapsids that contain incompletely matured genomes from participating in budding and secretion. We provide here evidence that binding of hepadnavirus core particles from the cytosol to their target membranes is a distinct step in morphogenesis, discriminating among different populations of intracellular capsids. Using the duck hepatitis B virus (DHBV) and a flotation assay, we found about half of the intracellular capsids to be membrane associated due to an intrinsic membrane-binding affinity. In contrast to free cytosolic capsids, this subpopulation contained largely mature, double-stranded DNA genomes and lacked core protein hyperphosphorylation, both features characteristic for secreted virions. Against expectation, however, the selective membrane attachment observed did not require the presence of the large DHBV envelope protein, which has been considered to be crucial for nucleocapsid-membrane interaction. Furthermore, removal of surfaceexposed phosphate residues from nonfloating capsids by itself did not suffice to confer membrane affinity and, finally, hyperphosphorylation was absent from nonenveloped nucleocapsids that were released from DHBVtransfected cells. Collectively, these observations argue for a model in which nucleocapsid maturation, involving the viral genome, capsid structure, and capsid dephosphorylation, leads to the exposure of a membrane-binding signal as a step crucial for selecting the matured nucleocapsid to be incorporated into the capsid-independent budding of virus particles.
Hepadnavirus genome replication involves cytoplasmic and nuclear stages, requiring balanced targeting of cytoplasmic nucleocapsids to the nuclear compartment. In this study, we analyze the signals determining capsid compartmentalization in the duck hepatitis B virus (DHBV) animal model, as this system also allows us to study hepadnavirus infection of cultured primary hepatocytes. Using fusions to the green fluorescent protein as a functional assay, we have identified a nuclear localization signal (NLS) that mediates nuclear pore association of the DHBV nucleocapsid and nuclear import of DHBV core protein (DHBc)-derived polypeptides. The DHBc NLS mapped is unique. It bears homology to repetitive NLS elements previously identified near the carboxy terminus of the capsid protein of hepatitis B virus, the human prototype of the hepadnavirus family, but it maps to a more internal position. In further contrast to the hepatitis B virus core protein NLS, the DHBc NLS is not positioned near phosphorylation target sites that are generally assumed to modulate nucleocytoplasmic transport. In functional assays with a knockout mutant, the DHBc NLS was found to be essential for nuclear pore association of the nucleocapsid. The NLS was found to be also essential for virus production from the full-length DHBV genome in transfected cells and from hepatocytes infected with transcomplemented mutant virus. Finally, the DHBc additionally displayed activity indicative of a nuclear export signal, presumably counterbalancing NLS function in the productive state of the infected cell and thereby preventing nucleoplasmic accumulation of nucleocapsids.Viruses that replicate in the nucleus have evolved means to transport their infecting genome through the cytoplasm towards and into the nucleus. As described previously for adenovirus and herpesviruses, viruses typically exploit the preexisting cellular nucleocytoplasmic transport machinery for targeting the nucleocapsid to the nuclear pore, where the genome is released and imported as a more or less complex nucleoprotein, as exemplified by lentiviruses or influenza viruses (12,17,21,42). Nuclear targeting of the nucleocapsid is of particular importance in the case of the hepadnaviruses (hepatitis B viruses [HBVs]), small, enveloped animal viruses, which replicate their circular, partially double-stranded DNA genome only in part in the nucleoplasm. There, the incoming genome matures into an extrachromosomal, covalently closed, circular DNA molecule, the template for genomic RNA synthesis through the cellular transcription machinery (10, 29). The following steps, reverse transcription and plus-strand DNA synthesis, occur inside cytoplasmic nucleocapsids, giving rise to mature core particles. Of these, a fraction is diverted from the major pathway leading to the export of the enveloped virion, thereby serving to establish an elevated intranuclear pool of genome copies early on and to replenish this pool throughout persistent infection (41). For duck HBV (DHBV), it is well established that nuclea...
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