Virus replication is possible in many cultured cells. There are, however, several examples in which cells do not provide the machinery for efficient import of some viruses, leading to a partial or total block of infection. Hepadnaviruses serve as an example of this phenomenon, as they are fully capable of replicating in a variety of hepatoma cells but are incapable of infecting the same cells. As determined previously, hepadnaviruses efficiently bind to primary hepatocytes, followed by viral uptake (3
). Productive infection, however, is poor, indicating a block in virus release after entry.
The method of LMCT allowed us to circumvent the entry steps of endocytosis and efficiently initiate productive infection. The time course of infection was shown to be in accordance with the data known from infection of primary cells with duck hepatitis B virus (DHBV) (3
) and HBV (5
). First, the partially double-stranded viral DNA was converted to cccDNA, followed by amplification of intracellular DNA. Surface protein secretion started at a time when intracellular viral DNA had already accumulated, and consequently, virus secretion was delayed. After the onset of surface antigen and virus secretion, the number of intracellular HBV genomes remained at a level similar to that observed upon infection of primary duck hepatocytes by DHBV (39
), which shows the same replication strategy as HBV. As in the in vivo situation, more surface proteins than virus particles were expressed.
Total virus production and genome multiplication were strikingly higher than in primary cells (5
) or than reported for the susceptible cell line HepaRG (11
). While these systems secreted only a few viruses, generally fewer than those to which the cells were subjected, LMCT led to a significant secretion of progeny HBV and multiplied the number of initially incorporated HBV genomes ~110-fold. The efficiency and quantity of the hepadnaviral infection markers by LCC-mediated transfer were very similar to those from experiments using chimeric adenoviruses with an HBV genome (36
). Therefore, LMCT may serve as a system for analyzing the replication of HBV directly from patients' sera without cloning and use of a vector. Amplification and cloning may lead to the loss of minor populations within a pool. In addition, LMCT is easier to handle and acts much faster, allowing, e.g., application for phenotype drug resistance assays.
The transfer by lipids was not significantly inhibited by actin-depolymerizing drugs. Thus, the entry pathway of macropinocytosis, phagocytosis, or caveolae-mediated endocytosis did not significantly participate in LCC uptake. These observations are in accordance with infections of primary duck hepatocytes in which actin inhibitors were shown not to block DHBV infection (3
The addition of chlorpromazine excluded an entry via clathrin-mediated endocytosis that usually targets viral cargos to the late endosome where acidification occurs. Apparently, LMCT circumvented this passage by direct fusion of the lipid with the plasma membrane, which has been described to be the physiological entry mechanism for the human immunodeficiency virus and herpesviruses (herpes simplex virus and cytomegalovirus) (47
). The observation that HBV capsids, after this mode of entry, were still capable of initiating infection is in accordance with the finding that hepatitis B viruses, in contrast to influenza and parvo- and adenoviruses (8
), do not require acidification for infection (13
A receptor-independent uptake by fusion may also explain why LMCT is so efficient in induction of infection. First, it allows virtually every cell to take up the capsids. In addition, it circumvents the release from the endosome that was shown to be the limiting step for parvoviruses (38
). Receptor-independent uptake makes it possible to load high numbers of capsids per cell irrespective of the cell type. This allows the analysis of early infection events by microscopy.
We showed that the capsids were rapidly transported to the nucleus and that generation of intranuclear viral DNA occurred as early as 15 min p.l. Such a short time span can be explained only by an active transport, since passive diffusion was calculated to take 1 h (35
). Accordingly, inhibitor experiments showed that the microtubule transport system is required for detectable capsid accumulation at the nuclear envelope and for generation of intranuclear capsid-released virus DNA. Capsids were found to be localized with the microtubule network that is the dominant intracytoplasmic long-distance cellular transporter (35
). Binding assays with digitonin-permeabilized cells and coimmune precipitation assays showed that this binding does not require vesicles.
These studies indicate that the capsids are actively transported toward the nucleus using the cellular microtubule transport system, as was previously shown for herpesviruses (35
). Such a capsid-mediated transport seems to reflect the physiological intracellular infection pathway. During infection, DHBV capsids undergo intracytoplasmic transport toward the nucleus, as suggested by Köck et al. (20
) and for HBV by Rabe et al. (29
), followed by a rapid release of the genome into the karyoplasm (29
). The occurrence of released viral DNA exclusively inside the nucleus indicates that disassembly of HBV capsids is not random but is subject to a tight regulation. Apparently, this regulation is a well-conserved phenomenon throughout mammalian cells, as is indicated by the same observations for both hepatoma and cervix carcinoma cell lines.
As shown by our results, LMCT may provide a potent tool in viral research. LMCT is suitable to address questions about early infection events for a variety of viruses, regardless of whether they replicate in the cytoplasm or in the nucleus of nondividing cells.