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

Abstract

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.

FIG. 1.

Time course of an HBV infection induced by LCC in HuH-7 cells. Markers of LMCT-initiated infection. The cells were incubated with 2.5 LCC per cell for 4 h. Unbound LCC were then removed, and bound LCC that had not been internalized were inactivated by washing with serum containing DMEM, followed by trypsin treatment. The cells were seeded onto new dishes and cultivated for various times. The medium was changed after 17 h and every 3 days thereafter. Cells were harvested at the indicated time points. Viral genomes were quantified by real-time PCR, and surface proteins were detected by enzyme-linked immunosorbent assay. The columns show the total mean numbers of viral DNA and cccDNA (left log scale) and nanograms of HBsAg (right log scale) per 10-cm dish at different time points p.l. The absolute numbers are given at the top of each column, and the numbers of HBV DNA in the medium and HBsAg in the medium are shown in a cumulative form. The bars on top of the columns represent the ranges of the results. Detection limits were as follows: total intracellular HBV DNA, 1.8 × 10²; intracellular cccDNA, 5.6 × 10²; virus-related DNA (medium), 2.4 × 10³; surface proteins (medium), 39 pg. Further details are given in the text.

FIG. 2.

Low magnification of LMCT into HuH-7 cells at 17 h p.l. The cells were lipofected with 2.5 × 10⁵ LCC per cell. Capsids and NPC were detected by confocal laser scan microscopy after indirect immune staining. Negative, unlipofected cells; positive, capsid-lipofected cells. All cells showed an intracellular capsid fluorescence of similar intensity.

FIG. 3.

Localization of HBV capsids and released genomes after LMCT. Using 2.5 × 10⁵ LCC per cell, the fate of capsids and viral genomes could be visualized at different time points. Capsids and NPC were detected by indirect immune staining. Released viral genomes were visualized by NFISH. (A) Unlipofected HuH-7 cells and HuH-7 cells at (B) 15 min p.l., (C) 1 h p.l., and (D) 17 h p.l. Capsids accumulated as early as 15 min p.l. at the cytoskeleton and at the nuclear membrane, and released viral genomes were seen as spotty structures in the karyoplasm. Viral genomes accumulated in the karyoplasm within 1 h. Intranuclear capsids became observable at 17 h p.l. (E) HeLa cells at 17 h p.l. As in HuH-7 cells, capsids accumulated at the cytoskeleton, at the nuclear membrane, and within the nucleus. Released viral genomes were detected in defined areas of the karyoplasm.

FIG. 4.

Markers of LMCT-initiated HBV infection in the presence of different inhibitors. The columns show the total mean numbers of intracellular HBV DNA and cccDNA per 10-cm dish at 0 h p.l. and 17 h p.l. The HuH-7 cells were pretreated with the respective inhibitor for 30 min before 2.5 LCC per cell were added. The inhibitors were present during all incubation steps. pos. control, lipofected cells without inhibitor treatment. Detection limits were as follows: total HBV DNA, 1.4 × 10²; cccDNA, 6.6 × 10³.

FIG. 5.

Cytoskeletons of untreated and inhibitor-treated HuH-7 cells. Cells were treated either with the actin-depolymerizing drug cytochalasin D or latrunculin B (A) or with the microtubule-destabilizing drug nocodazole (B) for 30 min. Cells were then stained with an antiactin antibody (A) or an antitubulin antibody (B). To allow localization within the cell, a control antibody against the NPC was added. Both primary antibodies were visualized by a Cy3 secondary antibody. All samples showed a confocal section on the equatorial level of the nuclei, as indicated by the rim-like anti-NPC stain of the nuclei. (A) The untreated control showed the actin cortex at the plasma membrane, the actin network, and some dotty stain in the cytoplasm. Cells treated with cytochalasin D or latrunculin B were devoid of an actin network and the actin cortex. (B) The untreated control showed the filamentous structure of microtubules, while the nocodazole-treated cells exhibited a granular and diffuse pattern of tubulin, indicating a disruption of microtubules.

FIG. 6.

Localization of HBV capsids and released genomes in inhibitor-treated HuH-7 cells at 15 min p.l. (A) Unlipofected cells, (B) lipofected control, and cells treated with (C) chlorpromazine, (D) cytochalasin D, (E) latrunculin B, or (F) nocodazole. The same staining pattern was observed at 1 h p.l. Lipofection and staining were done as described in the legend to Fig. 3.

FIG. 7.

Interaction of HBV capsids with microtubules. Capsids were incubated with HuH-7 cells as LCC by LMCT (A and B) or with digitonin-permeabilized HuH-7 cells without lipids (C and D). Microtubules (A and C) and capsids (B and D) were stained by indirect immune fluorescence. In both assays, capsids and tubulin showed a colocalization (arrowheads). (E) Coimmune precipitation of capsids and tubulin. The capsids were preincubated with a HuH-7 cell lysate for 1 h at 37°C. Capsids that were precipitated by anticapsid antibody-saturated biomagnetic beads coprecipitated tubulin from the cell lysate (right lane). The negative control (left lane), which contains the cell lysate but no capsids, showed that anticapsid antibody-saturated beads are unable to precipitate tubulin. The positive control (middle lane) showed tubulin precipitation with antitubulin antibody-saturated beads. The tubulin proteins were visualized by immune blotting after SDS-PAGE.