Spread of Hepatitis B Viruses In Vitro Requires Extracellular Progeny and May Be Codetermined by Polarized Egress

Abstract

Viruses can spread by different mechanisms: via intracellular particles through cell junctions to neighboring cells or via secreted virions to adjacent or remote cells. The observation of clusters of hepadnavirus-infected cells both in vivo and in primary hepatocytes neither proves the first mechanism nor excludes the second. In order to test which mechanism, if not both, is used by hepatitis B viruses in order to spread, we used primary duck hepatocytes and duck hepatitis B virus (DHBV) as an infection model. If extracellular progeny virus alone determines spreading, neutralizing antisera or drugs blocking virus binding to hepatocytes should abolish secondary infection. In order to test this, we used DHBV envelope-specific neutralizing antisera, as well as suramin, a known inhibitor of infection. Both reagents strongly reduced hepatocellular attachment of viral particles and almost completely abolished primary infection, whereas an ongoing intracellular infection was not affected as long as no progeny virus was released. In contrast, incubation of infected primary hepatocytes with these reagents during release of progeny virus completely prevented secondary infection. Moreover, the combination of electron and immunofluorescence microscopy analyses revealed the residence of viral particles in cytoplasmic vesicles preferentially located near the basolateral membrane of infected hepatocytes. Taken together, these data strongly suggest that hepatitis B viruses mainly spread by secreted, extracellular progeny and point to polarized egress of viral particles into intercellular compartments, which restricts their diffusion and favors transmission of virus to adjacent cells.

The Promoter of Human Telomerase Reverse Transcriptase Is Activated During Liver Regeneration and Hepatocyte Proliferation

“…21 Chromatin immunoprecipitation. ChIP experiments were performed as described by Weinmann et al 22 .…”

Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles

“…We then permeabilized the paraformaldehyde-fixed cells with 0.1% Triton-X-100 for 10 minutes prior to immunofluorescence analysis. 25 We analyzed the staining with a confocal laser scanning microscope (LSM 510 META; Zeiss) and processed the images using Zeiss LSM Image Browser.…”

“…25 The PDHs were seeded into 6-well or 12-well plates at a density of about 1 ϫ 10 6 or 5 ϫ 10 5 liver cells per well, respectively. We performed the experiments 3 to 7 days postplating if not otherwise indicated.…”

“…25,30 We performed Immunogold staining on paraformaldehyde-fixed sections of PDHs using corespecific and L-specific antisera as described. 30 We acquired and processed the images using DigitalMicrograph (Gatan).…”

pH-independent entry and sequential endosomal sorting are major determinants of hepadnaviral infection in primary hepatocytes

“…Fetal PDHs were prepared and cultivated as previously described. 10 PDHs were seeded in 6-or 12-well plates at a density of about 10 6 or 5 ϫ 10 5 liver cells per well, respectively. PDHs were infected as previously described.…”