Patterns of Single- and Double-stranded Hepatitis B Virus DNA and Viral Antigen Accumulation in Infected Liver Cells

Abstract

SUMMARYLiver sections from five patients with persistent hepatitis B virus (HBV) infection and active cirrhosis were shown to contain intracellular HBV DNA by in situ hybridization using cloned 3H-labelled HBV DNA probes. Two classes of infected cells, with different distributions throughout the liver, were distinguished: (i) cells containing a low copy number of double-stranded HBV nucleotide sequences, confined to the cell nucleus and thought to represent HBV DNA, and (ii) cells containing large amounts (estimated to be greater than 10 or 15 genome copies per cell) of HBV DNA, much of it in a single-stranded form and largely confined to the cell cytoplasm; these single-stranded regions represented widely separated regions of the HBV genome, in contrast to the structure of the DNA in mature virions. It is likely that these latter cells may be supporting viral DNA synthesis. Cells with large amounts of cytoplasmic HBV DNA invariably contained hepatitis B surface antigen (HBsAg) and in addition contained either no detectable hepatitis B core antigen (HBcAg), or cytoplasmic HBcAg or nuclear HBcAg in that order of frequency. Cytoplasmic HBcAg was highly predictive of the presence of large amounts of cytoplasmic HBV DNA in the same cell, while either nuclear HBcAg, or cytoplasmic HBsAg, were often seen both in cells with and without such levels of DNA. These patterns, relating HBV DNA and antigen content in naturally occurring asynchronous infection in a heterogeneous cell population, should provide a background to further studies of the virus replication cycle with a defined experimental system, when such a system becomes available. INTRODUCTIONPersistent infection with hepatitis B virus (HBV) in man is associated with different histological types of chronic liver disease in different patients, that reflect varying levels of active hepatocyte damage and inflammation, together with varying degrees of disturbance of liver architecture.In general, (i) healthy patients with minimal hepatocyte damage tend to have significant expression of hepatitis B surface antigen (HBsAg) within hepatocyte cytoplasm, (ii) immunosuppressed patients with minimal liver damage show abundant intranuclear hepatitis B core antigen (HBcAg) together with cytoplasmic membrane expression of HBsAg and (iii) patients with active liver damage show 'spotty' expression of both HBcAg and HBsAg (Gudat et al., 1975;Gerber & Thung, 1979). Three additional markers have been postulated to identify those patients with a higher general level of HBV replication within infected tissues. These are (i) the presence of detectable HBV DNA (or HBV virions) in a patient's serum (Werner et al., 1977;Bonino et al., 1981 ; Brechot et al., 1981 b), (ii) the presence of non-integrated 'free' HBV DNA in DNA extracts from infected liver (Brechot et al., 1981 a, b) and (iii) the presence of hepatitis B e antigen (HBeAg), rather than hepatitis B e antibody (anti-HBe), in a patient's serum. In different patients, the above markers of virus 'replication' tend to coexist, and...

Detection of hepatitis B virus-DNA in liver tissues in chronic type B liver diseases and its relevancy to viral antigens. A study by in situ hybridization using a biotinylated probe and streptavidin-alkaline phosphatase.

“…These findings were not confirmed in the present study. The result on the predominant cytoplasmic detection of HBV-DNA in hepatocytes was consistent with other reports on the detection of HBV-DNA by in situ hybridization using radiolabeled probes (3,7,10,11,17). It is thought that the replication cycle of HBV-DNA in hepatocytes resembles in some ways retroviruses replicating in the cytoplasm (22).…”

“…It is likely that assay by in situ hybridization is not so highly sensitive as by Southern or dot blot hybridization using radiolabeled or biotinylated probes (6,10,11,18). Therefore in situ hybridization for the detection of HBV-DNA in hepatocytes seems to be detectable in the hepatocytes containing a large amount of free replicating form of HBV-DNA (10,11,18). It was described in one report that HBV-DNA was not only detected in the hepatocytes but also bile duct epithelial cells and vascular elements (2).…”

Hepatitis B virus transcription in the infected liver.

“…From the intensity of this band relative to standards of known concentration we estimated that on average -50 molecules of HBV DNA per cell were present in the piece of liver analysed. The number of HBV DNA molecules in hepatocytes supporting HBV replication has been estimated by in situ hybridization to be several hundreds to several thousands (Burrell et al, 1982;Blum et al, 1983), but it has also been observed that not all hepatocytes supported HBV replication (Gowans et al, 1983). Thus it is likely that only a fraction of the hepatocytes in the liver biopsy used for DNA and RNA extraction was productively infected, and that the number of HBV genomes (-50) and HBV transcripts (20-50) per infected cell is in fact much higher.…”

Cytoplasmic (but not Nuclear) Hepatitis B Virus (HBV) Core Antigen Reflects HBV DNA Synthesis at the Level of the Infected Hepatocyte

“…We have previously examined several liv ers with markers of virus replication by in situ hybridization; since our original observation [2] that the bulk of HBV DNA in infected hepatocytes was localized in the cytoplasm, we have shown that much of this viral DN A is single-stranded and probably represents re plicative intermediates [3]. Furthermore, nuclei that were strongly positive for HBcAg by immunofluorescence contained little, if any, detectable viral DNA, although the cytoplasm of the same cells was often positive for viral DNA [3].…”

“…Furthermore, nuclei that were strongly positive for HBcAg by immunofluorescence contained little, if any, detectable viral DNA, although the cytoplasm of the same cells was often positive for viral DNA [3]. However, in that study HBcAg was detected by direct immunofluorescence, a technique lacking in sensitivity [4], By using a recently available high-titer rabbit anti-HBc in indirect immu nofluorescence or in peroxidase-antiperoxi dase (PAP) reactions, we have detected more widespread cytoplasmic HBcAg than was previously found by direct immunofluores cence [4].…”