Clonal expansion of hepatocytes during chronic woodchuck hepatitis virus infection

Abstract

Chronic hepadnavirus infections cause liver damage with ongoing death and regeneration of hepatocytes. In the present study we set out to quantify the extent of liver turnover by measuring the clonal proliferation of hepatocytes by using integrated viral DNA as a genetic marker for individual hepatocyte lineages. Liver tissue from woodchucks with chronic woodchuck hepatitis virus (WHV) infection was assayed for randomly integrated viral DNA by using inverse PCR. Serial endpoint dilution of viral-cell junction fragments into 96-well plates, followed by nested PCR and DNA sequencing, was used to determine the copy number of specific viral cell junctions as a measure of the clonal distribution of infected cell subpopulations. The results indicated that the livers contained a minimum of 100,000 clones of >1,000 cells containing integrated DNA, representing at least 0.2% of the hepatocyte population of the liver. Because cells with integrated WHV DNA comprised only 1-2% of total liver cells, it is likely that the total number of clones far exceeds this estimate, with as much as one-half of the liver derived from high copy clones of >1,000 cells. It may be inferred that these clones have a strong selective growth or survival advantage. The results provide evidence for a large amount of hepatocyte proliferation and selection having occurred during the period of chronic WHV infection ( approximately 1.5 years) in these animals.

Fig. 1.

Inverse PCR assay for detection of viral–cell junctions. DNA fragments containing viral–cell junctions were released by SacI digestion (S), ligated to produce circular DNAs, and converted to linear DNAs by digestion with HindIII (H). These fragments were diluted to microtiter plates, digested with AflII (A), and amplified by nested PCR by using primers specific to viral sequences at the left and right ends (arrows).

Fig. 2.

Analysis of liver fragments for clonal expansion of cells by serial dilution. DNA was extracted from pieces of liver containing 8.7 × 10⁴ cells (wc371 liver fragment 1a, Right) and 1.1 × 10⁵ cells (wc371 liver fragment 5a, Left) based on DNA recovery and assuming a woodchuck genome size of 2.5 pg. Following SacI cleavage, ligation, and HindIII digestion, aliquots were distributed to microtiter trays for nested PCR, followed by gel electrophoresis. Lanes A1 to A12 were from reactions containing in combination 2.5% (Right) or 22% (Left) of the total DNA, and subsequent groups of 12 contained serial 3-fold dilutions of the DNA. The marker (m) at the left and middle of each panel is HindIII cut bacteriophage λ-DNA mixed with a 1-kbp DNA ladder. Three occurrences of common band in the wc371 liver fragment 1a are marked with asterisks.

Fig. 3.

Size distribution of cell clones detected by end point dilution inverse PCR. Clones exceeding 27 cells in size were detected by end point dilution inverse PCR, as illustrated in Fig. 2. The results obtained with 15 liver fragments from wc370, 5 from wc371, and 15 from wc372, containing in total 2.5 × 10⁶, 5.0 × 10⁵, and 3.0 × 10⁶ cells, respectively, are summarized in the three panels.

Fig. 4.

Analysis of liver tissue for variability in levels of WHV infection. Consecutive sections of liver biopsies collected from wc359 (A, B, and F) and wc370 (C–E) at ≈15 months of age were analyzed for the distribution of hepatocytes expressing WHV core antigen (A, C, and E) and nucleic acids (B and D). F shows a plasmid (negative) control for hybridization specificity. A and B show consecutive liver sections from wc359 containing a patch of cells with low or undetectable WHV core antigen and nucleic acids (arrows). C and D similarly show consecutive sections of liver wc370 with variability in levels of WHV core antigen and nucleic acids across the lobule. E shows an area of wc370 with low levels of WHV core antigen. For comparison, G and H show WHV core antigen detection in liver tissue collected 7 and 11 weeks after infection during a transient infection (wc38; ref. 23) that was fully resolved by 21 weeks after infection. Magnification: ×120. (Scale bar, 100 μm.)