Hepatitis D virus isolates with low replication and epithelial-mesenchymal transition-inducing activity are associated with disease remission

Abstract

Clearance of hepatitis D virus (HDV) viremia leads to disease remission. Large hepatitis delta antigen (L-HDAg) has been reported to activate transforming growth factor β, which may induce epithelial-mesenchymal transition (EMT) and fibrogenesis. This study analyzed serum HDV RNA "quasispecies" in HDV-infected patients at two stages of infection: before and after alanine aminotransferase (ALT) elevations. Included in the study were four patients who went into remission after ALT elevation and three patients who did not go into remission and progressed to cirrhosis or hepatocellular carcinoma. Full-length HDV cDNA clones were obtained from the most abundant HDV RNA species at the pre- and post-ALT elevation stages. Using an in vitro model consisting of Huh-7 cells transfected with cloned HDV cDNAs, the pre- or post-ALT elevation dominant HDV RNA species were characterized for (i) their replication capacity by measuring HDV RNA and HDAg levels in transfected cells and (ii) their capacity to induce EMT by measuring the levels of the mesenchymal-cell-specific protein vimentin, the EMT regulators twist and snail, and the epithelial-cell-specific protein E-cadherin. Results show that in patients in remission, the post-ALT elevation dominant HDV RNA species had a lower replication capacity in vitro and lower EMT activity than their pre-ALT elevation counterparts. This was not true of patients who did not go into remission. The expression of L-HDAg, but not small HDAg, increased the expression of the EMT-related proteins. It is concluded that in chronically infected patients, HDV quasispecies with a low replication capacity and low EMT activity are associated with disease remission.

Fig 1

Replacement of the original dominant species by novel dominant species during the disease courses of three patients chronically infected with different genotypes of HDV. At least 100 HDV colonies were randomly selected by large-scale screening carried out using quasispecies-specific RFLP analysis at each time point for each case. The original dominant HDV strain and the novel dominant strain are indicated by the symbols □ and △, respectively. The values on each curve are relative percentages of the original and novel dominant HDV species. The percentages of the novel dominant species gradually increased as the percentages of the original dominant species decreased after ALT elevations (U/L, units per liter). The periods of IFN-α treatment are marked by the hatched bars below the graphs.

Fig 2

Correlation of clinical courses with HDV replication and HDAg expression of novel dominant HDV variants after ALT elevation in patients in remission. The clinical courses of patient II (A) and patient III (B) are shown, and arrows mark the time points of blood sampling to detect the original and novel dominant HDV quasispecies. Comparison of intracellular HDV RNA replication, HDAg expression, and secreted HDV virions in culture medium of original and novel dominant HDV variants isolated from patient II (C) and patient III (D) at early and late time points on days 3, 6, and 9 posttransfection with an HBV expression plasmid. The equal loading of RNA and protein samples was assessed by hybridization with a DIG-labeled DNA probe for G3PDH and a monoclonal antibody specific for heat shock protein Hsc70, respectively.

Fig 3

Correlation of clinical courses with HDV replication and HDAg expression of novel dominant HDV species after elevation of ALT levels in patient I, who went into disease remission. (A) The clinical course of patient I is shown. Arrows mark the time points of blood sampling to detect the dominant HDV species at the first, early, and late time points of the disease course. The period of IFN-α treatment is marked by the hatched bar below the graph. (B) Comparison of intracellular HDV RNA replication, HDAg expression, and secreted HDV virions in culture medium among the dominant HDV species isolated from patient I at different time points on days 3, 6, and 9 posttransfection with an HBV expression plasmid. The equal loading of RNA and protein samples was assessed by hybridization with a DIG-labeled DNA probe for G3PDH and a monoclonal antibody specific for heat shock protein Hsc70, respectively.

Fig 4

Correlation of clinical courses with HDV replication and HDAg expression of novel dominant HDV variants after ALT elevation in patients with persistently elevated ALT levels and adverse outcomes. Shown are the clinical courses of patient V (A) and patient VII (B) and a comparison of intracellular HDV RNA replication, HDAg expression, and secreted HDV virions in culture medium of the original and novel dominant HDV quasispecies isolated from patient V (C) and patient VII (D) at the early and late time points on days 3, 6, and 9 posttransfection.

Fig 5

Expression profiles of EMT markers determined by Western blotting. (A) EMT factor protein expression in Huh-7 cells transfected with the empty vector (pcDNA3.1) or the original or novel dominant HDV expression plasmid. (B) S-HDAg- or L-HDAg-expressing plasmids. Quantification of TGF-β1 was done with supernatants incubated under 2% FBS collected at the end of day 3. The basal level of TGF-β1 in culture medium without incubation with Huh-7 cells was 210 pg/ml. Intensities of bands shown for each EMT marker panel are indicated by asterisks. Statistical significance, P < 0.05 compared with the control, was determined by the two-tailed Student t test.

Fig 6

Expression levels of induced EMT markers at different doses of L-HDAg-expressing plasmids. Snail, twist, E-cadherin, and vimentin levels in Huh-7 cells transfected with the empty vector (pcDNA3.1) and different doses of L-HDAg-expressing plasmids from three different HDV genotypes (A) and triplicates of the same genotype (B) were analyzed by Western blotting. Heat shock protein Hsc70 was used as a loading control in each lane for the quantification of TGF-β1 in the supernatants collected after 3 days of incubation under 2% FBS. The basal level of TGF-β1 in culture medium without incubation with Huh-7 cells was 210 pg/ml. Intensities of bands shown for each EMT marker panel are indicated by asterisks. Statistical significance, P < 0.05 compared with control, was determined by the two-tailed Student t test.

Fig 7

Immunofluorescence staining of expressed EMT markers in Huh-7 cells transfected with large or small HDAg expression plasmids. S-HDAg and L-HDAg expression plasmids were transfected to Huh-7 cells (middle and right panels, respectively). The cells were doubly stained with human anti-HDV antibody and mouse or rabbit anti-EMT markers (E-cadherin, β-catenin, vimentin, twist, snail) after 3 days of transfection. Rhodamine-conjugated rabbit anti-human IgG, FITC-conjugated sheep anti-mouse IgG, or FITC-conjugated goat anti-rabbit IgG secondary antibodies were used to show the HDAg and EMT markers in red and green immunofluorescence stainings. Photographs (original magnification, ×630) were taken with a confocal fluorescence microscope (Axiovert 200M; Zeiss).