Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection

Background and Aims: The expression of hepatitis B viral (HBV) antigens in liver tissue reflects the replicative status of chronic HBV infection. We h ave previously recognized a novel marginal pattern of hepatitis B surface a ntigen (HBsAg) in hepatocytes, which usually clusters in groups and emerges at the late non-replicative phase. This study was designed to investigate whether the marginal-type HBsAg represented the gene product of a specific HBV-surface mutant. Methods: Microdissection of cirrhotic nodules homogeneously expressing marg inal HBsAg was performed on two of 12 resected livers from HBsAg-seropositi ve patients with hepatocellular carcinoma. The gene presumably encoding mar ginal HBsAg was polymerase chain reaction (PCR)-cloned, sequenced and analy sed. In vitro transfection and expression of the cloned surface mutant plas mids were performed on the Huh7 cell line to illustrate intrahepatic HBsAg expression. Results: Immunohistochemical staining revealed that the marginal HBsAg was positive for pre-S1 and thus contained large surface proteins. The PCR clon ing and sequencing of the genes presumably encoding marginal-type HBsAg in both cases revealed the same deletion at the 5' terminus (nt 2-55) of pre-S 2. A point mutation on the small-surface (S) antigen was also found in one case. The pre-S2 deletion sequence and the mutation sites of the S gene coi ncide with human lymphocyte antigen-restricted T- and/or B-cell epitopes. I n vitro transfection of the mutant plasmid revealed a blot-like retention o r accumulation of HBsAg in the cytoplasm or at the periphery of hepatocytes , accompanied by a decreased secretion of HBsAg in the culture supernatant, mimicking intrahepatic expression. Conclusion: A natural pre-S2 deletion mutant was identified in hepatocytes expressing a novel marginal pattern of HBsAg, which probably contains mutan t, large, surface proteins. The biological significance of the pre-S2 delet ion mutant should be interesting in view of the clustering proliferation of hepatocytes expressing marginal HBsAg. (C) 2000 Blackwell Science Asia Pty Ltd.