Volume 130, Issue 4, Pages 1153-1168 (April 2006) High Prevalence and Mapping of Pre-S Deletion in Hepatitis B Virus Carriers With Progressive Liver Diseases  Bing–Fang Chen, Chun–Jen Liu, Guey–Mei Jow, Pei–Jer Chen, Jia–Horng Kao, Ding–Shinn Chen  Gastroenterology  Volume 130, Issue 4, Pages 1153-1168 (April 2006) DOI: 10.1053/j.gastro.2006.01.011 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Immune epitopes and functional domains within the HBV pre-S region. The pre-S region consists of the pre-S1 and pre-S2 domains. The pre-S1 domain contains 119 amino acids (used in this study) and is further divided into 2 parts: N half (aa 1–57) and C half (aa 58–119). The pre-S2 domain contains 55 amino acids. The pre-S domain contains many B or T epitopes and exerts multiple functions as shown. N half of pre-S1 contains a hepatocyte-binding site essential for infection. C half of pre-S1 contains a heat shock protein 70 (Hsc70)-binding site and cytosolic anchorage determinant (CAD) important for dual topology of L proteins and a nucleocapsid-binding site for virion morphogenesis. C half of pre-S1 also contains an S-promoter and a CCAAT binding factor (CBF)-binding site necessary for expression of S gene. Pre-S2 domain has a polymerized human serum albumin (pHSA)-binding site. Solid triangle, myristylation at second amino acid; open triangle, N-link glycosylation at N-4 of the M protein; shaded triangle, O-link glycosylation at T-37 of the M protein. Gastroenterology 2006 130, 1153-1168DOI: (10.1053/j.gastro.2006.01.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Prevalence of pre-S deletion mutants in 152 patients infected with genotype B, C, or mixed genotypes (genotype B, C, and/or recombinants). The frequency of pre-S deletion increased with advancing clinical stages in both genotypes B- and C-infected patients (2.5% and 25% in chronic carriers to 50.0% and 57.1% in hepatocellular carcinoma patients for genotypes B and C, respectively). Genotype C has a higher frequency of pre-S deletion than genotype B. The number shown in each bar represents the number of cases in each category. CC, chronic carrier; CH, chronic hepatitis; LC, cirrhosis; HCC, hepatocellular carcinoma. Gastroenterology 2006 130, 1153-1168DOI: (10.1053/j.gastro.2006.01.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 The amino acid sequences of the 88 pre-S1/S2 deletion mutants from 3 chronic carriers, 9 chronic hepatitis, 7 cirrhosis (3A), and 25 hepatocellular carcinoma patients (3B). Mapping of the wild-type pre-S1/S2 domain is shown at the top, and the number above the map indicates the amino acid site of the defined domain. Shaded box indicates the deleted region; dot box indicates frame shift mutation, and the number in the box indicates the length of this box. *Nonsense mutation, and ▵ indicates point mutation of the start codon. Deletions were more often in the C half of pre-S1 (45 of 88, 51.1%) and the N terminus of pre-S2 domain (60 of 88, 68.2%). The deletion of 5 functional sites are indicated and shown at right hand column. S, T, N, M, and P indicate 5 functional sites: S-promoter, topology (CAD or Hsc70 binding site), nucleocapsid binding site, the start codon of M protein, and pHSA site, respectively. +, −, and ▵ indicate presence of deletion, absence of deletion, and point mutation, respectively. Gastroenterology 2006 130, 1153-1168DOI: (10.1053/j.gastro.2006.01.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 The amino acid sequences of the 88 pre-S1/S2 deletion mutants from 3 chronic carriers, 9 chronic hepatitis, 7 cirrhosis (3A), and 25 hepatocellular carcinoma patients (3B). Mapping of the wild-type pre-S1/S2 domain is shown at the top, and the number above the map indicates the amino acid site of the defined domain. Shaded box indicates the deleted region; dot box indicates frame shift mutation, and the number in the box indicates the length of this box. *Nonsense mutation, and ▵ indicates point mutation of the start codon. Deletions were more often in the C half of pre-S1 (45 of 88, 51.1%) and the N terminus of pre-S2 domain (60 of 88, 68.2%). The deletion of 5 functional sites are indicated and shown at right hand column. S, T, N, M, and P indicate 5 functional sites: S-promoter, topology (CAD or Hsc70 binding site), nucleocapsid binding site, the start codon of M protein, and pHSA site, respectively. +, −, and ▵ indicate presence of deletion, absence of deletion, and point mutation, respectively. Gastroenterology 2006 130, 1153-1168DOI: (10.1053/j.gastro.2006.01.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 The nucleotide sequences of the pre-S clones obtained from 3 patients. RB2-4, RB2-1, and RB2-5 isolated from chronic carrier RB2; D6-2, D6-5, and D6-8 from HCC patient D6; G2-3, G2-9, G2-1, and G2-8 from HCC patient G2. Reference sequences of genotype B isolate (AY293309) and genotype C isolate (AF233236) are shown at the top. Positions of nucleotide sequences of the pre-S region and the start codon of pre-S1 and pre-S2 genes are indicated above the reference sequence. The dashes indicate identical sequences compared with the reference sequences of genotype B isolate. The slashes indicate deletion. RB2-4, D6-2, and G2-3 are genotype B; D6-5 and G2-9 are genotype C; RB2-1, RB2-5, D6-8, G2-1, and G2-8 are recombinants between HBV genomes of genotypes B and C. Gastroenterology 2006 130, 1153-1168DOI: (10.1053/j.gastro.2006.01.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Plots of similarity (generated by SimPlot) of a set of reference sequences to those of (A) RB2-1, (B) RB2-5, (C) D6-8, (D) G2-1, and (E) G2-8 clones. The reference sequences involved in recombination are genotype B (AY293309, black line) and genotype C (AF233236, gray line) and an out group sequence genotype F (AB036910, dotted line). Each curve is a comparison between the query sequence (pre-S region) and a reference sequence. Each point plotted is the percentage identity within a sliding window 100 base pairs wide centered on the position plotted, with a step size between points of 20 base pairs, and GapStrip off. The horizontal bars above plots are a cartoon of the pre-S regions of these recombinants. The colors are consistent with those used for the similarity curves and indicate the genotype to which that part of genome is most similar based on the adjacent similarity plot. Gastroenterology 2006 130, 1153-1168DOI: (10.1053/j.gastro.2006.01.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 The proposed model for the generation of deletion mutants and their possible roles in liver damage and carcinogenesis. Gastroenterology 2006 130, 1153-1168DOI: (10.1053/j.gastro.2006.01.011) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions