Sequence analysis of integrated hepatitis B virus DNA during HBeAg-seroconversion

Figures & data

Fig. 1 Clone sizes of hepatocytes containing virus-cell junctions in in vivo datasets.

Hepatocyte clones detected in HBeAg-positive (n = 22) and HBeAg-negative (n = 13) groups in Tu et al.¹⁸ and Mason et al.^17,19 were estimated by copy number of repeated virus-cell junctions contained in these cellular clones. Only accurately determined clone sizes using repeated virus-cell junctions detected in liver fragments (and not from liver slide sections or laser-microdissected material) were included for this comparison. Geometric mean ± SD; **p < 0.01, two-sided Mann–Whitney test

Fig. 2 Analysis workflow.

Flowcharts describing the analysis workflow to identify unique virus-cell junctions in HBeAg-positive and HBeAg-negative patients (a), and integration in the proximity of various cellular genomic features, including structural and functional regions (b)

Patient numbers per group in the datasets used in this study

Publication	In silico	In vitro	HBeAg(+)	HBeAg(−)
Tu et al.^Citation14	10,000^a	15^b	0	0
Mason et al.^Citation19	–	–	19	7
Tu et al.^Citation18	–	–	3	10
Mason et al.^Citation17	–	–	0	5
Number of unique integration events after filtration	883	161	364	192

^aIndependent integrations generated in in silico simulation prior to filtering

^bIndependent infections

HBeAg(+) HBeAg-positive, HBeAg(−) HBeAg-negative

Fig. 3 Chromosomal distribution of virus-cell junctions.

Distribution of the integration breakpoints across human chromosomes and in the HBV genome. Each line represents an integration event at a particular locus in the HBV and human genome (hg38) in the in silico dataset (a), in vitro dataset (b), HBeAg-positive patients (c), and HBeAg-negative patients (d). Chromosome numbers are shown on the outer rim. Viral integration breakpoints were randomly produced in the in silico model based on the frequency distribution of HBV junctions observed in the in vitro and in vivo datasets. It should be noted that the HBV genome has been expanded in scale and cropped to the area analyzed by invPCR to show more detailed positional information

Fig. 4 Cellular structural features in proximity to HBV DNA integration junctions.

Percentages of HBV integration junctions in each dataset [in silico (gold), in vitro (gray), HBeAg-positive (blue), and HBeAg-negative (green)] were calculated with respect to (a) occurrence in chromosomal fragile sites (CFS), b proximity to S/MAR, and c occurrence in early-/late-replication timing regions (early replication (ER), mid replication (MR) and late replication (LR) regions of the host cell genome, and not distinguished (ND)). **p < 0.01 and ***p < 0.001, Normal approximation z-test

HBV DNA integration into cellular repeat regions

Type	In silico	In vitro	HBeAg(+)	HBeAg(−)
Tandem repeats	0.49	2.31	0.90	0.86
DNA transposons	2.25	3.24	2.26	2.16
Retrotransposons
LTR	6.15	6.94	9.05	7.33
SINE	5.76	6.48	7.69	6.90
LINE	16.60	19.44	11.09	15.09
SVA	–	0.46	0.23	–
Others	0.88	1.39	2.94	0.86

All figures are given in percentages of the total number of integrations (after quality control filtration as shown in Fig. 2a) for each dataset

HBeAg(+) HBeAg-positive, HBeAg(−) HBeAg-negative, LTR long terminal repeat, SINE short interspersed nuclear elements, LINE long interspersed nuclear elements, SVA SINE-VNTR-Alu

Fig. 5 Cellular functional features in proximity to HBV DNA integration junctions.

Percentages of HBV integration junctions in each dataset [in silico (gold), in vitro(gray), HBeAg-positive (blue), and HBeAg-negative (green)] were calculated with respect to occurrence in functional regions [separated into intergenic, intronic, exonic regions, promoters, UTRs and non-coding RNAs (ncRNA) (a)]. We also measured the distance from the transcriptional start site (TSS) of the closest gene (b) and the nearest CpG island (both upstream and downstream) (c). The frequency is shown as a percentage of all integration events per dataset. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, Normal approximation z-test

Fig. 6 Tissue expression of genes containing HBV DNA integrations.

Tissue transcriptional expression levels [mean fragments per kilobase per million mapped reads (FPKM) ± SD] of genes containing HBV DNA integration is shown for 9 normal tissues (liver, kidney, lung, colon, thyroid, breast, brain, heart and white blood cells) and Huh7 cells infected with HBV (a). Outliers were excluded using Robust regression and Outlier removal (ROUT) method. Percentages of HBV integrations in genes that were either expressed or not expressed in Huh7 (in silico and in vitro datasets) and liver tissue (in silico and in vivo datasets) (b). **p < 0.01, Normal approximation z-test

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