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\begin{document}

\title{Multi-Omics Panoramic~ Analysis of HBV Integration, Transcriptional
Regulation, and Epigenetic~ Modifications in PLC/PRF/5 Cell Line~~~~~~~~}



\author[1]{Guiwen Guan}%
\author[1]{Abudurexiti Abulaiti}%
\author[1]{Chia-Chen Chen}%
\author[1]{Zhiqiang Gu}%
\author[2]{Jing Yang}%
\author[1]{Ting Zhang}%
\author[3]{Xiaojie Chen}%
\author[1]{Zhao Zhou}%
\author[1]{Fengmin Lu}%
\author[1]{Xiangmei Chen}%
\affil[1]{Peking University School of Basic Medical Sciences}%
\affil[2]{Guangdong Provincial People's Hospital}%
\affil[3]{Capital Medical University Affiliated Beijing Friendship Hospital}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
\textbf{Background:~}The clearance or transcriptional silencing of
integrated HBV DNA is crucial for achieving a functional cure in
patients with chronic hepatitis B (CHB) and reducing the risk of
hepatocellular carcinoma (HCC) development. The PLC/PRF/5 cell line is
commonly used as an in vitro model for studying HBV integration. In this
study, we employed a range of multi-omics techniques to gain a panoramic
understanding of the characteristics of HBV integration in PLC/PRF/5
cells and to reveal the transcriptional regulatory mechanisms of
integrated HBV DNA.

\textbf{Methods:} Transcriptome long-read sequencing (ONT) was conducted
to analyze and characterize the transcriptional activity of different
HBV DNA integration sites in PLC/PRF/5 cells. Additionally, we collected
data related to epigenetic regulation, including whole-genome bisulfite
sequencing (WGBS), histone chromatin immunoprecipitation sequencing
(ChIP-seq), and assays for transposase-accessible chromatin using
sequencing (ATAC-seq), to explore the potential mechanisms involved in
the transcriptional regulation of integrated HBV DNA.

\textbf{Result:} Long-read RNA sequencing analysis revealed significant
transcriptional differences at various integration sites in the
PLC/PRF/5 cell line, with higher HBV DNA transcription levels at
integration sites on chr11, chr13, and the chr13/chr5 fusion chromosome
t (13:5). Combining long-read DNA and RNA sequencing results, we found
that transcription of integrated HBV DNA generally starts downstream of
the SP1, SP2, or XP promoters. ATAC-seq data confirmed that chromatin
accessibility has limited influence on the transcription of integrated
HBV DNA in the PLC/PRF/5 cell line. Analysis of whole-genome bisulfite
sequencing data showed that the methylation intensity of integrated HBV
DNA was highly negatively correlated with its transcription level
(\emph{r} = -0.8929, \emph{p} = 0.0123). After AzaD treatment, the
transcription level of integrated HBV DNA significantly increased,
especially for the integration chr17, which had the highest level of
methylation. Through ChIP-seq data, we observed the association between
histone modification of H3K4me3 and H3K9me3 with the transcription of
integrated HBV DNA.%
\end{abstract}%



\sloppy


\textbf{Authors}

Guiwen Guan\textsuperscript{1\#*}, Abudurexiti
Abulaiti\textsuperscript{1,2\#}, Chia-Chen Chen\textsuperscript{1,3\#},
Zhiqiang Gu\textsuperscript{1}, Jing Yang\textsuperscript{4}, Ting
Zhang\textsuperscript{1}, Xiaojie Chen\textsuperscript{5,6}, Zhao
Zhou\textsuperscript{1}, Fengmin Lu\textsuperscript{1,7*}, Xiangmei
Chen\textsuperscript{1*}

\textbf{Affiliations}

\textsuperscript{1}Department of Microbiology \& Infectious Disease
Center, School of Basic Medical Sciences, Peking University, Beijing,
100191, China.

\textsuperscript{2}Department of Cell Biology, School of Basic Medical
Sciences, Peking University, Beijing, 100191, China.

\textsuperscript{3} National Heart and Lung Institute Faculty of
Medicine (NHLI), Imperial College

22 London, Hammersmith campus, W12 0NN, London, UK

\textsuperscript{4} Medical Research Institute, Guangdong Provincial
People's Hospital (Guangdong Academy of Medical Sciences), Southern
Medical University, Guangzhou, 510080, China.

\textsuperscript{5} Liver Transplantation Center, National Clinical
Research Center for Digestive Diseases, Beijing Friendship Hospital,
Capital Medical University, Beijing 100050, China.

\textsuperscript{6} Clinical Research Center for Pediatric Liver
Transplantation of Capital Medical University, Beijing 100050, China.

\textsuperscript{7} Beijing Key Laboratory of Hepatitis C and
Immunotherapy for Liver Diseases, Peking University Hepatology
Institute, Peking University People's Hospital, Beijing, 100044, China.

\textsuperscript{\#} These authors contributed equally to this study

\textsuperscript{*} \textbf{Corresponding author:}

Xiangmei Chen:

Department of Microbiology \& Infectious Disease Center, School of Basic
Medical Sciences, Peking University, Beijing, 100191, China.

Tel: +86-10-82802807; Fax: +86-10-82805136;

E-mail:\emph{\href{mailto:xm_chen6176@bjmu.edu.cn}{\nolinkurl{xm\_chen6176@bjmu.edu.cn}}}

Fengmin Lu:

Department of Microbiology \& Infectious Disease Center, School of Basic
Medical Sciences, Peking University, Beijing, 100191, China.

Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver
Diseases, Peking University Hepatology Institute, Peking University
People's Hospital, Beijing, 100044, China.

Tel: 86-10-8280-5136; Fax: 86-10-8280-5136

E-mail:
\href{mailto:lu.fengmin@hsc.pku.edu.cn}{\nolinkurl{lu.fengmin@hsc.pku.edu.cn}}

Guiwen Guan

Department of Microbiology \& Infectious Disease Center, School of Basic
Medical Sciences, Peking University, Beijing, 100191, China.

Tel: 86-188-1017-2170

E-mail:
\href{mailto:guanguiwen@bjmu.edu.cn}{\nolinkurl{guanguiwen@bjmu.edu.cn}}

\textbf{Keywords:} Hepatitis B virus; Viral integration; PLC/PRF/5;
Multi-omics; Cripsr-cas9; RNA Interference.

\textbf{Financial Support}

This work was supported by the National Natural Science Foundation of
China {[}grant numbers: 82272315{]} and Beijing Municipal Natural
Science Foundation {[}grant numbers: 7222108{]}.

\textbf{Background}

Globally, approximately 296 million individuals suffered from chronic
HBV infection in 2019, resulting in over 820,000
deaths\textsuperscript{1}. Upon infecting the liver cells of the host,
HBV not only generates covalently closed circular DNA (cccDNA) within
the cells' nuclei but also integrates its double-stranded linear DNA
(dslDNA) into the human genome\textsuperscript{2}. The presence of
integrated HBV contributes to the instability of the chromosomal
structure in patients and allows for the transcription and translation
of viral proteins, including HBsAg and truncated
HBx\textsuperscript{3,4}. Research has demonstrated that integration is
the primary contributor to the presence of HBsAg in CHB patients with
HBeAg negative and low HBV load\textsuperscript{5,6}. The clearance or
transcriptional silencing of integrated HBV DNA is crucial for achieving
a functional cure in patients with CHB and reducing the risk of HCC.
However, the regulation mechanism of the transcription of the integrated
HBV DNA has not yet been fully elucidated.

Although some researchers have tried to simulate HBV DNA integration
through DNA insertion techniques, these methods significantly differ
from natural HBV DNA integration. Among cell models with integrated HBV
DNA, Hep3B and Huh-1 have only a few integration sites, while PLC/PRF/5,
the first discovered cell line integrated with HBV
DNA\textsuperscript{7}, possesses a large number of integration sites,
making it the most classical model for studying HBV DNA integration.
Furthermore, PLC/PRF/5 cells, unlike primary liver cells infected with
HBV, do not exhibit HBV replication. This characteristic enables
researchers to eliminate interference from cccDNA. However, the lack of
comprehensive understanding of the complex HBV DNA integration patterns
and transcription characteristics within PLC/PRF/5 cells limited the
research on HBV DNA integration based on the PLC/PRF/5 cell line.

The availability of various high-throughput sequencing technologies and
publicly accessible data in PLC/PRF/5 cells facilitate combined analysis
of multi-omics data when investigating the transcriptional regulation
mechanism of HBV DNA integration. In our previous study, we analyzed the
HBV DNA integration sites in the PLC/PRF/5 cell line using long-read DNA
sequencing technology\textsuperscript{8} and constructed a map of
integrated HBV DNA in PLC/PRF/5. In this study, we aim to investigate
the transcriptional activity of each integrated HBV DNA and explore the
transcriptional regulatory mechanisms of integrated HBV DNA by combining
DNA long-read sequencing, RNA long-read sequencing, WGBS, ChIP-seq, and
ATAC-seq. The findings from this study have the potential to enhance our
understanding of integrated HBV in PLC/PRF/5 cells and provide a
theoretical foundation for the future development of drugs targeting HBV
DNA integration.

\textbf{Materials and Methods}

\textbf{Cell line and Cell culture}

PLC/PRF/5 cells were purchased from the American Type Culture Collection
(Manassas, VA, USA). Cells were cultured in Dulbecco's Modified Eagle's
Medium (DMEM) supplemented with 10\% fetal bovine serum (Gibco, Life
Technologies, Carlsbad, CA, USA) in a humidified incubator maintained at
37\selectlanguage{ngerman}°C and 5\% CO\textsubscript{2}.

\textbf{~}

\textbf{RNA extraction and cDNA preparation}

Total RNA was extracted from the tissue using TRIzol reagent (Takara,
Kyoto, Japan). The purity of RNA was tested using the Nano Photometer
spectrophotometer (IMPLEN, Westlake Village, USA). cDNA libraries were
constructed from 1 \selectlanguage{greek}μ\selectlanguage{english}g of total RNA of PLC/PRF/5 cells using a cDNA-PCR
Sequencing Kit (SQK-PCS109) according to the manufacturer's protocol.
Briefly, reverse transcriptase was used to enrich full-length cDNAs and
add defined PCR adapters to both ends of the first-strand cDNA, followed
by 14 cycles of cDNA PCR using LongAmp Taq DNA polymerase (New England
Biolabs, Ipswich, MA, USA) with an 8-minute elongation time. The PCR
products were then subjected to ONT adaptor ligation using T4 DNA ligase
(New England Biolabs, Ipswich, MA, USA). Agencourt XP beads (Beckman
Coulter, Brea, USA) were used for DNA purification. The final cDNA
libraries were loaded onto FLO-MIN109 flow cells and analyzed on a
PromethION platform at Biomarker Technology Company (Beijing, China).

~

\textbf{Oxford Nanopore Technologies long read processing}

The data analysis methods are consistent with previous
literature\textsuperscript{9}. Briefly, raw reads were first filtered
with a minimum average read quality score of 7 and a minimum read length
of 500 bp. Ribosomal RNA was discarded after mapping to the rRNA
database. Next, full-length, non-chimeric (FLNC) transcripts were
identified by searching for the primer at both ends of the reads.
Clusters of FLNC transcripts were obtained after mapping to the
reference genome with minimap2 (V2.24)\textsuperscript{10}, and
consensus isoforms were generated after polishing within each cluster by
pinfish. The raw FASTQ data was subjected to quality control and
trimming using the built-in quality control tool of Porechop, with
default parameters. Low-quality sequences and adapter contamination were
removed from the data. The quality of ONT sequencing reads was evaluated
using Nanoplot V 1.33.0 \textsuperscript{11}, with default parameters.
FLNC transcripts were aligned to 2\selectlanguage{ngerman}× genotype A (PLC/PRF/5 cells) HBV
genome and human genome reference hg19 using minimap2. Transcripts
aligned to both HBV and human genomes were considered to originate from
integrated HBV DNA. Finally, the identified HBV transcripts were
visualized using Integrative Genomics Viewer software, version
2.11.4\textsuperscript{12}.

~

\textbf{Processing of WGBS data}

Raw sequencing FASTQ files were assessed for quality, adapter content,
and duplication rates with FastQC v0.11.7, then trimmed using
trim\_galore. Data alignment was performed using Bismark
(V0.24.0)\textsuperscript{13} to align the sequencing data to the human
hg19 and HBV reference genomes, generating SAM files with default
parameters. For methylation analysis, the
bismark\_methylation\_extractor was used to generate a report on global
genomic cytosine methylation. Then, the GlobalMethLev function in the
viewBS (V0.1.11)\textsuperscript{14} was used to compare the overall
methylation levels of the integrated HBV DNA with the host genome in
PLC/PRF/5 cells. The MethOneRegion function in viewBS was used to
visualize the methylation levels of the integrated HBV genome. The
GlobalMethLev function in viewBS was used to calculate the average
genomic methylation levels of different regions around the integrated
sites, and the R software (V4.2.2) pheatmap was used to visualize the
methylation results.

~

\textbf{Processing of ATAC-seq data}

Raw sequencing FASTQ files were assessed for quality, adapter content,
and duplication rates with FastQC v0.11.7, trimmed using trim\_galore,
and aligned with bowtie2 (v2.6.0)\textsuperscript{15} (parameters: -N 1
-X 2000) to the HBV and human genomes. PCR duplicates bias was removed
using the samtools markdup. ATAC-seq data were assessed for quality
control using the ATACseqQC\textsuperscript{16}, and the
factorFootprints function was used to detect and visualize the signal
changes around the motif regions where transcription factors bind.
MACS2\textsuperscript{17} software was used for peak calling with
parameters set as `--shift -100 --extsize 200'. BAM files were converted
to BigWig format, with key parameters `--binSize 50 --normalizeUsing
RPM', by deeptools\textsuperscript{18}. The deeptools plotProfile tool
was used to calculate ATAC-seq signal and visualize the chromatin
accessibility levels. The HINT-ATAC tool was used to predict potential
transcription factor information in PLC/PRF/5 cells.

~

\textbf{Processing of ChIP-seq data}

Raw sequencing FASTQ files were assessed for quality, adapter content,
and duplication rates with FastQC v0.11.7, trimmed using trim\_galore,
and aligned with bowtie2 (default parameters) to the HBV and human
genomes to generate BAM files. The markdup function in Samtools was used
to remove PCR-amplified duplicate sequences from the BAM files. Next,
chimeric reads were extracted using Samtools and aligned to the
integrated HBV DNA using bowtie2 with the `--no-softclip' parameter to
calculate the number of chimeric reads from different integration sites.
Chimeric read counts were normalized using RPM, and differences between
different samples were compared.

~

\textbf{Comparison of histone modification levels between groups}

The GSE113879 dataset WIG files were downloaded, representing the
histone modification levels of each position of HBV DNA normalized to
RPM. A custom script was used to correct the start site to the EcoRI
site. Next, the WIG file was converted to BigWig format using the UCSC
wigToBigWig software. The average signal values of the replicate
experiments were calculated using the deeptools bigwigCompare function
with the `---operation mean' parameter. Deeptools bigwigCompare function
was further used to calculate fold enrichment over the input of averaged
HBV RPM, setting the `--operation' parameter set to log\textsubscript{2}
ratio. Calculation of histone modification levels near the integration
site: The binary BigWig files of the GSM6341171, GSM6341172, and
GSM6341173 datasets were downloaded from the GEO database. The deeptools
plotProfile function was used to calculate the histone modification
levels in the region near the integration site.

\textbf{~}

\textbf{Real-time Reverse Transcription (RT)-PCR}

Real-time RT-PCR was performed as described
previously\textsuperscript{19}. \selectlanguage{greek}β-\selectlanguage{english}Actin was used as the reference gene
to determine gene expression. The primers used for real-time RT-PCR are
listed in \textbf{Table S1}.

~

\textbf{Western blot analysis}

Western blot analysis was performed as previously
described\textsuperscript{19}. Briefly, the lysed cell supernatant was
run on an SDS-PAGE and blotted with antibodies. The antibodies used in
western blot are anti-HBs (ab9193, Abcam, Cambridge, UK), anti-GAPDH
(AP7873b, Abcepta, Suzhou, China), and anti-\selectlanguage{greek}β-\selectlanguage{english}tubulin (AM1020b, Abgent,
CA, USA).

~

\textbf{Quantification of HBsAg}

The supernatant of the cultured cells was harvested at 48 h
post-treatment. Levels of hepatitis B surface antigen (HBsAg) in the
supernatant were quantified using an HBsAg quantitative determination
kit (Shenzhen New Industries Biomedical Engineering Co., Ltd., Shenzhen,
China) on a MAGLUMI X3 series automatic chemiluminescence immunoassay
analyzer according to the manufacturer's instructions.

\par\null

\textbf{Results}

\textbf{Research design}

The PLC/PRF/5 cell line is a well-established \emph{in vitro} cell model
for studying hepatitis B virus (HBV) integration and hepatocellular
carcinoma\textsuperscript{7}. Therefore, we initially collected the
high-throughput sequencing datasets associated with PLC/PRF/5 cells from
GEO (https://www.ncbi.nlm.nih.gov/geo/) and SRA databases
(https://www.ncbi.nlm.nih.gov/sra).

Our previous work has identified the multiple HBV integration in
PLC/PRF/5 cells using long-read whole-genome sequencing technology
(PacBio)\textsuperscript{8}. By integrating our previous PLC/PRF/5 DNA
sequencing data obtained with the PacBio platform and the data acquired
through targeted DNA long-read sequencing techniques in studies by Meng,
Ricardo Ramirez's studies\textsuperscript{20,21}, we constructed a
reference genome for HBV integration in the PLC/PRF/5 cell line.
Furthermore, we conducted transcriptome long-read sequencing (ONT) to
identify transcriptional characteristics of integrated HBV DNA in
PLC/PRF/5 cells. Based on transcriptomic analysis results, we further
employed epigenetic-related sequencing data, including WGBS, ChIP-seq,
and ATAC-seq, to determine the relationship between the epigenetic
modification and the transcription of integrated HBV DNA. Cellular
experiments were conducted to validate the regulatory mechanisms
exerting integrated HBV DNA transcription in PLC/PRF/5 cells. The
analysis flow chart of this study is illustrated in \textbf{Fig. 1}. The
datasets used in this study are summarized in \textbf{Table S2}.

\textbf{~}

\textbf{The transcriptional landscape of integrated HBV DNA in PLC/PRF/5
cells}

Due to limitations of low throughput and short read lengths, neither
Sanger sequencing nor second-generation high-throughput sequencing can
accurately characterize the transcriptional features of integrated HBV
DNA in PLC/PRF/5 cells. Therefore, we employed long-read transcriptome
sequencing base on ONT platform to sequence the transcripts from
PLC/PRF/5 cells and investigated the transcriptional features of
integrated HBV DNA.

A total of 7,238,100 cleaning reads was obtained, with an average length
of 806 bp. Among these reads, 1630 were identified as chimeric fragments
of the HBV DNA and human genome (\textbf{Fig. S1}). Initially, chimeric
reads were aligned to the genome of each integrated HBV (previously
acquired using PacBio WGS sequencing by our research group, as well as
in the studies by Meng and Ricardo Ramirez) to assign a transcriptional
signature to each integration event. As depicted in \textbf{Fig. 2}, the
integrated HBV DNA on chr4 and t (1;8) (chromosomal fusion between
chromosome 1 and 8) did not initiate transcription, while the remaining
integrated HBV DNA fragments did. Specifically, the integrated HBV DNA
on chr11, chr12, chr13, chr16, and chr17 utilized HBV promoters, while
those on chr3 and chr17 employed host promoters.

\textbf{~}

\textbf{The characteristic of HBV chimeric transcripts derived from
different integrated HBV DNA}

To investigate the transcriptional activity of HBV DNA at various
integration sites in PLC/PRF/5 cells, we quantified chimeric transcripts
originating from distinct integration sites using long-read
transcriptome sequencing. The results showed that the transcription
levels of HBV DNA on different integration sites vary substantially. The
highest number of transcripts were derived from integrated HBV DNA on
chr11, chr13, and t (5;13) (\textbf{Fig. 3A}), suggesting that these
three integration sites exhibit more active HBV DNA transcription and
are the primary sources of viral protein expression in the PLC/PRF/5
cells.

Next, we focus on the transcripts from integrated HBV DNA on different
chromosomes in PLC/PRF/5 cells, revealing transcriptional features of
the integrated HBV DNA. We discovered that the majority of integrated
HBV DNA utilized promoters SP1, SP2, and XP to initiate transcription.
Additionally, human polyA served as transcription terminal signals,
generating chimeric PreS1, PreS2/S, and HBx transcripts (\textbf{Fig.
3B}). While the HBV integrated fragment contains multiple promoters
(chr13, t (13:5)), the transcription of integrated HBV exhibits promoter
selectivity \textbf{(Fig. 3C \& Fig. S2B)}. In addition, the RNA
originating from the integrated HBV DNA further exhibited splicing
properties (chr12), forming fusion transcripts with host exons (chr12.
\textbf{Fig. 3D}). Furthermore, we observed that certain integrated HBV
DNA initiate transcription with the host promoter (chr3), and
transcription ceased via HBV canonical poly(A) (\textbf{Fig 3E}).
Besides, multiple transcriptional modes can arise from the same
integrated HBV DNA (\textbf{Fig. 3F \& Fig. S2A}). Due to the presence
of host homologous sequences between different integration sites, the
origins of two types of transcripts cannot be determined with certainty
(\textbf{Fig. S2C \& S2D}).

To verify the accuracy of long-read sequencing results, we conducted
reverse transcription-polymerase chain reaction (RT-PCR) experiments to
detect HBV chimeric transcripts with the highest expression from chr13
\& t (5;13) and the lowest expression from chr16. The locations of the
primers and the sizes of the amplified fragments are shown in
\textbf{Fig. 3G and Table S3}. The RT-PCR results reveal that a
prominent amplification band of approximately 500 bp is visible for the
chimeric transcript from chr13 \& t (5;13), while the chimeric
transcript from chr16 is nearly undetectable (\textbf{Fig. 3H}). This
confirms that the transcription level of integrated HBV DNA on chr13 \&
t (5;13) is higher than that on chr16. Furthermore, we performed Sanger
sequencing on the PCR amplification product of the HBV chimeric
transcript from chr13 \& t (5;13), and the obtained sequence consists
ONT long-read sequencing (\textbf{Fig. 3I}), further validating the
accuracy of the results of PLC/PRF/5 cell transcriptome long-read
sequencing.

Taken together, these results suggest significant differences of
transcriptional level and characteristic of integrated HBV DNA on
various site and the preservation of promoters on integrated HBV DNA
fragments is important for the transcription of integrated HBV DNA.

\textbf{~}

\textbf{The effect of chromatin accessibility on the transcription of
integrated HBV DNA is limited in PLC/PRF/5 cells}

The accessibility of chromatin is an essential mechanism to regulate
gene transcription\textsuperscript{22}. To explore the correlation
between chromatin states around HBV integration sites in PLC/PRF/5 cells
and the transcriptional activity of integrated HBV DNA, we further
analyzed the ATAC-seq dataset (GSM4217243) from PLC/PRF/5 cells. We
divided the integrated HBV into two groups based on transcriptional
activity and compared the chromatin accessibility levels around the
integration sites (within 100 kb upstream and downstream). HBV DNA
integrated into chr3, chr11, chr12, and chr13 is comparatively active in
transcription and has been defined as the transcriptionally active
group, whereas HBV DNA integrated into chr4 and chr8 shows low levels of
transcription and has been defined as the transcriptionally inactive
group. The results show that in some regions around transcriptional
active integration sites, ATAC-seq signal are significantly higher than
those around transcriptional inactive integration sites (\textbf{Fig.
4A}). Furthermore, we used the log2CPM of mapped reads in a certain
region to normalize and measure the state of chromatin accessibility in
that region. The correlation analysis between this value and the
transcription level of HBV DNA at each integration site showed a
positive correlation trend, but the correlation was not statistically
significant (\emph{r} = 0.3739, \emph{p} = 0.3216) (\textbf{Fig. 4B}).
Therefore, the role of chromatin accessibility in regulating integrated
HBV DNA transcription is limited in PLC/PRF/5 cells.

The principle of ATAC-seq technology is based on the Tn5 enzyme binds
and cuts open chromatin. When transcription factors were bound to DNA,
it will prevent Tn5 cleavage in an otherwise nucleosome-free regions,
resulting in small regions called footprints\textsuperscript{23}. For
instance, in the case of the transcription factor NFYC, Tn5 cleavage in
these footprints decreases significantly within peak regions of high
cleavage probability (\textbf{Fig. 4C}). Based on this, we used
HINT-ATAC to predict transcription factors that potentially play a role
in transcription of integrated HBV DNA in PLC/PRF/5 cells. We selected
the 100 kb region upstream and downstream of the HBV integration site to
predict and enrich transcription factors that may play a role in the
vicinity of the integration site. As shown in \textbf{Fig. 4D,} NFYC,
KLF15, FOS, JUN, and other transcription factors are enriched around the
HBV integration site compared to randomly selected background regions.
Since previous studies have reported that those transcription factors
can bind to the HBV cccDNA promoter sequence and initiate HBV gene
transcription\textsuperscript{24-26}, we hypothesize that these
transcription factors may bind to integrated HBV DNA or host DNA in the
vicinity of the integration site, participating in the initiation of
integrated HBV DNA transcription.

~

\textbf{Methylation modification inhibits the transcription of
integrated HBV DNA in PLC/PRF/5 cells}

The methylation level of the genome is one of the important factors
affecting gene transcription\textsuperscript{27}. In order to explore
whether the transcription of integrated HBV DNA is influenced by
methylation levels, we firstly compared the average methylation levels
of integrated HBV DNA with the average methylation levels of the whole
human genome in PLC/PRF/5 cells. As shown in \textbf{Fig. 5A}, the
average methylation level of integrated HBV DNA was lower than that of
the human genome (HBV 19.7\% vs. human 32.9\%). Considering that the
transcriptional activity of cccDNA in the form of mini-chromosomes in
hepatocyte nuclei is also regulated by DNA methylation modifications, we
further analyzed the methylation levels of cccDNA extracted from
HBV-infected HepG2-NTCP cells (from dataset
SRR10426842)\textsuperscript{28} and compared them with the methylation
characteristics of integrated HBV DNA in the PLC/PRF/5 cells. The
results showed that the methylation levels of CpG island 1, CpG island
2, and CpG island 3 on integrated HBV DNA\textsuperscript{29} were
higher than those on the corresponding CpG islands of cccDNA
(\textbf{Fig. S3A, B, C}). This result suggests that the episomal HBV
genome itself may be at a low methylation level, and when HBV DNA is
integrated into the human genome, it is methylated by the host
methylation modification system, resulting in suppressed expression.
This high methylation modification of foreign integrated DNA may be one
of the mechanisms of host cell self-protection.

Next, we analyzed the differences in methylation levels of the host
chromosome at various distances from the HBV integration sites in
PLC/PRF/5 cells (500 bp, 1 kb, 2kb, 5 kb, 10kb, 20kb, and 50 kb). As
shown in \textbf{Fig. 5B}, the average methylation level within the 500
bp region nearby HBV integration sites were relatively low compared to
the genome at a distance from the integration site. Subsequently, we
performed a correlation analysis between the average methylation level
of the host genome within the 500 bp region nearby HBV integration sites
and the number of HBV chimeric transcripts from each integration site.
As shown in \textbf{Fig. 5C}, the transcription level of integrated HBV
DNA was strongly negatively correlated with the methylation level
(\emph{r} = -0.8929, \emph{p} = 0.0123). ~The host genome around the HBV
integration sites on chromosomes 5, 11, 12, and 13 was relatively
hypomethylated, as shown in \textbf{Fig. 5B}, and these sites exhibited
high transcriptional activity of HBV DNA. Conversely, the methylation
levels around the integration sites on chromosomes 3, 16, and 17 were
relatively higher (see \textbf{Fig. 5B}), correlating with low
transcriptional activity of the integrated HBV DNA. These results
suggest that methylation status of the adjacent host genome affect the
transcriptional level of integrated HBV DNA.~5-aza-2'-deoxycytidine
(AzaD) is a cytidine deoxynucleo-9 side analogue. It inhibits DNA
methyltransferases (DNMTs), leading to DNA hypomethylation including
gene promoter regions

\textsuperscript{30}. To further verify the effect of DNA methylation on
the transcription of integrated HBV DNA, we used AzaD to inhibit the DNA
methylation and detected the effects of AzaD on the expression of
integrated HBV DNA. The results showed that AzaD treatment significantly
upregulated the HBs RNA levels in PLC/PRF/5 cells, presenting a
dose-dependent effect, with significantly higher HBs RNA levels at
treatment concentrations of 1 \selectlanguage{greek}μ\selectlanguage{english}M and 4 \selectlanguage{greek}μ\selectlanguage{english}M (\textbf{Fig. 5D}). Consistent
with this result, the Western blot results also confirmed that AzaD
treatment upregulated the intracellular HBsAg protein levels in a
dose-dependent manner (\textbf{Fig. 5E}). In addition, we also analyzed
the transcription levels of HBV RNA at each integration site before and
after {AzaD} treatment using quantitative reverse transcriptase PCR
(qRT-PCR). The results showed that the transcription level of HBV DNA
integrated into chr17 significantly increased after AzaD treatment
(\textbf{Fig. 5F}), and this integration site has the highest level of
methylation among HBV integration sites in the PLC/PRF/5 cell line
(\textbf{Fig. 5B}). These results further confirm that the transcription
of integrated HBV in PLC/PRF/5 cells is regulated by methylation.

\textbf{~}

\textbf{Transcription of integrated HBV DNA is associated with histone
modifications in PLC/PRF/5 cells}

Previous studies have demonstrated that H3K4Me3 is presented at the
transcription start site (TSS) of actively transcribed genes, promoting
transcription by rapidly recruiting RNA polymerase for mRNA synthesis,
while H3K9Me3 is predominantly found in transcriptionally silent
heterochromatin, hindering RNA polymerase access to the promoter region
and inhibiting mRNA transcription\textsuperscript{31}. To investigate
whether the transcription of integrated HBV DNA is associated with
histone modifications in PLC/PRF/5 cells, we analyzed the effect of
modifications of H3K9Me3 and H3K9Me3 on transcription of integrated DNA
in the PLC/PRF/5 using Chip-seq data from GEO database.

Firstly, to comprehend the distinct deposition of histone
post-translational modifications (PTMs) on cccDNA and integrated HBV
DNA, we compared the deposition patterns of two histone modifications on
integrated HBV DNA in PLC/PRF/5 cells and cccDNA from liver biopsy
samples of HBeAg-positive (HBeAg\textsuperscript{+}) and HBeAg-negative
(HBeAg\textsuperscript{-}) CHB patient's liver biopsy samples
(\textbf{Fig. 6A \& Fig. S4}). We observed three deposition features:
first, the inhibitory H3K9me3 modification is less enriched on
integrated HBV DNA, HBeAg\textsuperscript{+} cccDNA, and HBeAg- cccDNA
compared to the H3K4me3; second, the deposition of H3K4me3 is relatively
highly enriched and exhibits a similar pattern on integrated HBV DNA and
HBeAg\textsuperscript{+} patient cccDNA, while its enrichment level is
lower on HBeAg- patient cccDNA; third, on integrated HBV DNA and
HBeAg\textsuperscript{+} cccDNA, the activating histone PTM H3K4me3 is
highly enriched around promoter SP1 and SP2 and the S gene ORF, while
the inhibitory histone PTM H3K9me3 is relatively less enriched. Taken
together, the deposition pattern of histone PTM on integrated HBV DNA
and HBeAg\textsuperscript{+} cccDNA is similar, with highly enriched
activating histone PTM H3K4me3 and relatively low enrichment of
inhibitory histone PTM H3K9me3, promoting active transcription of HBV
DNA.

Next, we explored the correlation between the activating histone PTM
H3K4me3 and the inhibitory histone PTM H3K9me3 on different integration
sites of HBV DNA and integrated HBV DNA transcriptional activity. Since
the ChIP-seq library was sequenced by NGS, limited by read length and
shared overlapping sequences of integrated HBV DNA, we could only select
histone PTM levels at the junction of HBV and the human genome to
represent the histone PTM status at each integration site, and use the
normalized read count of the HBV-host junction reads to represent the
enrichment level of histone PTM. The result showed that the activating
histone PTM H3K4me3 was positively correlated with integrated HBV DNA
transcription levels, although not statistically significant (\emph{r} =
0.6109, \emph{p} = 0.0878), while the inhibitory histone PTM H3K9me3
enrichment level was negatively correlated with transcription levels
(\emph{r} = -0.7806, \emph{p} = 0.0223) (\textbf{Fig. 6B \& 6C}).

Furthermore, we analyzed the histone modification levels of the host
genome within 5 kb upstream and downstream of the integration sites. The
results revealed that integration sites with active transcription
exhibited higher levels of activating histone PTMs, such as H3K4me1,
H3K4me3, and H3K27ac, compared to transcriptionally inactive sites.
Compared to H3K27ac, H3K4me1 and H3K4me3 had higher levels of activating
histone modifications around integration sites (\textbf{Fig. 6D}).
Furthermore, we analyzed the correlation between histone modification
levels around each integration site and integrated HBV DNA transcription
levels at each site. The analysis revealed a significant positive
correlation between H3K4me3 modification levels near integration sites
and integrated HBV DNA transcription levels (\emph{r} = 0.809, \emph{p}
= 0.0083), while no significant correlation was found for H3K4me1 and
H3K27ac modifications (\textbf{Fig. 6E}).

These findings suggest that histone modifications may play a crucial
role in regulating integrated HBV DNA transcription.

~

\textbf{Discussion}

Previous studies have shown that integrated HBV DNA, has the potential
to express HBsAg and truncated HBx protein, ultimately triggering
HCC\textsuperscript{3}. Despite numerous systemic studies revealed the
transcriptional characteristics of HBV cccDNA, detail research regarding
the transcriptional patterns of integrated HBV DNA remains insufficient.
This study is based on genomic architecture of integrated HBV DNA in
PLC/PRF/5 cells identified previously by WGS long read sequencing, and
is the first to comprehensively determine the transcriptional landscape
of each integrated HBV DNA in PLC/PRF/5 cells through RNA long-read
sequencing.

We conducted transcriptome sequencing and obtained the transcription
spectrum of integrated HBV DNA in PLC/PRF/5 cells for the first time.
Ascribing HBV chimeric transcripts to the integrated HBV genome, we
found that, the transcriptional activity of different integrated HBV DNA
varies significantly. The most active transcription of HBV DNA occurred
in those on chr11, chr13, and t (5;13). The integrated HBV DNA on chr4
cannot initiate transcription due to the absence of HBV promoters. This
implies that and the preservation of promoters on integrated HBV DNA
fragments is essential for the transcription of integrated HBV DNA.
However, even though the integrated HBV DNA on chr8 retains complete SP1
and SP2 promoters, it does not undergo transcription. Furthermore,
significant differences exist in the transcription levels of integrated
HBV DNA on various sites, even with complete promoter preservation. This
suggests that host factors may influence the transcriptional activity of
integrated HBV DNA beyond viral promoter sequences.

According to previous research, HBV is more likely to integrate into
transcriptionally active regions\textsuperscript{32}, indicating that
chromatin accessibility around integration sites may impact the
transcription of integrated HBV DNA. Through analyzing the ATAC-seq data
of PLC/PRF/5 cells, we discovered that the chromatin around
transcriptionally active integration sites were more accessible than
that around integration sites without transcription. However,
correlation analysis showed that the accessibility of chromatin near the
integrated HBV DNA does not have a significant correlation with its
transcription level, suggesting that the accessibility of chromatin has
limited regulatory effects on the transcription of integrated HBV DNA in
the PLC/PRF/5 cell line. Furthermore, some transcription factors were
predicted that may function around HBV integration sites, including
NFYC, KLF15, FOS, JUN, and JUNB by ATAC-seq data. Previous research has
shown that these transcription factors can bind to HBV promoters and
enhance their transcriptional activity. For instance, NFYC can bind to
HBV's Sp1 promoter\textsuperscript{26}, KLF15 can bind to HBV's Sp1 and
CP promoters\textsuperscript{24}, and JUN-FOS can bind to the Sp2
promoter\textsuperscript{25}. These results suggest that transcription
factors may regulate the transcription of integrated HBV DNA in the
PLC/PRF/5 cell line, but still need further experimental verification to
validate their actual functions on the transcription of integrated HBV.

In eukaryotic cells, CpG islands in the promoter regions of host genes
are modified under the action of methyltransferases, thereby inhibiting
gene transcription\textsuperscript{33}. The literature reports that
cccDNA contains three CpG islands, which also undergo methylation and
affect virus replication\textsuperscript{34}\begin{CJK}{UTF8}{gbsn}。\end{CJK}\selectlanguage{english}Watanabe' study has found
that the methylation level of integrated HBV DNA is strongly correlated
with the methylation level of the adjacent host
genome\textsuperscript{35}, Watanabe's study has found that the
methylation level of integrated HBV DNA is strongly correlated with the
methylation level of the adjacent host genome, but it is still unclear
whether the methylation of integrated HBV is regulated by the host\begin{CJK}{UTF8}{gbsn}。\end{CJK}\selectlanguage{english}Our
study found that the methylation level of integrated HBV DNA is higher
than that of cccDNA. This result suggests that the episomal HBV genome
itself may be at a low methylation level, and when HBV DNA is integrated
into the human genome, it is methylated by the host methylation
modification system, resulting in suppressed expression. This high
methylation modification of foreign integrated DNA may be one of the
mechanisms of host cell self-protection. However, it has not yet been
elucidated whether methylation affects the transcription of integrated
HBV. In this study, we found that the methylation level of integrated
HBV DNA was higher than that of cccDNA but lower than that of the host
genome. In addition, we observed a significant negative correlation
between methylation of host genome around integration sites and HBV DNA
transcription levels. These results suggest that the HBV genome is
methylated to inhibit transcription after it is integrated into the host
genome, and methylation modification is an important mechanism affecting
the replication of integrated HBV.

Histone PTMs is another epigenetic modification which plays an important
role in regulating gene transcription, occurring in both cccDNA and the
host genome\textsuperscript{36}. In this study, we found that the
H3K4me3 in the S gene region of integrated HBV DNA in PLC/PRF/5 cells
had a similar deposition pattern to that of cccDNA from
HBeAg\textsuperscript{+} patients, which suggests both integrated HBV
DNA in PLC/PRF/5 cells and cccDNA from HBeAg\textsuperscript{+} patients
have a higher transcription activity. Furthermore, we observed a highly
enrichment of inhibitory histone PTM H3K9me3 in integrated HBV DNA
without transcription on t (1;8), indicating that the chromatin around
this integration site was in a state of heterochromatin and consequently
inhibiting the transcription of HBV DNA. Additionally, we observed that
on the integrated HBV DNA in the PLC/PRF/5 cell line, the deposition of
H3K4me3 and H3K9me3 seems to be mutually exclusive. However, in the
liver tissues of patients with chronic hepatitis B, H3K4me3 and H3K9me3
are not always mutually exclusive, indicating that the epigenetic
modifications of HBV cccDNA in the human liver are more complex.

In addition to the promoter of integrated HBV, methylation levels, and
histone modification levels, we also focused on the impact of the cell
cycle on HBV transcription in PLC/PRF/5 cells. We found that, compared
to PLC/PRF/5 cells cultured with 10\% FBS, those cultured with 0.5\% FBS
experienced cell cycle arrest, and at the same time, the transcription
level of integrated HBV was increased (\textbf{Fig. S5}).

~~~~~~~ This study also has many limitations. In our analysis of the
correlation between histone modifications of integrated HBV DNA and its
transcription levels, we only included data for H3K4me3 and H3K9me3
histones. Although these two histones are representative, future studies
will need to analyze correlations with more histone modifications to
comprehensively validate the impact of epigenetic modifications on the
transcription of integrated HBV DNA. Additionally, HBV DNA fragments
that are too short or transcribed from homologous regions cannot be
accurately sourced, which will affect the precision of result analysis.
In this study, we focused solely on the transcriptional regulation of
HBV integration in the PLC/PRF/5 cell line. In the future, it is
necessary to analyze the transcriptional regulation of HBV integration
in primary hepatocytes under natural infection conditions using
single-cell multi-omics technologies.

In summary, through comprehensive analysis, we successfully constructed
an integration and transcription map of HBV integration in the PLC/PRF/5
cell line. Our research confirms that the structure of integrated HBV
DNA, methylation levels, and the modification levels of H3K4me3 and
H3K9me3 histones affect the transcription of integrated HBV DNA. These
results suggest that the regulation of epigenetic modifications in
integrated HBV DNA could lead to transcriptional silencing and provide
novel insights to improve the functional cure rate for CHB patients
while reducing the incidence of liver cancer.

\par\null

\textbf{Data Availability Statement}

The datasets generated during and/or analyzed during the current study
are available from the corresponding author on reasonable request. Other
datasets from public databases are listed in Table S2.

\textbf{Author contributions}

Study concept and design was performed by XC, FL, GG and AA. Analysis
and interpretation of data were performed by AA, GG, CC, ZG, JY, TZ, ZZ.
Administrative, technical, or material support was performed by XC and
FL. Study supervision was performed by XC. The first draft of the
manuscript was written by XC, AA, GG, CC, and XC, and all authors
commented on.

\textbf{Conflict of Interest statement}

The authors declare that they have no competing interests

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-11/figure-1-ya}
\caption{{\textbf{~Analysis flow chart of HBV integration in PLC/PRF/5 cells. ~}
{\label{277569}}%
}}
\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/figure-2-ya}
\caption{{\textbf{~Transcriptional~profiles of HBV integrations in PLC/PRF/5
cells.~The light blue and pink colors representing the human and HBV
genomes, respectively. The numbers depict the base position of the HBV
genome. The black arrows indicate the transcription start site and
direction, and the HBV promoters are represented by different colored
boxes. The integrated HBV DNA in PLC/PRF/5 cells on chr11, chr13, and t
(5;13) shares a nearly 230 bp homologous junction sequence of at the
flanking host genome sequence. We labeled the homologous sequence as
junction.}%
}}
\end{center}
\end{figure}

{}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/figure-3-ya}
\caption{{\textbf{Transcriptional~characteristics of integrated HBV DNA in
PLC/PRF/5. (A) Quantification of chimeric transcripts derived from
different integrated HBV DNA. The counts of chimeric HBV RNA reads
originating from different integration sites was used to represent the
transcription level of HBV DNA at different integration sites. (B) IGV
snapshot of HBV DNA transcription integrated on chr11. Pink is the HBV
sequence, light blue is the host sequence, and gray is the junction
sequence. Integration of HBV transcription is usually accompanied by the
addition of a poly(A) tail in humans, taking HBV integration in
chromosome 11 as an example. (C) IGV snapshot of transcription patterns
of HBV DNA on chr13.~ (D) IGV snapshot of HBV DNA transcription
integrated on chr12. The pink color in ONT reads is the HBV sequence
(3196\textasciitilde{}3221/1\textasciitilde{}458nt), the light blue is
the MVK gene exon sequence, the green represents the polyA, and the dark
blue is the RNA splicing information. (E) IGV snapshot of HBV DNA
transcription integrated on chr3. (F) Schematic representation of two
transcription patterns of integrated HBV DNA on chr17.}%
}}
\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/figure-4-ya}
\caption{{\textbf{Analysis of Chromatin Accessibility Around HBV Integration Sites
in PLC/PRF/5 Cells and Prediction of Related Transcription Factors.~(A)
The ATAC-seq signal density plots within 100 kb upstream and downstream
of transcriptional active (green) and transcriptional inactive (blue)
integration sites. (B) Dot plot of the ATAC-seq accessibility and
integrated HBV DNA transcription level. (C) The NFYC transcription
factor aggregates on chromosome 1 of PLC/PRF/5 cells and can impede Tn5
enzyme cleavage when it binds to DNA sequences, reducing the likelihood
of cutting in regions marked by NFYC. (D) Predicted transcription
factors that bind to the genome at 100 kb upstream and downstream near
the HBV integration site.}%
}}
\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/figure-5-ya}
\caption{{\textbf{DNA~methylation inhibits integrated HBV DNA transcription.~(A)
genome-wide methylation levels of integrated HBV DNA and host genome.CG,
CHG and CHH represent the three forms of C nucleotide distribution on
the genome (H represents A, T or C nucleotide). (B) Heat map of host
genome methylation levels around integration sites in the PLC/PRF/5 cell
line. (C) Correlation between host genome methylation levels within 500
bp upstream and downstream of HBV integration sites and the
transcriptional level of integrated HBV DNA. (Methylation levels of
genome around Integration sites on chromosome 4 and chromosome 8 with
severely outlying were excluded from the correlation analysis.) The
y-axis represents log2-transformed HBV chimeric read counts from each
integration site, indicating the transcriptional level of each
integration site. (D) RT-qPCR was used to detect the relative expression
of intracellular HBV RNA of PLC/PRF/5 cells before and after AzaD
treatment using primer that targeting S region (329-451 nt) of HBV. The
control group and experimental group were treated with DMSO and
different concentrations of AzaD, respectively, for 48 h before
harvesting. (E) Western Blot was used to detect the intracellular HBsAg
level of PLC/PRF/5 cells before and after AzaD treatment. (F)
Quantitative PCR detection of transcription levels at each HBV
integration sites in PLC/PRF/5 cells before and after AzaD treatment
using primer that targeting the specific sequence of each integrated
sites (Table S1). The expression level of genes was normalized using
\selectlanguage{greek}β-\selectlanguage{english}actin. * Indicates P \textless{} 0.05, **** indicates P \textless{}
0.0001.}%
}}
\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6/figure-6-ya}
\caption{{\textbf{H3K4me3~and H3K9me3 deposition on integrated HBV-DNA correlates
with viral transcription.~(A) Distribution of H3K4me3 and H3K9me3 along
the HBV genome in different samples. (B-C) Pearson correlation was used
to evaluate the association between histone PTMs H3K4me3 and H3K9me3 on
integrated HBV DNA and its transcription levels. The horizontal axis
represents histone modification levels of each HBV integrated site,
which were represented by taking the log2 ratio of Chip-seq RPM/input
RPM. The vertical axis represents the transcription levels of each
integrated site, which were represented by taking the log2 of the count
of HBV chimeric reads from each integrated site. (D) The green and blue
lines represent the ChIP-seq average signal for histone PTM within 5kb
upstream and downstream of the integration site, the with and without
transcriptional activity, respectively. (E) Pearson correlation between
average histone ChIP-seq signal of each integration site (5 kb upstream
and downstream) and its corresponding transcriptional activity. The
vertical axis represents the transcription levels of each integrated
site, which were determined by computing the log2 value of the counts of
reads from HBV chimeric reads from each respective integration site.}%
}}
\end{center}
\end{figure}

\selectlanguage{english}
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\end{document}

