Analysis of HBV Genomes Integrated into the Genomes of Human Hepatoma PLC/PRF/5 Cells by HBV Sequence Capture-Based Next-Generation Sequencing

Ishii, Tomotaka; Tamura, Akinori; Shibata, Toshikatsu; Kuroda, Kensuke; Sugiyama, Masaya; Mizokami, Masashi; Moriyama, Mitsuhiko

doi:10.3390/genes11060661

Cited by 21 publications

(18 citation statements)

References 49 publications

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“…As early as the 1980, researchers have found that PLC/PRF/5 cell line genome contains HBV genome integration (Edman et al, 1980). Since then, the HBV integration of PLC/PRF/5 cell line had been successively detected by Northern blot, FISH, Alu-PCR and NGS technology (Jiang et al, 2012;Li et al, 2013;Watanabe et al, 2015;Ishii et al, 2020). Although researchers used the HBV-specific probe capture technology to improve the sensitivity of HBV DNA detection and found that PLC/PRF/5 has HBV integration on chromosomes 3, 4, 5, 8, 10, 11, 12, 13, 16, 17, 19 (Li et al, 2013;Watanabe et al, 2015;Ishii et al, 2020), there were selective bias in this technology so that it is impossible to have a comprehensive analysis of the characteristics of PLC/PRF/5 HBV integration.…”

Section: Discussionmentioning

confidence: 99%

“…Since then, the HBV integration of PLC/PRF/5 cell line had been successively detected by Northern blot, FISH, Alu-PCR and NGS technology (Jiang et al, 2012;Li et al, 2013;Watanabe et al, 2015;Ishii et al, 2020). Although researchers used the HBV-specific probe capture technology to improve the sensitivity of HBV DNA detection and found that PLC/PRF/5 has HBV integration on chromosomes 3, 4, 5, 8, 10, 11, 12, 13, 16, 17, 19 (Li et al, 2013;Watanabe et al, 2015;Ishii et al, 2020), there were selective bias in this technology so that it is impossible to have a comprehensive analysis of the characteristics of PLC/PRF/5 HBV integration. Meanwhile, it is hard to analyze the complex variation of the genome sequence surrounding the HBV integration sites through NGS because the length of the sequencing fragment was only 100 bp.…”

Section: Discussionmentioning

confidence: 99%

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Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System

Chen

Guan

et al. 2021

Front. Mol. Biosci.

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The integration of HBV DNA is one of the carcinogenic mechanisms of HBV. The clearance of HBV integration in hepatocyte is of great significance to cure chronic HBV infection and thereby prevent the occurrence of HBV-related hepatocellular carcinoma (HCC). However, the low throughput of traditional methods, such as Alu-PCR, results in low detecting sensitivity of HBV integration. Although the second-generation sequencing can obtain a large amount of sequencing data, but the sequencing fragments are extremely short, so it cannot fully explore the characteristics of HBV integration. In this study, we used the third-generation sequencing technology owning advantages both in sequencing length and in sequencing depth to analyze the HBV integration characteristics in PLC/PRF/5 cells comprehensively. A total of 4,142,311 cleaning reads was obtained, with an average length of 18,775.6 bp, of which 84 reads were fusion fragments of the HBV DNA and human genome. These 84 fragments located in seven chromosomes, including chr3, chr4, chr8, chr12, chr13, chr16, and chr17. We observed lots of DNA rearrangement both in the human genome and in HBV DNA fragments surrounding the HBV integration site, indicating the genome instability causing by HBV integration. By analyzing HBV integrated fragments of PLC/PRF/5 cells that can potentially express HBsAg, we selected three combinations of sgRNAs targeting the integrated fragments to knock them out with CRISPR/Cas9 system. We found that the sgRNA combinations could significantly decrease the level of HBsAg in the supernatant of PLC/PRF/5 cells, while accelerated cell proliferation. This study proved the effectiveness of third-generation sequencing to detect HBV integration, and provide a potential strategy to reach HBsAg clearance for chronic HBV infection patients, but the knock-out of HBV integration from human genome by CRISPR/Cas9 system may have a potential of carcinogenic risk.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System

Chen

Guan

et al. 2021

Front. Mol. Biosci.

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“…Although integrated HBV DNA is thought to be a dead end in terms of viral replication, integrated HBV can produce viral proteins, creating an obstacle to complete viral antigen clearance. In addition, integration may lead to chromosomal instability, which could be a key contributor for progression to HCC although the mechanism is not clearly defined [8].…”

Section: J O U R N a L P R E -P R O O Fmentioning

confidence: 99%

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B

et al. 2022

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“…Berthillon et al infected the human hepatoma cell line, PLC/PRF/5 [ 71 ], which integrates HBV DNA and produces HBsAg, with the HAV CF53 strain [ 72 ]. The inhibition of HBsAg production in PLC/PRF/5 cells infected with HAV was observed, compared with those without HAV infection, demonstrating that HAV interferes with the expression of HBsAg from hepatocytes harboring integrated HBV DNA sequences [ 71 ].…”

Section: Coinfection Of Hav With Hbvmentioning

confidence: 99%

Co-Occurrence of Hepatitis A Infection and Chronic Liver Disease

Sasaki

Masuzaki

Takahashi

et al. 2020

IJMS

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Hepatitis A virus (HAV) infection occasionally leads to a critical condition in patients with or without chronic liver diseases. Acute-on-chronic liver disease includes acute-on-chronic liver failure (ACLF) and non-ACLF. In this review, we searched the literature concerning the association between HAV infection and chronic liver diseases in PubMed. Chronic liver diseases, such as metabolic associated fatty liver disease and alcoholic liver disease, coinfection with other viruses, and host genetic factors may be associated with severe hepatitis A. It is important to understand these conditions and mechanisms. There may be no etiological correlation between liver failure and HAV infection, but there is an association between the level of chronic liver damage and the severity of acute-on-chronic liver disease. While the application of an HAV vaccination is important for preventing HAV infection, the development of antivirals against HAV may be important for preventing the development of ACLF with HAV infection as an acute insult. The latter is all the more urgent given that the lives of patients with HAV infection and a chronic liver disease of another etiology may be at immediate risk.

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Analysis of HBV Genomes Integrated into the Genomes of Human Hepatoma PLC/PRF/5 Cells by HBV Sequence Capture-Based Next-Generation Sequencing

Cited by 21 publications

References 49 publications

Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System

Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B

Co-Occurrence of Hepatitis A Infection and Chronic Liver Disease

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